CA2765916A1 - Fuel compositions comprising isoprene derivatives - Google Patents

Abstract

The invention provides for methods, compositions and systems using bioisoprene derived from renewable carbon for production of a variety of hydrocarbon fuels and fuel additives.

Description

DEMANDE OU BREVET VOLUMINEUX

LA PRRSENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS

THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

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FUEL COMPOSITIONS COMPRISING ISOPRENE DERIVATIVES
CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/187,959, filed June 17, 2009, the disclosure of which is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION

[0002] The development of renewable transportation fuels is one of the key challenges of the twenty-first century. The current market is dominated by ethanol derived from yeast fermentation of sucrose and starch, and to a lesser extent by biodiesel (fatty acid esters) derived from triglycerides. Ethanol has limitations as a liquid fuel with a lower energy density relative to hydrocarbons. In addition, ethanol cannot be transported in conventional infrastructure due to its affinity for water and corrosive nature. Processes for the conversion of renewable carbon sources (biomass, sugars, oils) to hydrocarbon fuels offer an attractive alternative to bioethanol.

[0003] Isoprene (2-methyl-1,3-butadiene) is a key industrial chemical used primarily for the production of synthetic rubber. Currently isoprene is derived from petrochemical sources either directly by cracking of naphtha and other light petroleum fractions, or indirectly through chemical synthesis (See, for examples, H. Pommer and A. Nurrenbach, Industrial Synthesis of Terpene Compounds, Pure Appl. Chem., 1975, 43, 527-551; H. M. Weitz and E.
Loser, Isoprene, in Ullmann's Encyclopedia of Industrial Chemistry, Seventh Edition, Electronic Release, Wiley-VCH Verlag GMBH, Weinheim, 2005; and H.M. Lybarger, Isoprene in Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed., Wiley, New York (1995), 14, 934-952.) The resulting crude isoprene streams are typically subjected to extensive purification processes in order to remove numerous chemically similar impurities, many of which can interfere with subsequent transformation of isoprene to polymers and other chemicals.

[0004] In contrast, isoprene derived from biological sources contains very few hydrocarbon impurities and instead contains a number of oxygenated compounds such as ethanol, acetaldehyde and acetone. Many of these compounds can be easily removed by contact with water or passage through alumina or other adsorbents.

[0005] Industry relies on petrochemical feedstocks for isoprene production and extensive purification trains are needed before isoprene can be converted to polymers and other chemicals.
Cost effective methods are desirable for converting biologically produced isoprene to valuable

6 PCT/US2010/039088 chemical products taking advantage of the high purity and/or the unique impurity profiles of bioisoprene.
[0006] All patents, patent applications, documents, and articles cited herein are herein incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

[0007] Disclosed are methods and systems for producing fuel constituents from highly pure isoprene and fuel compositions produced from highly pure isoprene.

[0008] In one aspect, the invention provides a method for producing a fuel constituent from a bioisoprene composition comprising chemically transforming a substantial portion of the isoprene in the bioisoprene composition to non-isoprene compounds. In one embodiment, the bioisoprene composition is chemically transformed by subjecting the bioisoprene composition to heat or catalytic conditions suitable for isoprene dimerization to produce an isoprene dimer and then catalytically hydrogenating the isoprene dimer to forma saturated Cl0 fuel constituent. In another embodiment, the bioisoprene composition is chemically transformed by (i) partially hydrogenating the bioisoprene composition to produce an isoamylene, (ii) dimerizing the isoamylene with a mono-olefin selected from the group consisting of isoamylene, propylene and isobutene to form a dimate and (iii) completely hydrogenating the dimate to produce a fuel constituent. In some embodiments, at least about 95% of isoprene in the bioisoprene composition is converted to non-isoprene compounds during the chemical transformation. In some embodiments, the bioisoprene composition is heated to about 150 C to about 250 C to produce an unsaturated cyclic isoprene dimer and the unsaturated cyclic isoprene dimer is hydrogenated catalytically to produce a saturated cyclic isoprene dimer fuel constituent. In some embodiments, the method comprises: (i) contacting the bioisoprene composition with a catalyst for catalyzing cyclo-dimerization of isoprene to produce an unsaturated cyclic isoprene dimer and the unsaturated cyclic isoprene dimer is hydrogenated catalytically to produce a saturated cyclic isoprene dimer fuel constituent. In some embodiments, the catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of a nickel catalyst, iron catalysts and chromium catalysts. In some embodiments, the step of partially hydrogenating the bioisoprene composition comprises contacting the bioisoprene composition with hydrogen gas and a catalyst for catalyzing partial hydrogenation of isoprene. In some embodiments, the catalyst for catalyzing partial hydrogenation of isoprene comprises a palladium catalyst. In some embodiments, the step of dimerizing the isoamylene with a mono-olefin comprises contacting the isoamylene with the mono-olefin in the presence of a catalyst for catalyzing dimerization of mono-olefin. In some embodiments, the catalyst for catalyzing dimerization of mono-olefin comprises an acid catalyst. In some embodiments, the method further comprises purifying the isoprene from the bioisoprene composition prior to chemically transforming the bioisoprene composition to a fuel constituent.

[0009] In one aspect, the invention provides a system for producing a fuel constituent from a bioisoprene composition, wherein a substantial portion of the isoprene in the bioisoprene composition is chemically converted to non-isoprene compounds, the system comprising a bioisoprene composition and (a) (i) one or more chemicals capable of dimerizing isoprene in the bioisoprene composition or a source of heat capable of dimerizing isoprene in the bioisoprene composition; and (ii) a catalyst capable of hydrogenating the isoprene dimer to form a saturated C10 fuel constituent; or (b) (i) a chemical capable of partially hydrogenating isoprene in the bioisoprene composition to produce an isoamylene, (ii) a chemical capable of dimerizing the isoamylene with mono-olefins selected from the group consisting of isoamylene, propylene and isobutene to form a dimate and (iii) a chemical capable of completely hydrogenating the dimate to produce a fuel constituent.

[0010] In some embodiments of the system, the bioisoprene composition comprising greater than about 2 mg of isoprene and comprising greater than or about 99.94%
isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments of the system, the one or more chemicals capable of dimerizing isoprene comprises catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of ruthenium catalysts, nickel catalysts, iron catalysts and chromium catalysts. In some embodiments of the system, the catalyst for hydrogenating the unsaturated isoprene dimers comprises a catalyst selected from the group consisting of palladium catalysts, nickel catalysts, ruthenium catalysts and rhodium catalysts. In some embodiments of the system, the chemical capable of partially hydrogenating isoprene comprises a palladium catalyst. In some embodiments of the system, the chemical capable of dimerizing the isoamylene with mono-olefins comprises an acid catalyst.

[0011] In one aspect, the invention provides a fuel composition comprising a fuel constituent produced by the methods described herein. In some embodiments, the fuel composition is substantially free of isoprene. In some embodiments, the fuel composition has 813C value which is greater than -22% or within the range of -32% to -24%o.

[0012] In some aspects, the invention provides a system for producing a fuel constituent from isoprene comprising: (a) a commercially beneficial amount of highly pure isoprene; and (b) a fuel constituent produced from at least a portion of the highly pure isoprene;
wherein at least a portion of the commercially beneficial amount of highly pure isoprene undergoes a chemical transformation.

[0013] In some embodiments of the system, the commercially beneficial amount of highly pure isoprene comprises greater than about 2 mg of isoprene and comprising greater than or about 99.94% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the commercially beneficial amount of highly pure isoprene comprises greater than about 2 mg of isoprene and comprising one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the commercially beneficial amount of highly pure isoprene comprises greater than about 2 mg of isoprene and comprising one or more second compounds selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol, 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine; wherein the amount of the second compound relative to the amount of the isoprene is greater than or about 0.01 % (w/w). In some embodiments, the commercially beneficial amount of highly pure isoprene comprises greater than about 2 mg of isoprene and comprising less than or about 0.5 g/L per compound for any compound in the composition that inhibits the polymerization of isoprene. In some preferred embodiments, the commercially beneficial amount of highly pure isoprene is produced by a biological process.

[0014] In some embodiments of the system, the fuel constituent comprises one or more compounds selected from the group consisting of cyclic isoprene dimers and trimers, linear isoprene oligomers, aromatic and alicyclic isoprene derivatives, and oxygenated isoprene derivatives. In some embodiments, the oxygenated isoprene derivatives are compounds selected from the group consisting of alcohols, ketones, esters and ethers derived from isoprene.

[0015] In some embodiments, the fuel constituent comprises cyclic isoprene dimers and the chemical transformation comprises a dimerization reaction of isoprene. In some embodiments, the dimerization reaction is carried out by heating the commercially beneficial amount of highly pure isoprene. In some embodiments, the dimerization reaction of isoprene produces a product comprising unsaturated isoprene dimers and the chemical transformation further comprises a hydrogenation reaction of the unsaturated isoprene dimers. In some embodiments, the system further comprises a catalyst for catalyzing the hydrogenation reaction of the unsaturated isoprene dimers. In some embodiments, the catalyst for catalyzing the hydrogenation reaction of the unsaturated isoprene dimers comprising a catalyst selected from the group consisting of palladium catalysts, nickel catalysts, ruthenium catalysts and rhodium catalysts.

[0016] In some embodiments, the dimerization reaction is carried out by contacting the commercially beneficial amount of highly pure isoprene with a catalyst for catalyzing cyclo-dimerization of isoprene. In some embodiments, the catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of ruthenium catalysts, nickel catalysts, iron catalysts and chromium catalysts. In some embodiments, the catalyst for catalyzing cyclo-dimerization of isoprene is a nickel catalyst and the fuel constituent comprising one or more eight-membered ring dimers of isoprene.

[0017] In some embodiments, the fuel constituent comprises linear and/or cyclic trimers of isoprene and the chemical transformation comprises catalytic trimerization of isoprene.

[0018] In one aspect, the invention provides a method for producing a fuel constituent from isoprene comprising: (a) obtaining a commercially beneficial amount of highly pure isoprene;
and (b) chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent. In some embodiments, the commercially beneficial amount of highly pure isoprene comprises bioisoprene.

[0019] In some embodiments, the commercially beneficial amount of highly pure isoprene is obtained by the steps comprising: (i) culturing cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide under suitable culture conditions for the production of isoprene, wherein the cells (1) produce greater than about 400 nmole/gwcm/hr of isoprene, (2) convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, or (3) have an average volumetric productivity of isoprene greater than about 0.1 mg/Lbroth/hr of isoprene, and (ii) producing isoprene. In some embodiments, the cells further comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide or an MVA pathway polypeptide.

[0020] In some embodiments of the methods described herein, chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent comprises: (i) heating the commercially beneficial amount of highly pure isoprene to about 150 C to about 250 C; (ii) converting at least a portion of the commercially beneficial amount of highly pure isoprene to unsaturated cyclic isoprene dimers; (iii) hydrogenating the unsaturated cyclic isoprene dimers to produce saturated cyclic isoprene dimers; and (iv) producing the fuel constituent. In some embodiments, at least about 20% to about 100% of isoprene in the commercially beneficial amount of highly pure isoprene is converted to unsaturated cyclic isoprene dimers.

[0021] In some embodiments of the method, chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent comprises: (i) contacting the commercially beneficial amount of highly pure isoprene with a catalyst for catalyzing cyclo-dimerization of isoprene, (ii) converting at least a portion of the commercially beneficial amount of highly pure isoprene to cyclic isoprene dimers; and (iii) producing the fuel constituent.

[0022] In some embodiments of the method, chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent comprises: (i) contacting the commercially beneficial amount of highly pure isoprene with a catalyst for catalyzing cyclo-dimerization of isoprene, (ii) converting at least a portion of the commercially beneficial amount of highly pure isoprene to unsaturated cyclic isoprene dimers; (iii) hydrogenating the unsaturated cyclic isoprene dimers to produce saturated cyclic isoprene dimers; and (iv) producing the fuel constituent. In some embodiments of the method, the catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of a nickel catalyst, iron catalysts and chromium catalysts.

[0023] In some embodiments of the method, chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent comprises: (i) contacting the highly pure isoprene composition with a catalyst system; (ii) converting at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers; and (iii) hydrogenating the unsaturated dimers and/or trimers to produce saturated C10 and/or C15 hydrocarbons.

[0024] In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) converting at least a portion of the starting isoprene composition to oxygenated isoprene derivatives; and optionally (c) hydrogenating any unsaturated oxygenated isoprene derivatives to produce saturated oxygenates.
In some embodiments, the oxygenated isoprene derivatives are compounds selected from the group consisting of alcohols, ketones, esters and ethers derived from isoprene.

[0025] In some embodiments, any of the methods described herein further comprises purifying the commercially beneficial amount of highly pure isoprene prior to chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent.

[0026] In one aspect, provided is a continuous process for producing a fuel constituent from isoprene comprising: (a) continuously producing a commercially beneficial amount of highly pure isoprene; and (b) continuously transforming chemically at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent.
In some embodiments, the commercially beneficial amount of highly pure isoprene comprising a gas phase comprising isoprene. In some embodiments, the method further comprises passing the gas phase comprising isoprene to a reactor for chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent.
In a preferred embodiment, the commercially beneficial amount of highly pure isoprene comprises bioisoprene.

[0027] Also provided is a fuel composition comprising a fuel constituent produced by any of the methods described herein. In some embodiments, the fuel constituent comprises less than or about 0.5 g/L a product from a C5 hydrocarbon other than isoprene after undergoing the steps according to the methods described herein. In some embodiments, the fuel constituent comprises one or more product from one or more compound selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol, 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3 -methyl-3 -buten-l-yl acetate, 3 -methyl-2-buten-l-yl acetate, 3 -hexen- l -ol, 3 -hexen-l-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene and 2,3-cycloheptenolpyridine after undergoing the steps according to the methods described herein.

[0028] In some embodiments, the fuel composition comprises a fuel composition having 813C
value which is greater than -22%o. In some embodiments, the fuel composition has 813C value which is within the range of -22%o to -10% or -34%o to -24%o. In some embodiments, the fuel composition has fm value which is greater than 0.9. Also provided is a blend of any of the fuel compositions described herein with a petroleum based fuel in the amount of from about I% to about 95% by weight or volume, based on the total weight or volume of the total fuel composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the nucleotide sequence of a kudzu isoprene synthase gene codon-optimized for expression in E. coli (SEQ ID NO: 1). The atg start codon is in italics, the stop codon is in bold and the added Pstl site is underlined.

[0030] Figure 2 is a map of pTrcKudzu.

[0031] Figures 3A-C are the nucleotide sequence of pTrcKudzu (SEQ ID NO:2).
The RBS is underlined, the kudzu isoprene synthase start codon is in bold capitol letters and the stop codon is in bold, capital letters. The vector backbone is pTrcHis2B.

[0032] Figure 4 is a map of pETNHisKudzu.

[0033] Figures 5A-C are the nucleotide sequence of pETNHisKudzu (SEQ ID NO:3).

[0034] Figure 6 is a map of pCL-lac-Kudzu.

[0035] Figures 7A-C are the nucleotide sequence of pCL-lac-Kudzu (SEQ ID
NO:4).

[0036] Figure 8A is a graph showing the production of isoprene in E. coli BL21 cells with no vector.

[0037] Figure 8B is a graph showing the production of isoprene in E. coli BL21 cells with pCL-lac-Kudzu

[0038] Figure 8C is a graph showing the production of isoprene in E. coli BL21 cells with pTrcKudzu.

[0039] Figure 8D is a graph showing the production of isoprene in E. coli BL21 cells with pETN-HisKudzu.

[0040] Figure 9A is a graph showing OD over time of fermentation of E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

[0041] Figure 9B is a graph showing isoprene production over time of fermentation of E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

[0042] Figure 1 OA is a graph showing the production of isoprene in Panteoa citrea. Control cells without recombinant kudzu isoprene synthase. Grey diamonds represent isoprene synthesis, black squares represent OD600.

[0043] Figure 10B is a graph showing the production of isoprene in Panteoa citrea expressing pCL-lac Kudzu. Grey diamonds represent isoprene synthesis, black squares represent OD600.

[0044] Figure I OC is a graph showing the production of isoprene in Panteoa citrea expressing pTrcKudzu. Grey diamonds represent isoprene synthesis, black squares represent OD600.

[0045] Figure 11 is a graph showing the production of isoprene in Bacillus subtilis expressing recombinant isoprene synthase. BG3594comK is a B. subtilis strain without plasmid (native isoprene production). CF443 is B. subtilis strain BG3594comK with pBSKudzu (recombinant isoprene production). IS on the y-axis indicates isoprene.

[0046] Figures 12A-C are the nucleotide sequence of pBS Kudzu #2 (SEQ ID
NO:5).

[0047] Figure 13 is the nucleotide sequence of kudzu isoprene synthase codon-optimized for expression in Yarrowia (SEQ ID NO:6).

[0048] Figure 14 is a map of pTrex3g comprising a kudzu isoprene synthase gene codon-optimized for expression in Yarrowia.

[0049] Figures 15A-C are the nucleotide sequence of vector pSPZl(MAP29Spb) (SEQ ID
NO:7).

[0050] Figure 16 is the nucleotide sequence of the synthetic kudzu (Pueraria montana) isoprene gene codon-optimized for expression in Yarrowia (SEQ ID NO:8).

[0051] Figure 17 is the nucleotide sequence of the synthetic hybrid poplar (Populus alba x Populus tremula) isoprene synthase gene (SEQ ID NO:9). The ATG start codon is in bold and the stop codon is underlined.

[0052] Figure 18A (Figures 18A1 and 18A2) shows a schematic outlining construction of vectors pYLA 1, pYLI and pYL2 (SEQ ID NO:75, 73, 72, 71, 70, 69).

[0053] Figure 18B shows a schematic outlining construction of the vector pYLA(POP1) (SEQ
ID NO:68, 69).

[0054] Figure 18C shows a schematic outlining construction of the vector pYLA(KZ1)

[0055] Figure 18D shows a schematic outlining construction of the vector pYLI(KZ1) (SEQ
ID NO:66, 67)

[0056] Figure 18E shows a schematic outlining construction of the vector pYLI(MAP29)

[0057] Figure 18F shows a schematic outlining construction of the vector pYLA(MAP29)

[0058] Figure 19A shows the MVA and DXP metabolic pathways for isoprene (based on F.
Bouvier et al., Progress in Lipid Res. 44: 357-429, 2005). The following description includes alternative names for each polypeptide in the pathways and a reference that discloses an assay for measuring the activity of the indicated polypeptide (each of these references are each hereby incorporated by reference in their entireties, particularly with respect to assays for polypeptide activity for polypeptides in the MVA and DXP pathways). Mevalonate Pathway:
AACT;
Acetyl-CoA acetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184:
2116-2122, 2002;
HMGS; Hydroxymethylglutaryl-CoA synthase, MvaS, EC 2.3.3.10. Assay: J.
Bacteriol., 184:
4065-4070, 2002; HMGR; 3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC
1.1.1.34.
Assay: J. Bacteriol., 184: 2116-2122, 2002; MVK; Mevalonate kinase, ERG12, EC
2.7.1.36.
Assay: Curr Genet 19:9-14, 1991. PMK; Phosphomevalonate kinase, ERGS, EC
2.7.4.2, Assay:
Mol Cell Biol., 11:620-631, 1991; DPMDC; Diphosphomevalonate decarboxylase, MVD1, EC
4.1.1.33. Assay: Biochemistry, 33:13355-13362, 1994; IDI; Isopentenyl-diphosphate delta-isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem. 264:19169-19175, 1989. DXP
Pathway:
DXS; 1-Deoxyxylulose-5-phosphate synthase, dxs, EC 2.2.1.7. Assay: PNAS, 94:12857-62, 1997; DXR; 1-Deoxy-D-xylulose 5-phosphate reductoisomerase, dxr, EC 2.2.1.7.
Assay: Eur. J.
Biochem. 269:4446-4457, 2002; MCT; 4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD, EC 2.7.7.60. Assay: PNAS, 97: 6451-6456, 2000; CMK; 4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC 2.7.1.148. Assay: PNAS, 97:1062-1067, 2000; MCS; 2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase, IspF, EC 4.6.1.12. Assay:
PNAS, 96:11758-11763, 1999; HDS; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, ispG, EC
1.17.4.3. Assay: J. Org. Chem., 70:9168 -9174, 2005; HDR; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, IspH, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.

[0059] Figure 19B illustrates the classical and modified MVA pathways. 1, acetyl-CoA
acetyltransferase (AACT); 2, HMG-CoA synthase (HMGS); 3, HMG-CoA reductase (HMGR);
4, mevalonate kinase (MVK); 5, phosphomevalonate kinase (PMK); 6, diphosphomevalonate decarboxylase (MVD or DPMDC); 7, isopentenyl diphosphate isomerase (IDI); 8, phosphomevalonate decarboxylase (PMDC); 9, isopentenyl phosphate kinase (IPK).
The classical MVA pathway proceeds from reaction 1 through reaction 7 via reactions 5 and 6, while a modified MVA pathway goes through reactions 8 and 9. P and PP in the structural formula are phosphate and pyrophosphate, respectively. This figure was taken from Koga and Morii, Microbiology and Mol. Biology Reviews, 71:97-120, 2007, which is incorporated by reference in its entirety, particular with respect to nucleic acids and polypeptides of the modified MVA
pathway. The modified MVA pathway is present, for example, in some archaeal organisms, such as Methanosarcina mazei.

[0060] Figure 20 (Figures 20A and 20B) shows graphs representing results of the GC-MS
analysis of isoprene production by recombinant Y. lipolytica strains without (left) or with (right) a kudzu isoprene synthase gene. The arrows indicate the elution time of the authentic isoprene standard.

[0061] Figure 21 is a map of pTrcKudzu yIDI DXS Kan.

[0062] Figures 22A-D are the nucleotide sequence of pTrcKudzu yIDI DXS Kan (SEQ ID
NO:10).

[0063] Figure 23A is a graph showing production of isoprene from glucose in BL21/pTrcKudzukan. Time 0 is the time of induction with IPTG (400 mol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene ( g/L
headspace or specific productivity ( g/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity ( g/L) and squares represent specific productivity of isoprene ( g/L/OD).

[0064] Figure 23B is a graph showing production of isoprene from glucose in BL21/pTrcKudzu yIDI kan. Time 0 is the time of induction with IPTG (400 mol).
The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene ( g/L
headspace or specific productivity ( g/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity ( g/L) and squares represent specific productivity of isoprene ( g/L/OD).

[0065] Figure 23C is a graph showing production of isoprene from glucose in BL21/pTrcKudzu DXS kan. Time 0 is the time of induction with IPTG (400 mol).
The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene ( g/L
headspace or specific productivity ( g/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity ( g/L) and squares represent specific productivity of isoprene ( g/L/OD).

[0066] Figure 23D is a graph showing production of isoprene from glucose in BL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of induction with IPTG (400 mol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene ( g/L headspace or specific productivity ( g/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity ( g/L) and squares represent specific productivity of isoprene ( g/L/OD).

[0067] Figure 23E is a graph showing production of isoprene from glucose in BL21/pCL
PtrcKudzu. Time 0 is the time of induction with IPTG (400 mol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene ( g/L headspace or specific productivity ( g/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity ( g/L) and squares represent specific productivity of isoprene ( g/L/OD).

[0068] Figure 23F is a graph showing production of isoprene from glucose in BL21/pCL
PtrcKudzu yIDI. Time 0 is the time of induction with IPTG (400 mol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene ( g/L headspace or specific productivity ( g/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity ( g/L) and squares represent specific productivity of isoprene ( g/L/OD).

[0069] Figure 23G is a graph showing production of isoprene from glucose in BL21/pCL
PtrcKudzu DXS. Time 0 is the time of induction with IPTG (400 mol). The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene ( g/L headspace or specific productivity ( g/L headspace/OD). Diamonds represent OD600, circles represent total isoprene productivity ( g/L) and squares represent specific productivity of isoprene ( g/L/OD).

[0070] Figure 23H is a graph showing production of isoprene from glucose in BL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of induction with IPTG
(400 mol).
The x-axis is time after induction; the y-axis is OD600 and the y2-axis is total productivity of isoprene ( g/L headspace or specific productivity ( g/L headspace/OD). Black diamonds represent OD600, black triangles represent isoprene productivity ( g/L) and white squares represent specific productivity of isoprene ( g/L/OD).

[0071] Figure 24 is a map of pTrcKKDyIkIS kan.

[0072] Figures 25A-D are a nucleotide sequence of pTrcKKDyIkIS kan (SEQ ID NO:
11).

[0073] Figure 26 is a map of pCL PtrcUpperPathway.

[0074] Figures 27A-27D is a nucleotide sequence of pCL PtrcUpperPathway (SEQ
ID
NO: 12).

[0075] Figure 28 shows a map of the cassette containing the lower MVA pathway and yeast idi for integration into the B. subtilis chromosome at the nprE locus. nprE
upstream/downstream indicates 1 kb each of sequence from the nprE locus for integration. aprE
promoter (alkaline serine protease promoter) indicates the promoter (-35, -10, +1 transcription start site, RBS) of the aprE gene. MVKl indicates the yeast mevalonate kinase gene. RBS-PMK
indicates the yeast phosphomevalonate kinase gene with a Bacillus RBS upstream of the start site.
RBS-MPD
indicates the yeast diphosphomevalonate decarboxylase gene with a Bacillus RBS
upstream of the start site. RBS-IDI indicates the yeast idi gene with a Bacillus RBS
upstream of the start site.
Terminator indicates the terminator alkaline serine protease transcription terminator from B.
amyliquefaciens. SpecR indicates the spectinomycin resistance marker. "nprE
upstream repeat for amp." indicates a direct repeat of the upstream region used for amplification.

[0076] Figures 29A-D are a nucleotide sequence of cassette containing the lower MVA
pathway and yeast idi for integration into the B. subtilis chromosome at the nprE locus (SEQ ID
NO:13).

[0077] Figure 30 is a map of p9796-poplar.

[0078] Figures 3lA-B are a nucleotide sequence of p9796-poplar (SEQ ID NO:14).

[0079] Figure 32 is a map of pTrcPoplar.

[0080] Figures 33A-C are a nucleotide sequence of pTrcPoplar (SEQ ID NO:15).

[0081] Figure 34 is a map of pTrcKudzu yIDI Kan.

[0082] Figures 35A-C are a nucleotide sequence of pTrcKudzu yIDI Kan (SEQ ID
NO: 16).

[0083] Figure 36 is a map of pTrcKudzuDXS Kan.

[0084] Figures 37A-C are a nucleotide sequence of pTrcKudzuDXS Kan (SEQ ID
NO:17).

[0085] Figure 38 is a map of pCL PtrcKudzu.

[0086] Figures 39A-C are a nucleotide sequence of pCL PtrcKudzu (SEQ ID
NO:18).

[0087] Figure 40 is a map of pCL PtrcKudzu A3.

[0088] Figures 41A-C are a nucleotide sequence of pCL PtrcKudzu A3 (SEQ ID
NO:19).

[0089] Figure 42 is a map of pCL PtrcKudzu yIDI.

[0090] Figures 43A-C are a nucleotide sequence of pCL PtrcKudzu yIDI (SEQ ID
NO:20).

[0091] Figure 44 is a map of pCL PtrcKudzu DXS.

[0092] Figures 45A-D are a nucleotide sequence of pCL PtrcKudzu DXS (SEQ ID
NO:21).

[0093] Figures 46A-E show graphs representing isoprene production from biomass feedstocks.
Panel A shows isoprene production from corn stover, Panel B shows isoprene production from bagasse, Panel C shows isoprene production from softwood pulp, Panel D shows isoprene production from glucose, and Panel E shows isoprene production from cells with no additional feedstock. Grey squares represent OD600 measurements of the cultures at the indicated times post-inoculation and black triangles represent isoprene production at the indicated times post-inoculation.

[0094] Figure 47A shows a graph representing isoprene production by BL21 (),DE3) pTrcKudzu yIDI DXS (kan) in a culture with no glucose added. Squares represent OD600, and triangles represent isoprene produced ( g/ml).

[0095] Figure 47B shows a graph representing isoprene production from 1%
glucose feedstock invert sugar by BL21 (? DE3) pTrcKudzu yIDI DXS (kan). Squares represent OD600, and triangles represent isoprene produced ( g/ml).

[0096] Figure 47C shows a graph representing isoprene production from 1%
invert sugar feedstock by BL21 (? DE3) pTrcKudzu yIDI DXS (kan). Squares represent OD600, and triangles represent isoprene produced ( g/ml).

[0097] Figure 47D shows a graph representing isoprene production from 1% AFEX
corn stover feedstock by BL21 (XDE3) pTrcKudzu yIDI DXS (kan). Squares represent OD6oo, and triangles represent isoprene produced ( g/ml).

[0098] Figures 48A-C show graphs demonstrating the effect of yeast extract of isoprene production. Panel A shows the time course of optical density within fermentors fed with varying amounts of yeast extract. Panel B shows the time course of isoprene titer within fermentors fed with varying amounts of yeast extract. The titer is defined as the amount of isoprene produced per liter of fermentation broth. Panel C shows the effect of yeast extract on isoprene production in E. coli grown in fed-batch culture.

[0099] Figures 49A-C show graphs demonstrating isoprene production from a 500 L
bioreactor with E. coli cells containing the pTrcKudzu + yIDI + DXS plasmid.
Panel A shows the time course of optical density within the 500-L bioreactor fed with glucose and yeast extract.
Panel B shows the time course of isoprene titer within the 500-L bioreactor fed with glucose and yeast extract. The titer is defined as the amount of isoprene produced per liter of fermentation broth. Panel C shows the time course of total isoprene produced from the 500-L
bioreactor fed with glucose and yeast extract.

[0100] Figure 50 is a map of pJMupperpathway2.

[0101] Figures 51A-C are the nucleotide sequence of pJMupperpathway2 (SEQ ID
NO:22).

[0102] Figure 52 is a map of pBS Kudzu #2.

[0103] Figure 53A is a graph showing growth during fermentation time of Bacillus expressing recombinant kudzu isoprene synthase in 14 liter fed batch fermentation. Black diamonds represent a control strain (BG3594comK) without recombinant isoprene synthase (native isoprene production) and grey triangles represent CF443, Bacillus strain BG3594comK with pBSKudzu (recombinant isoprene production).

[0104] Figure 53B is a graph showing isoprene production during fermentation time of Bacillus expressing recombinant kudzu isoprene synthase in 14 liter fed batch fermentation.
Black diamonds represent a control strain (BG3594comK) without recombinant isoprene synthase (native isoprene production) and grey triangles represent CF443, Bacillus strain BG3594comK with pBSKudzu (recombinant isoprene production).

[0105] Figure 54 is a time course of optical density within the 15-L
bioreactor fed with glucose.

[0106] Figure 55 is a time course of isoprene titer within the 15-L bioreactor fed with glucose.
The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0107] Figure 56 is a time course of total isoprene produced from the 15-L
bioreactor fed with glucose.

[0108] Figure 57 is a time course of optical density within the 15-L
bioreactor fed with glycerol.

[0109] Figure 58 is a time course of isoprene titer within the 15-L bioreactor fed with glycerol.
The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0110] Figure 59 is a time course of total isoprene produced from the 15-L
bioreactor fed with glycerol.

[0111] Figures 60A-60C are the time courses of optical density, mevalonic acid titer, and specific productivity within the 150-L bioreactor fed with glucose.

[0112] Figures 61A-61C are the time courses of optical density, mevalonic acid titer, and specific productivity within the 15-L bioreactor fed with glucose.

[0113] Figures 62A-62C are the time courses of optical density, mevalonic acid titer, and specific productivity within the 15-L bioreactor fed with glucose.

[0114] Figure 63A-63C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

[0115] Figures 64A-64C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

[0116] Figures 65A-65C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

[0117] Figures 66A-66C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

[0118] Figure 67A-67C are the time courses of optical density, isoprene titer, and specific productivity within the 15-L bioreactor fed with glucose.

[0119] Figure 68 is a graph of the calculated adiabatic flame temperatures for Series A as a function of fuel concentration for various oxygen levels. The figure legend lists the curves in the order in which they appear in the graph. For example, the first entry in the figure legend (isoprene in air at 40 C) corresponds to the highest curve in the graph.

[0120] Figure 69 is a graph of the calculated adiabatic flame temperatures for Series B as a function of fuel concentration for various oxygen levels with 4% water. The figure legend lists the curves in the order in which they appear in the graph.

[0121] Figure 70 is a graph of the calculated adiabatic flame temperatures for Series C as a function of fuel concentration for various oxygen levels with 5% C02. The figure legend lists the curves in the order in which they appear in the graph.

[0122] Figure 71 is a graph of the calculated adiabatic flame temperatures for Series D as a function of fuel concentration for various oxygen levels with 10% C02. The figure legend lists the curves in the order in which they appear in the graph.

[0123] Figure 72 is a graph of the calculated adiabatic flame temperatures for Series E as a function of fuel concentration for various oxygen levels with 15% C02. The figure legend lists the curves in the order in which they appear in the graph.

[0124] Figure 73 is a graph of the calculated adiabatic flame temperatures for Series F as a function of fuel concentration for various oxygen levels with 20% C02. The figure legend lists the curves in the order in which they appear in the graph.

[0125] Figure 74 is a graph of the calculated adiabatic flame temperatures for Series G as a function of fuel concentration for various oxygen levels with 30% C02. The figure legend lists the curves in the order in which they appear in the graph.

[0126] Figure 75A is a table of the conversion of the CAFT Model results from weight percent to volume percent for series A.

[0127] Figure 75B is a graph of the flammability results from the CAFT model for Series A in Figure 68 plotted as volume percent.

[0128] Figure 76A is a table of the conversion of the CAFT Model results from weight percent to volume percent for series B.

[0129] Figure 76B is a graph of the flammability results from the CAFT model for Series B in Figure 69 plotted as volume percent.

[0130] Figure 77 is a figure depicting the flammability test vessel.

[0131] Figure 78A is a graph of the flammability Curve for Test Series 1: 0%
Steam, 0 psig, and 40 C.

[0132] Figure 78B is a table summarizing the explosion and non-explosion data points for Test Series 1.

[0133] Figure 78C is a graph of the flammability curve for Test Series 1 compared with the CAFT Model.

[0134] Figure 79A is a graph of the flammability curve for Test Series 2: 4%
Steam, 0 psig, and 40 C.

[0135] Figure 79B is a table summarizing the explosion and non-explosion data points for Test Series 2.

[0136] Figure 79C is a graph of the flammability curve for Test Series 2 compared with the CAFT Model.

[0137] Figures 80A-B are a table of the detailed experimental conditions and results for Test Series 1.

[0138] Figure 81 is a table of the detailed experimental conditions and results for Test Series 2.

[0139] Figure 82 is a graph of the calculated adiabatic flame temperature plotted as a function of fuel concentration for various nitrogen/oxygen ratios at 3 atmospheres of pressure.

[0140] Figure 83 is a graph of the calculated adiabatic flame temperature plotted as a function of fuel concentration for various nitrogen/oxygen ratios at 1 atmosphere of pressure.

[0141] Figure 84 is a graph of the flammability envelope constructed using data from Figure 82 and following the methodology described in Example 13. The experimental data points (circles) are from tests described herein that were conducted at 1 atmosphere initial system pressure.

[0142] Figure 85 is a graph of the flammability envelope constructed using data from Figure 83 and following the methodology described in Example 13. The experimental data points (circles) are from tests described herein that were conducted at 1 atmosphere initial system pressure.

[0143] Figure 86A is a GC/MS chromatogram of fermentation off-gas.

[0144] Figure 86B is an expansion of Fig 86A to show minor volatiles present in fermentation off-gas.

[0145] Figure 87A is a GC/MS chromatogram of trace volatiles present in off-gas following cryo-trapping at -78 C.

[0146] Figure 87B is a GC/MS chromatogram of trace volatiles present in off-gas following cryo-trapping at -196 C.

[0147] Figure 87C is an expansion of Figure 87B.

[0148] Figure 87D is an expansion of Figure 87C.

[0149] Figures 88A-B are GC/MS chromatogram comparing C5 hydrocarbons from petroleum-derived isoprene (Figure 88A) and biologically produced isoprene (Figure 88B). The standard contains three C5 hydrocarbon impurities eluting around the main isoprene peak (Figure 88A). In contrast, biologically produced isoprene contains amounts of ethanol and acetone (run time of 3.41 minutes) (Figure 88A).

[0150] Figure 89 is a graph of the analysis of fermentation off-gas of an E.
coli BL21 (DE3) pTrcIS strain expressing a Kudzu isoprene synthase and fed glucose with 3 g/L
yeast extract.

[0151] Figure 90 shows the structures of several impurities that are structurally similar to isoprene and may also act as polymerization catalyst poisons.

[0152] Figure 91 is a map of pTrcHis2AUpperPathway (also called pTrcUpperMVA).

[0153] Figures 92A-92C are the nucleotide sequence of pTrcHis2AUpperPathway (also called pTrcUpperMVA) (SEQ ID NO:23).

[0154] Figure 93 is a time course of optical density within the 15-L
bioreactor fed with glucose.

[0155] Figure 94 is a time course of isoprene titer within the 15-L bioreactor fed with glucose.
The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0156] Figure 95 is a time course of total isoprene produced from the 15-L
bioreactor fed with glucose.

[0157] Figure 96 is a time course of optical density within the 15-L
bioreactor fed with invert sugar.

[0158] Figure 97 is a time course of isoprene titer within the 15-L bioreactor fed with invert sugar. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0159] Figure 98 is a time course of total isoprene produced from the 15-L
bioreactor fed with invert sugar.

[0160] Figure 99 is a time course of optical density within the 15-L
bioreactor fed with glucose.

[0161] Figure 100 is a time course of isoprene titer within the 15-L
bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0162] Figure 101 is a time course of isoprene specific activity from the 15-L
bioreactor fed with glucose.

[0163] Figure 102 is a map of pCLPtrcUpperPathwayHGS2.

[0164] Figures 103A-103C are the nucleotide sequence of pCLPtrcUpperPathwayHGS2 (SEQ
ID NO:24).

[0165] Figure 104 is a time course of optical density within the 15-L
bioreactor fed with glucose.

[0166] Figure 105 is a time course of isoprene titer within the 15-L
bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0167] Figure 106 is a time course of total isoprene produced from the 15-L
bioreactor fed with glucose.

[0168] Figure 107 is a map of plasmid MCM330 (FRT-cm-FRT-gil.2-KKDy at attTn7).

[0169] Figures 108A-108C are the nucleotide sequence of plasmid MCM330 (SEQ ID
NO:25).

[0170] Figure 109 is a map of pET24D-Kudzu.

[0171] Figures 1 l0A-B are the nucleotide sequence of pET24D-Kudzu (SEQ ID
NO:26).

[0172] Figure l 1 lA is a time course of optical density within the 15-L
bioreactor fed with glucose.

[0173] Figure 11 lB is a time course of isoprene titer within the 15-L
bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0174] Figure 111 C is a time course of specific productivity of isoprene in the 15-L bioreactor fed with glucose.

[0175] Figure 112A is a map of the M. mazei archaeal Lower Pathway operon.

[0176] Figures 112B-C are the nucleotide sequence of the M. mazei archaeal lower Pathway operon (SEQ ID NO:27).

[0177] Figure 113A is a map of MCM382 - pTrcKudzuMVK(mazei).

[0178] Figures 113B-C are the nucleotide sequence of MCM382 -pTrcKudzuMVK(mazei) (SEQ ID NO:28).

[0179] Figure 114A is a map of MCM376 - MVK from M. mazei archaeal Lower in pET200D.

[0180] Figures 114B-C are the nucleotide sequence of MCM376 - MVK from M.
mazei archaeal Lower in pET200D (SEQ ID NO:29).

[0181] Figures 115A-115D demonstrate that over-expression of MVK and isoprene synthase results in increased isoprene production. Accumulated isoprene and CO2 from MCM401 and MCM343 during growth on glucose in 100 mL bioreactors with 100 and 200 M IPTG
induction of isoprene production was measured over a 22 hour time course.
Figure l 15A is a graph of the accumulated isoprene (%) from MCM343. Figure 115B is a graph of the accumulated isoprene (%) from MCM401. Figure 115C is a graph of the accumulated CO2 (%) from MCM343. Figure 115D is a graph of the accumulated CO2 (%) from MCM401.

[0182] Figure 116 is a time course of optical density within the 15-L
bioreactor fed with glucose.

[0183] Figure 117 is a time course of isoprene titer within the 15-L
bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0184] Figure 118 is a time course of total isoprene produced from the 15-L
bioreactor fed with glucose.

[0185] Figure 119 is a graph of the total carbon dioxide evolution rate (TCER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.

[0186] Figure 120 is a graph of the cell viability during isoprene production within the 15-L
bioreactor fed with glucose. TVC/OD is the total viable counts (colony forming units) in 1 mL
of broth per optical density unit (OD550)=

[0187] Figure 121 is a time course of optical density within the 15-L
bioreactor fed with glucose.

[0188] Figure 122 is a time course of isoprene titer within the 15-L
bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0189] Figure 123 is a time course of total isoprene produced from the 15-L
bioreactor fed with glucose.

[0190] Figure 124 is a time course of volumetric productivity within the 15-L
bioreactor fed with glucose. The volumetric productivity is defined as the amount of isoprene produced per liter of broth per hour.

[0191] Figure 125 is a time course of instantaneous yield within the 15-L
bioreactor fed with glucose. The instantaneous yield is defined as the amount of isoprene (gram) produced per amount of glucose (gram) fed to the bioreactor (w/w) during the time interval between the data points.

[0192] Figure 126 is a graph of the total carbon dioxide evolution rate (TCER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.

[0193] Figure 127 is cell viability during isoprene production within the 15-L
bioreactor fed with glucose. TVC/OD is the total viable counts (colony forming units) in 1 mL
of broth per optical density unit (OD550)=

[0194] Figure 128 is a time course of optical density within the 15-L
bioreactor fed with glucose.

[0195] Figure 129 is a time course of isoprene titer within the 15-L
bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth.

[0196] Figure 130 is a time course of total isoprene produced from the 15-L
bioreactor fed with glucose.

[0197] Figure 131 is a graph of total carbon dioxide evolution rate (TCER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.

[0198] Figure 132 is a graph showing that a transient decrease in the airflow to the bioreactor caused a spike in the concentration of isoprene in the off-gas that did not cause a dramatic decrease in metabolic activity (TCER). TCER, or metabolic activity, is the total carbon dioxide evolution rate.

[0199] Figure 133 is a graph of the cell viability during isoprene production within the 15-L
bioreactor fed with glucose. TVC/OD is the total viable counts (colony forming units) in 1 mL
of broth per optical density unit (OD550)=

[0200] Figure 134 is a time course of optical density within the 15-L
bioreactor fed with glucose. Dotted vertical lines denote the time interval when isoprene was introduced into the bioreactor at a rate of 1 g/L/hr.

[0201] Figure 135 is total carbon dioxide evolution rate (TCER), or metabolic activity profile, within the 15-L bioreactor fed with glucose. Dotted vertical lines denote the time interval when isoprene was introduced into the bioreactor at a rate of 1 g/L/hr.

[0202] Figure 136 is cell viability during isoprene production within the 15-L
bioreactor fed with glucose. TVC/OD is the total viable counts (colony forming units) in 1 mL
of broth per optical density unit (OD550). Dotted vertical lines denote the time interval when isoprene was introduced into the bioreactor at a rate of 1 g/L/hr.

[0203] Figures 137A-B are the sequence of Populus alba pET24a: isoprene synthase gene highlighted in bold letters (SEQ ID NO:30).

[0204] Figures 137C-D are the sequence of Populus nigra pET24a: isoprene synthase gene highlighted in bold letters (SEQ ID NO:3 1).

[0205] Figures 137E-F are the sequence of Populus tremuloides pET24a (SEQ ID
NO:32).

[0206] Figure 137G is the amino acid sequence of Populus tremuloides isoprene synthase gene (SEQ ID NO:33).

[0207] Figures 137H-I are the sequence of Populus trichocarpa pET24a: isoprene synthase gene highlighted in bold letters (SEQ ID NO:34).

[0208] Figures 137J-K are the sequence of Populus tremula x Populus alba pET24a: isoprene synthase gene highlighted in bold letters (SEQ ID NO:35).

[0209] Figure 137L is a map of MCM93 which contains the kudzu IspS coding sequence in a pCR2.1 backbone.

[0210] Figures 137M-N are the sequence of MCM93 (SEQ ID NO:36).

[0211] Figure 1370 is a map of pET24D-Kudzu.

[0212] Figures 137P-Q are the sequence of pET24D-Kudzu (SEQ ID NO:37).

[0213] Figure 138 is isoprene synthase expression data for various poplar species as measured in the whole cell head space assay. Y-axis is g/L/01) of isoprene produced by 0.2 mL of a culture induced with IPTG.

[0214] Figure 139 is relative activity of Poplar isoprene synthase enzymes as measured by DMAPP assay. Poplar enzymes have significantly higher activity than the isoprene synthase from Kudzu. Poplar [alba x tremula] only had traces (< I%) of activity and is not shown in the plot.

[0215] Figure 140 is a map of pDONR22l :19430 - hybrid_HGS.

[0216] Figure 141 is the nucleotide sequence of pDONR22l :19430 - hybrid HGS, the sequence of Kudzu isoprene synthase codon-optimized for yeast (SEQ ID NO:38).

[0217] Figure 142A is a map of pDW14.

[0218] Figures 142B-C are the complete nucleotide sequence of pDW14 (SEQ ID
NO:39).

[0219] Figure 143 shows induced INVSc-l strains harboring pDW14 or pYES-DEST52.
Figure 143A. A 4-12% bis tris gel (Novex, Invitrogen) of lysates generated from INVSc-1 strains induced with galactose and stained with SimplyBlue SafeStain (Invitrogen). Figure 143B.
Western blot analysis of the same strains using the WesternBreeze kit (Invitrogen). Lanes are as follows: 1, INVSc-l + pYES-DEST52; 2, INVSc-l + pDW14 (isolate 1); 3, INVSc-l + pDW14 (isolate 2). MW (in kDa) is indicated (using the SeeBlue Plus2 molecular weight standard).

[0220] Figure 144 (Figures 144A and 144B) shows induced INVSc-l strains harboring pDW14 or pYES-DEST52. Figure 144A. OD600 of galactose-induced strains prior to lysis. The y-axis is OD600. Figure 144B. DMAPP assay of isoprene synthase headspace in control and isoprene synthase-harboring strains. Specific activity was calculated as g HG/L/OD. Samples are as follows: Control, INVSc-1 + pYES-DEST52; HGS-1, INVSc-1 + pDW14 (isolate 1);
HGS-2, INVSc-l + pDW14 (isolate 2).

[0221] Figure 145A is a map of codon optimized isoprene synthase fluo-opt2v2.

[0222] Figure 145B is the nucleotide sequence of codon optimized isoprene synthase fluo-opt2v2 (SEQ ID NO:40).

[0223] Figure 146A is a map of pBBR1MCS5.

[0224] Figures 146B-C are the nucleotide sequence of pBBR1MCS5 (SEQ ID NO:41).

[0225] Figure 147A is a map of pBBR5HGSOpt2_2.

[0226] Figures 147B-C are the nucleotide sequence of pBBR5HGSOpt2_2 (SEQ ID
NO:42).

[0227] Figure 148 is a graph of CER versus fermentation time for strain MCM401, uninduced, induced with IPTG (4 x 50 mol) or IPTG (2 x 100 mol).

[0228] Figure 149 shows concentration of glucose in sugar cane solutions, pH
adjusted or not, as a function of the number of autoclaving cycles (one cycle = 30 min).

[0229] Figure 150 shows growth curves (OD600 as a function of time) of Pseudomonas putida Fl and Pseudomonasfluorescens ATCC13525 on glucose, sugar cane, and inverted sugar cane.

[0230] Figure 151 shows growth curves (OD600 as a function of time) of E. coli BL21(DE3), MG1655, ATCC 11303 and B REL 606 on glucose, sugar cane, and inverted sugar cane.

[0231] Figure 152 is a map of plasmid pET24 P. alba HGS.

[0232] Figure 153A-B are the nucleotide sequence of plasmid pET24 P. alba HGS
(SEQ ID
NO:43).

[0233] Figure 154 is a schematic diagram showing restriction sites used for endonuclease digestion to construct plasmid EWL230 and compatible cohesive ends between BspHI and Ncol sites.

[0234] Figure 155 is a map of plasmid EWL230.

[0235] Figures 156A-B are the nucleotide sequence of plasmid EWL230 (SEQ ID
NO:44).

[0236] Figure 157 is a schematic diagram showing restriction sites used for endonuclease digestion to construct plasmid EWL244 and compatible cohesive ends between Nsil and Pstl sites.

[0237] Figure 158 is a map of plasmid EWL244.

[0238] Figures 159A-B are the nucleotide sequence of plasmid EWL244 (SEQ ID
NO:45).

[0239] Figure 160A is a map of the M. mazei archaeal Lower Pathway operon.

[0240] Figures 160B-C are the nucleotide sequence of the M. mazei archaeal Lower Pathway operon (SEQ ID NO:46).

[0241] Figure 161A is a map of MCM376-MVK from M. mazei archaeal Lower in pET200D.

[0242] Figures 161B-C are the nucleotide sequence of MCM376-MVK from M. mazei archaeal Lower in pET200D (SEQ ID NO:47).

[0243] Figure 162 is a map of plasmid pBBRCMPGIl.5-pgl.

[0244] Figures 163A-B are the nucleotide sequence of plasmid pBBRCMPGIl.5-pgl (SEQ ID
NO:48).

[0245] Figures 164A-F are graphs of isoprene production by E. coli strain expressing M. mazei mevalonate kinase, P. alba isoprene synthase, and pgl (RHM 111608-2), and grown in fed-batch culture at the 15-L scale. Figure 164A shows the time course of optical density within the 15-L
bioreactor fed with glucose. Figure 164B shows the time course of isoprene titer within the 15-L
bioreactor fed with glucose. The titer is defined as the amount of isoprene produced per liter of fermentation broth. Method for calculating isoprene: cumulative isoprene produced in 59 hrs, g/Fermentor volume at 59 hrs, L [=] g/L broth. Figure 164C also shows the time course of isoprene titer within the 15-L bioreactor fed with glucose. Method for calculating isoprene:

f (Instantaneous isoprene production rate, g/L/hr)dt from t = 0 to 59 hours [=] g/L broth. Figure 164D shows the time course of total isoprene produced from the 15-L bioreactor fed with glucose. Figure 164E shows volumetric productivity within the 15-L bioreactor fed with glucose. Figure 164F shows carbon dioxide evolution rate (CER), or metabolic activity profile, within the 15-L bioreactor fed with glucose.

[0246] Figures 165A-B are graphs showing analysis of off-gas from fermentation in 15L
bioreactors. Sample A is strain RM111608-2 sampled at 64.8 hours. Sample B is strain EWL256 was E. coli BL21 (DE3), pCL upper, cmR-gil.2-yKKDyI, pTrcAlba-mMVK
sampled at 34.5 hours. Hydrogen is detected above the baseline (0.95 x 10-8 torn) for both samples.

[0247] Figure 166A shows an exemplary BioisopreneTM recovery unit.

[0248] Figure 166B shows an exemplary BioisopreneTM desorption/condensation setup.

[0249] Figure 167 shows a GC/FID chromatogram of a BioisopreneTM product. The material was determined to be 99.7% pure.

[0250] Figure 168A-C show the GC/FID chromatograms of a BioisopreneTM sample before (A) and after treatment with alumina (B) or silica (C). The isoprene peak is not shown in these chromatograms.

[0251] Figure 169 shows a diagram of a process and associated apparatus for purifying isoprene from a fermentation off-gas.

[0252] Figure 170 shows GC/FID chromatogram of partially hydrogenated BioIsopreneTM
monomer. Compound 1 (RT = 12.30 min) = 3-methyl-l-butene, compound 2 (RT =
12.70 min) = 2-methylbutane, compound 3 (RT = 13.23 min) = 2-methyl-l-butene, compound 4 (RT =
13.53 min) = isoprene, compound 5 (RT = 14.01 min) = 2-methyl-2-butene).

[0253] Figure 171 shows the GC/MS Total Ion Chromatogram for products derved from the Amberlyst- 15 acid resin-catalyzed dimerization of 2-methyl-2-butene.

[0254] Figure 172 shows the GC/MS Total Ion Chramatogram for products derived from Amberlyst 15 acid resin-catalyzed oligomerization of BioIsopreneTM monomer.

[0255] Figure 173 shows a process flow diagram for the conversion of a C5 stream into a C10/Cl5 product stream using a dimerization reactor. The C5 stream comprises BioIsopreneTM
monomer and/or C5 derivatives of BioIsopreneTM monomer.

DETAILED DESCRIPTION OF THE INVENTION

[0256] The invention provides, inter alia, compositions and methods for producing a fuel constituent from isoprene. Provided herein are fuel constituents or additives, for example, cyclic isoprene dimers and trimers, linear isoprene oligomers, aromatic and alicyclic isoprene derivatives, and oxygenated isoprene derivatives. The fuel constituent can be produced by chemical transformations of a starting material comprising a commercially beneficial amount of highly pure isoprene. In one aspect, the commercially beneficial amount of highly pure isoprene comprises bioisoprene. In another aspect, a commercially beneficial amount of highly pure isoprene can be bioisoprene. In another aspect, a commercially beneficial amount of highly pure isoprene can be highly pure isoprene compositions produced by culturing cells expressing a heterologous isoprene synthase enzyme. In other aspects, highly pure isoprene undergoes oligomerization to form unsaturated isoprene oligomers such as cyclic dimers or trimers and linear oligomers. The unsaturated oligomers may be hydrogenated to produce saturated hydrocarbon fuel constituent. In some embodiment, reaction of highly pure isoprene with alcohols in the presence of an acid catalyst produces fuel oxygenates. In another aspect, the highly pure isoprene is partially hydrogenated to produce isoamylenes. In some embodiments, an isoamylene product derived from the highly pure isoprene undergoes dimerization to form isodecenes. In some embodiments, isoamylene products derived from the highly pure isoprene react with alcohols in the presence of an acid catalyst to produce fuel oxygenates.

[0257] Bioisoprene derived from renewable carbon can be converted to a variety of hydrocarbon fuels by chemical catalysis. Provided herein are methods for recovering isoprene from fermentation and subsequent conversion to hydrocarbon fuels by chemical catalysis to compounds of higher molecular weight. These methods include, but are not limited to, recovering and purifying isoprene from fermentation off-gas and subsequent gas or liquid phase catalysis to provide compounds with fuel value. Both continuous and batch mode processes are contemplated within the scope of the invention.

[0258] As further detailed herein, a bioisoprene composition is distinguished from a petro-isoprene composition in that a bioisoprene composition is substantially free of any contaminating unsaturated C5 hydrocarbons that are usually present in petro-isoprene compositions, such as, but not limited to, 1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne. If any contaminating unsaturated C5 hydrocarbons are present in the bioisoprene starting material described herein, they are present in lower levels than that in petro-isoprene compositions. Accordingly, any fuel products derived from bioisoprene compositions described herein is essentially free of, or contains at lower levels than that in fuel products derived from petro-isoprene, any contaminating unsaturated C5 hydrocarbons or products derived from such contaminating unsaturated C5 hydrocarbons. In addition, the sulfur levels in a bioisoprene composition are lower than the sulfur levels in petro-isoprene compositions. Fuels products derived from bioisoprene compositions contain lower levels of sulfur than that in fuel products derived from petro-isoprene.

[0259] Bioisoprene is distinguished from petro-isoprene in that bioisoprene is produced with other bio-byproducts (compounds derived from the biological sources and/or associated the biological processes that are obtained together with bioisoprene) that are not present or present in much lower levels in petro-isoprene compositions, such as alcohols, aldehydes, ketone and the like. The bio-byproducts may include, but are not limited to, ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis-and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-1-ol), 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). Fuel products derived from bioisoprene contain one or more of the bio-byproducts or compounds derived from any of the bio-byproducts. In addition, fuel products derived from bioisoprene may contain compounds formed from these bio-byproducts during subsequent chemical conversion.
Examples of such compounds include those derived from Diels-Alder cycloaddition of dienophiles to isoprene or fuel derivatives thereof, the oxidation of isoprene or fuel derivatives.

[0260] Further, bioisoprene is distinguished from petro-isoprene by carbon finger-printing. In one aspect, bioisoprene has a higher radioactive carbon-14 (14C) content or higher 14C/12C ratio than petro-isoprene. Bioisoprene is produced from renewable carbon sources, thus the 14C
content or the 14C/12C ratio in bioisoprene is the same as that in the present atmosphere. Petro-isoprene, on the other hand, is derived from fossil fuels deposited thousands to millions of years ago, thus the 14C content or the 14C/12C ratio is diminished due to radioactive decay. As discussed in greater detail herein, the fuel products derived from bioisoprene has higher 14C
content or 14C/12C ratio than fuel products derived from petro-isoprene. In one embodiment, a fuel product derived from bioisoprene described herein has a 14C content or 14C/12C ratio similar to that in the atmosphere. In another aspect, bioisoprene can be analytically distinguished from petro-isoprene by the stable carbon isotope ration (13C/12C), which can be reported as "delta values" represented by the symbol d13C. For examples, for isoprene derived from extractive distillation of C5 streams from petroleum refineries, 813C is about -22%o to about -24%0. This range is typical for light, unsaturated hydrocarbons derived from petroleum, and products derived from petroleum-based isoprene typically contain isoprenic units with the same 813C.
Bioisoprene produced by fermentation of corn-derived glucose (813C -10.73%o) with minimal amounts of other carbon-containing nutrients (e.g., yeast extract) produces isoprene which can be polymerized into polyisoprene with 813C -14.66%o to -14.85%0. Products produced from such bioisoprene are expected to have 813C values that are less negative than those derived from petroleum-based isoprene.

[0261] Compounds made by these methods include cyclic isoprene dimers and trimers, linear oligomers, aromatic and alicyclic derivatives. Diisoamylenes are made by methods comprising partial hydrogenation of bioisoprene compositons. These chemical derivatives of isoprene are useful as liquid transportation fuels (IsoFuelsTM) and as fuel additives.

[0262] Also provided herein are methods for the production of oxygenated derivatives of isoprene including alcohols, ketones, esters and ethers. Methods for the synthesis of oxygenated derivatives of isoprene can also be performed in liquid or gas phase, using homogeneous and heterogeneous catalysts. Compounds of this chemical class are also useful as liquid transportation fuels, and can be used in fuel blends as fuel oxygenates for emissions reduction and as fuel modifiers, for example as cetane boosters for diesel.

[0263] While isoprene can be obtained by fractionating petroleum, the purification of this material is expensive and time-consuming. Petroleum cracking of the C5 stream of hydrocarbons produces only about 15% isoprene. Isoprene is also naturally produced by a variety of microbial, plant, and animal species. In particular, two pathways have been identified for the biosynthesis of isoprene: the mevalonate (MVA) pathway and the non-mevalonate (DXP) pathway. Genetically engineered cell cultures in bioreactors have produced isoprene more efficiently, in larger quantities, in higher purities and/or with unique impurity profiles, e.g. as described in U.S. provisional patent application Nos. 61/013,386 and 61/013,574, filed on December 13, 2007, WO 2009/076676, U.S. provisional patent application Nos.
61/134,094, 61/134,947, 61/134,011 and 61/134,103, filed on July 2, 2008, WO 2010/003007, U.S.
provisional patent application No. 61/097,163, filed on September 15, 2008, WO
2010/031079, U.S. provisional patent application No. 61/097,186, filed on September 15, 2008, WO
2010/031062, U.S. provisional patent application No. 61/097,189, filed on September 15, 2008, WO 2010/031077, U.S. provisional patent application No. 61/097,200, filed on September 15, 2008, WO 2010/031068, U.S. provisional patent application No. 61/097,204, filed on September 15, 2008, WO 2010/031076, U.S. provisional patent application No.61/141,652, filed on December 30, 2008, PCT/US09/069862, U.S. patent application No. 12/335,071, filed December 15, 2008 (US 2009/0203102 Al) and U.S. patent application No.
12/429,143, filed April 23, 2009 (US 2010/0003716 Al), which are incorporated by reference in their entireties.
Definitions

[0264] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. All documents cited are, in relevant part, incorporated herein by reference. However, the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

[0265] As used herein, the singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.

[0266] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0267] The term "isoprene" refers to 2-methyl-1,3-butadiene (CAS# 78-79-5 ), which is the direct and final volatile C5 hydrocarbon product from the elimination of pyrophosphate from 3,3-dimethylallyl pyrophosphate (DMAPP), and does not involve the linking or polymerization of [an] IPP molecule(s) to [a] DMAPP molecule(s). The term "isoprene" is not generally intended to be limited to its method of production unless indicated otherwise herein.

[0268] As used herein, "biologically produced isoprene" or "bioisoprene" is isoprene produced by any biological means, such as produced by genetically engineered cell cultures, natural microbials, plants or animals.

[0269] A "bioisoprene composition" refers to a composition that can be produced by any biological means, such as systems (e.g., cells) that are engineered to produce isoprene. It contains isoprene and other compounds that are co-produced (including impurities) and/or isolated together with isoprene. A bioisoprene composition usually contains fewer hydrocarbon impurities than isoprene produced from petrochemical sources and often requires minimal treatment in order to be of polymerization grade. As detailed herein, bioisoprene composition also has a different impurity profile from a petrochemically produced isoprene composition.

[0270] As used herein, "at least a portion of the isoprene starting composition" can refer to at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% of the isoprene starting composition undergoing chemical transformation.

[0271] As used herein, IsoFuelsTM refers to fuels including liquid transportation fuels that are derived from isoprene. BioIsoFuelsTM refers to fuels including liquid transportation fuels that are derived from bioisoprene.

[0272] The term "oligomerization" as used herein refers to a chemical process for combining two or more monomer units. "Oligomerization" of isoprene produces a derivative of isoprene derived from two or more molecules of isoprene, such as linear dimers of isoprene, cyclic dimers of isoprene, linear trimers of isoprene, cyclic trimers of isoprene and the like.

[0273] "Complete hydrogenation", "Completely hydrogenate" or "Fully hydrogenate" is defined as the addition of hydrogen (H2), typically in the presence of a hydrogenation catalyst, to all unsaturated functional groups, such as carbon-carbon double bonds, within a precursor compound to give fully saturated product compounds. For example, complete hydrogenation of isoprene forms isopentane whereby 2 moles of H2 is comsumed per mole of isoprene.

[0274] "Partial hydrogenation" or "Partially hydrogenate" is defined as the addition of hydrogen (H2), typically in the presence of a hydrogenation catalyst, to at least one, but not all unsaturated functional groups, such as carbon-carbon double bonds, within a precursor compound. The product(s) of partial hydrogenation can be further completely hydrogenated to give fully saturated product compounds. Partial hydrogenation of a diene forms one or more mono-olefins. For example, partial hydrogenation of isoprene can give 3 isomeric isopentenes (2-methylbut-l-ene, 2-methylbut-2-ene and 3-methylbut-l-ene) whereby 1 mole of H2 is comsumed per mole of isoprene.

[0275] "Selective hydrogenation" or "Selectively hydrogenate" is defined as the addition of hydrogen (H2), typically in the presence of a hydrogenation catalyst, to at least one, but not all unsaturated functional groups, such as carbon-carbon double bonds, within a precursor compound whereby certain unsaturated functional groups are preferentially hydrogenated over other unsaturated groups under the chosen conditions. For example, selective hydrogenation of isoprene may form preferentially 2-methyl-2-butene, 2-methyl-l-butene, 3-methyl-l-butene or a mixture thereof.

[0276] As used herein, the term "polypeptides" includes polypeptides, proteins, peptides, fragments of polypeptides, and fusion polypeptides.

[0277] As used herein, an "isolated polypeptide" is not part of a library of polypeptides, such as a library of 2, 5, 10, 20, 50 or more different polypeptides and is separated from at least one component with which it occurs in nature. An isolated polypeptide can be obtained, for example, by expression of a recombinant nucleic acid encoding the polypeptide.

[0278] By "heterologous polypeptide" is meant a polypeptide whose amino acid sequence is not identical to that of another polypeptide naturally expressed in the same host cell. In particular, a heterologous polypeptide is not identical to a wild-type polypeptide that is found in the same host cell in nature.

[0279] "Codon degeneracy" refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid for improved expression in a host cell, it is desirable in some embodiments to design the nucleic acid such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

[0280] As used herein, a "nucleic acid" refers to two or more deoxyribonucleotides and/or ribonucleotides covalently joined together in either single or double-stranded form.

[0281] By "recombinant nucleic acid" is meant a nucleic acid of interest that is free of one or more nucleic acids (e.g., genes) which, in the genome occurring in nature of the organism from which the nucleic acid of interest is derived, flank the nucleic acid of interest. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA, a genomic DNA
fragment, or a cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.

[0282] By "heterologous nucleic acid" is meant a nucleic acid whose nucleic acid sequence is not identical to that of another nucleic acid naturally found in the same host cell. In particular, a heterologous nucleic acid is not identical to a wild-type nucleic acid that is found in the same host cell in nature.

[0283] As used herein, a "vector" means a construct that is capable of delivering, and desirably expressing one or more nucleic acids of interest in a host cell.
Examples of vectors include, but are not limited to, plasmids, viral vectors, DNA or RNA
expression vectors, cosmids, and phage vectors.

[0284] As used herein, an "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid of interest. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. An "inducible promoter" is a promoter that is active under environmental or developmental regulation. The expression control sequence is operably linked to the nucleic acid segment to be transcribed.

[0285] The term "selective marker" or "selectable marker" refers to a nucleic acid capable of expression in a host cell that allows for ease of selection of those host cells containing an introduced nucleic acid or vector. Examples of selectable markers include, but are not limited to, antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/or nucleic acids that confer a metabolic advantage, such as a nutritional advantage on the host cell. Exemplary nutritional selective markers include those markers known in the art as amdS, argB, and pyr4.
Compositions and Systems

[0286] Isoprene derived from petrochemical sources usually is an impure C5 hydrocarbon fraction which requires extensive purification before the material is suitable for polymerization or other chemical transformations. Several impurities are particularly problematic given their structural similarity to isoprene and the fact that they can act as polymerization catalyst poisons.
Such compounds include, but are not limited to, 1,3-cyclopentadiene, cis- and trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne. As detailed below, biologically produced isoprene can be substantially free of any contaminating unsaturated C5 hydrocarbons without undergoing extensive purification. Some biologically produced isoprene compositions contain ethanol, acetone, and C5 prenyl alcohols. These components are more readily removed from the isoprene stream than the isomeric C5 hydrocarbon fractions that are present in isoprene compositions derived from petrochemical sources. Further, these impurities can be managed in the bioprocess, for example by genetic modification of the producing strain, carbon feedstock, alternative fermentation conditions, recovery process modifications and additional or alternative purification methods.

[0287] In one aspect, the invention features compositions and systems for producing a fuel constituent from isoprene comprising: (a) a commercially beneficial amount of highly pure isoprene starting composition; and (b) a fuel constituent produced from at least a portion of the highly pure isoprene starting material; where at least a portion of the commercially beneficial amount of highly pure isoprene starting composition undergoes a chemical transformation. A
highly pure isoprene starting material is subjected to chemical reactions to produce a commercially beneficial amount of product that is useful for making fuels. In one aspect, a commercially beneficial amount of highly pure isoprene comprises bioisoprene.
In one aspect, a commercially beneficial amount of highly pure isoprene can be bioisoprene.

Exemplary starting isoprene compositions

[0288] In some embodiments, the commercially beneficial amount of highly pure isoprene starting composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the starting isoprene composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. In some embodiments, the starting isoprene composition comprises greater than or about 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 kg of isoprene. In some embodiments, the amount of isoprene in the starting composition is between about 2 to about 5,000 mg, such as between about 2 to about 100 mg, about 100 to about 500 mg, about 500 to about 1,000 mg, about 1,000 to about 2,000 mg, or about 2,000 to about 5,000 mg. In some embodiments, the amount of isoprene in the starting composition is between about 20 to about 5,000 mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg, about 200 to about 1,000 mg, about 300 to about 1,000 mg, or about 400 to about 1,000 mg. In some embodiments, the amount of isoprene in the starting composition is between about 2 to about 5,000 g, such as between about 2 to about 100 g, about 100 to about 500 g, about 500 to about 1,000 g, about 1,000 to about 2,000 g, or about 2,000 to about 5,000 g. In some embodiments, the amount of isoprene in the starting composition is between about 2 to about 5,000 kg, about 10 to about 2,000 kg, about 20 to about 1,000 kg, about 20 to about 500 kg, about 30 to about 200 kg, or about 40 to about 100 kg. In some embodiments, greater than or about 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% (w/w) of the volatile organic fraction of the starting composition is isoprene.

[0289] In some embodiments, the highly pure isoprene starting composition comprises greater than or about 98.0, 98.5, 99.0, 99.5, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the highly pure isoprene starting composition comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100%
isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the starting composition has a relative detector response of greater than or about 98.0, 98.5, 99.0, 99.5, or 100% for isoprene compared to the detector response for all C5 hydrocarbons in the starting composition. In some embodiments, the starting composition has a relative detector response of greater than or about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.99, or 100% for isoprene compared to the detector response for all C5 hydrocarbons in the starting composition. In some embodiments, the starting isoprene composition comprises between about 98.0 to about 98.5, about 98.5 to about 99.0, about 99.0 to about 99.5, about 99.5 to about 99.8, about 99.8 to 100%
isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the starting isoprene composition comprises between about 99.90 to about 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96, about 99.96 to about 99.98, about 99.98 to 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition.

[0290] In some embodiments, the highly pure isoprene starting composition comprises less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-l-yne) by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the starting composition has a relative detector response of less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for C5 hydrocarbons other than isoprene compared to the detector response for all C5 hydrocarbons in the starting composition. In some embodiments, the starting composition has a relative detector response of less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001%
for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-l-yne compared to the detector response for all C5 hydrocarbons in the starting composition. In some embodiments, the highly pure isoprene starting composition comprises between about 0.02 to about 0.04%, about 0.04 to about 0.06%, about 0.06 to 0.08%, about 0.08 to 0.10%, or about 0.10 to about 0.12% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-l-yne) by weight compared to the total weight of all C5 hydrocarbons in the starting composition.

[0291] In some embodiments, the highly pure isoprene starting composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a compound that inhibits the polymerization of isoprene for any compound in the starting composition that inhibits the polymerization of isoprene. In some embodiments, the starting isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 g/L
of a compound that inhibits the polymerization of isoprene for any compound in the starting composition that inhibits the polymerization of isoprene. In some embodiments, the starting isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a hydrocarbon other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne). In some embodiments, the starting isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 g/L of a hydrocarbon other than isoprene.
In some embodiments, the starting isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a protein or fatty acid (such as a protein or fatty acid that is naturally associated with natural rubber).

[0292] In some embodiments, the highly pure isoprene starting composition comprises less than or about 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of alpha acetylenes, piperylenes, acetonitrile, or 1,3-cyclopentadiene. In some embodiments, the starting isoprene composition comprises less than or about 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of sulfur or allenes. In some embodiments, the starting isoprene composition comprises less than or about 30, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes (such as 1-pentyne, 2-pentyne, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne).
In some embodiments, the starting isoprene composition comprises less than or about 2000, 1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of isoprene dimers, such as cyclic isoprene dimers (e.g., cyclic C10 compounds derived from the dimerization of two isoprene units).

[0293] In some embodiments, the highly pure isoprene starting composition includes ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), or any two or more of the foregoing. In particular embodiments, the starting isoprene composition comprises greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 g/L of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), or any two or more of the foregoing. In some embodiments, the isoprene composition comprises between about 0.005 to about 120, such as about 0.01 to about 80, about 0.01 to about 60, about 0.01 to about 40, about 0.01 to about 30, about 0.01 to about 20, about 0.01 to about 10, about 0.1 to about 80, about 0.1 to about 60, about 0.1 to about 40, about 5 to about 80, about 5 to about 60, or about 5 to about 40 g/L of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol, or any two or more of the foregoing.

[0294] In some embodiments, the highly pure isoprene starting composition includes one or more of the following components: 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). In various embodiments, the amount of one of these components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w).
In some embodiments, the relative detector response for the second compound compared to the detector response for isoprene is greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%. In various embodiments, the amount of one of these components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is between about 0.01 to about 105 % (w/w), such as about 0.01 to about 90, about 0.01 to about 80, about 0.01 to about 50, about 0.01 to about 20, about 0.01 to about 10, about 0.02 to about 50, about 0.05 to about 50, about 0.1 to about 50, or 0.1 to about 20% (w/w).

[0295] In some embodiments, at least a portion of the highly pure isoprene starting composition is in a gas phase. In some embodiments, at least a portion of the highly pure isoprene starting composition is in a liquid phase (such as a condensate). In some embodiments, at least a portion of the highly pure isoprene starting composition is in a solid phase. In some embodiments, at least a portion of the highly pure isoprene starting composition is absorbed to a solid support, such as a support that includes silica and/or activated carbon.
In some embodiments, the starting isoprene composition is mixed with one or more solvents. In some embodiments, the starting isoprene composition is mixed with one or more gases.

[0296] In other embodiments, the commercially beneficial amount of highly pure isoprene starting composition is produced by a biological process. In some preferred embodiments, the highly pure isoprene starting composition is a bioisoprene composition produced by culturing cells that produce greater than about 400 nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/gwcm/hr) of isoprene. In one embodiment, the bioisoprene composition is produced by culturing cells that convert more than about 0.002% of the carbon in a cell culture medium into isoprene. In other embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide, e.g. a naturally-occurring polypeptide from a plant such as Pueraria, and (ii) is operably linked to a promoter, e.g. a T7 promoter. Other isoprene synthase polypeptides, for example, from poplar and variants of naturally-occurring as well as parent isoprene synthase, can be used to produce bioisoprene. Examples of isoprene synthase and its variants that can be used are described in U.S. Appl. No. 12/429,143, which is incorporated herein in its entirety.

[0297] In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions. In some embodiment, the cells further comprise a heterologous nucleic acid encoding an IDI polypeptide. In some embodiments, the cells further comprise a heterologous nucleic acid encoding an MDV pathway polypeptide. In some embodiment, the starting isoprene composition is an isoprene composition as descried or is produced by culturing any of the cells described in U.S. provisional patent application Nos. 61/134,094, filed on July 2, 2008, WO 2010/003007, and U.S. patent application No. 12/335,071, filed December 15, 2008 (US
2009/0203102 Al), which are incorporated by reference in their entireties.

[0298] In some embodiments, the highly pure isoprene starting composition comprises a gas phase (off-gas) produced by cells in culture that produces isoprene. In some embodiments, the gas phase has a nonflammable concentration of isoprene. In some embodiments, the gas phase comprises less than about 9.5 % (volume) oxygen. In some embodiments, the gas phase comprises greater than or about 9.5 % (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit.
In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 100% (volume) oxygen, such as between about 10% to about 100%
(volume) oxygen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 99% (volume) nitrogen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 1% to about 50% (volume)

[0299] In some embodiments, the highly pure isoprene starting composition includes one or more of the following: an alcohol, an aldehyde, a ketone, or an ester (such as any of the alcohols, aldehydes, ketones or esters described herein). In some embodiments, the isoprene composition includes (i) an alcohol and an aldehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) an alcohol, an aldehyde, and a ketone. In some embodiments, any of the isoprene compositions further includes an ester.

[0300] In some embodiments, the highly pure isoprene starting composition derived from a biological source (such as a cell culture) contains one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole. In some embodiments, the starting isoprene composition contains 1 ppm or more of one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole. In some embodiments, the concentration of more of one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole, is between about 1 to about 10,000 ppm in a starting isoprene composition (such as off-gas before it is purified). In some embodiments, the starting isoprene composition (such as off-gas after it has undergone one or more purification steps) includes one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole, at a concentration between about 1 to about 100 ppm, such as about 1 to about 10 ppm, about 10 to about 20 ppm, about 20 to about 30 ppm, about 30 to about 40 ppm, about 40 to about 50 ppm, about 50 to about 60 ppm, about 60 to about 70 ppm, about 70 to about 80 ppm, about 80 to about 90 ppm, or about 90 to about 100 ppm. In some embodiments, the starting isoprene composition contains less than 1 ppm of methanethiol (a potent catalyst poison and a source of sulfur in the final fuel product) Volatile organic compounds from cell cultures (such as volatile organic compounds in the headspace of cell cultures) can be analyzed using standard methods such as those described herein or other standard methods such as proton transfer reaction-mass spectrometry (see, for example, Bunge et al., Applied and Environmental Microbiology, 74(7):2179-2186, 2008 which is hereby incorporated by reference in its entirety, particular with respect to the analysis of volatile organic compounds).

[0301] The invention also contemplates the use of highly pure isoprene starting composition that is derived from a biological source (such as a cell culture) the co-produces isoprene and hydrogen. In some embodiments, the starting bioisoprene compositions comprise isoprene and hydrogen in ratios ranging from at least one molar percent of isoprene for every three molar percent of hydrogen to at least one molar percent of isoprene for every four molar percent of hydrogen. In some embodiments, the starting bioisoprene compositions comprise isoprene and hydrogen in molar ratios of about 1 to 9, 2 to 8, 3 to 7, 4 to 6, 5 to 5, 6 to 4, 7 to 3, 8 to 2, or 9 to 1. In some embodiments, the composition further comprises from 1 to 11 molar percent isoprene and from 4 to 44 molar percent hydrogen. In some embodiments, the composition further comprises oxygen, carbon dioxide, or nitrogen. In some embodiments, the composition further comprises from 0 to 21 molar percent oxygen, from 18 to 44 molar percent carbon dioxide, and from 0 to 78 molar percent nitrogen. In some embodiments, the composition further comprises 1.0 x 10-4 molar percent or less of non-methane volatile impurities. In some embodiments, the non-methane volatile impurities comprise one or more of the following: 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). In some embodiments, the non-methane volatile impurities comprise one or more of the following: the isoprene composition includes one or more of the following: an alcohol, an aldehyde, or a ketone (such as any of the alcohols, aldehydes, or ketones described herein). In some embodiments, the isoprene composition includes (i) an alcohol and an aldehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) an alcohol, an aldehyde, and a ketone. In some embodiments, the non-methane volatile impurities comprise one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole.

[0302] Techniques for producing isoprene in cultures of cells that produce isoprene are described in U.S. provisional patent application Nos. 61/013,386 and 61/013,574, filed on December 13, 2007, WO 2009/076676, U.S. provisional patent application Nos.
61/134,094, 61/134,947, 61/134,011 and 61/134,103, filed on July 2, 2008, WO 2010/003007, U.S.
provisional patent application No. 61/097,163, filed on September 15, 2008, WO
2010/031079, U.S. provisional patent application No. 61/097,186, filed on September 15, 2008, WO
2010/031062, U.S. provisional patent application No. 61/097,189, filed on September 15, 2008, WO 2010/031077, U.S. provisional patent application No. 61/097,200, filed on September 15, 2008, WO 2010/031068, U.S. provisional patent application No. 61/097,204, filed on September 15, 2008, WO 2010/031076, U.S. provisional patent application No.61/141,652, filed on December 30, 2008, PCT/US09/069862, U.S. patent application No. 12/335,071, filed December 15, 2008 (US 2009/0203102 Al) and U.S. patent application No.
12/429,143, filed April 23, 2009 (US 2010/0003716 Al), the teachings of which are incorporated herein by reference for the purpose of teaching techniques for producing and recovering isoprene by such a process. In any case, U.S. provisional patent application Nos. 61/013,386 and 61/013,574, filed on December 13, 2007, WO 2009/076676, U.S. provisional patent application Nos.
61/134,094, 61/134,947, 61/134,011 and 61/134,103, filed on July 2, 2008, WO 2010/003007, U.S.
provisional patent application No. 61/097,163, filed on September 15, 2008, WO
2010/031079, U.S. provisional patent application No. 61/097,186, filed on September 15, 2008, WO
2010/031062, U.S. provisional patent application No. 61/097,189, filed on September 15, 2008, WO 2010/031077, U.S. provisional patent application No. 61/097,200, filed on September 15, 2008, WO 2010/031068, U.S. provisional patent application No. 61/097,204, filed on September 15, 2008, WO 2010/031076, U.S. provisional patent application No.61/141,652, filed on December 30, 2008, PCT/US09/069862, U.S. patent application No. 12/335,071, filed December 15, 2008 (US 2009/0203102 Al) and U.S. patent application No.
12/429,143, filed April 23, 2009 (US 2010/0003716 Al) teach compositions and methods for the production of increased amounts of isoprene in cell cultures. U.S. patent application No.
12/335,071, filed December 15, 2008 and US 2009/0203102 Al further teaches compositions and methods for co-production of isoprene and hydrogen from cultured cells. In particular, these compositions and methods compositions and methods increase the rate of isoprene production and increase the total amount of isoprene that is produced. For example, cell culture systems that generate 4.8 x 104 nmole/gwcm/hr of isoprene have been produced (Table 1). The efficiency of these systems is demonstrated by the conversion of about 2.2% of the carbon that the cells consume from a cell culture medium into isoprene. As shown in the Examples and Table 2, approximately 3 g of isoprene per liter of broth was generated. If desired, even greater amounts of isoprene can be obtained using other conditions, such as those described herein. In some embodiments, a renewable carbon source is used for the production of isoprene. In some embodiments, the production of isoprene is decoupled from the growth of the cells. In some embodiments, the concentrations of isoprene and any oxidants are within the nonflammable ranges to reduce or eliminate the risk that a fire may occur during production or recovery of isoprene. The compositions and methods are desirable because they allow high isoprene yield per cell, high carbon yield, high isoprene purity, high productivity, low energy usage, low production cost and investment, and minimal side reactions. This efficient, large scale, biosynthetic process for isoprene production provides an isoprene source for synthetic isoprene-based products such as rubber and provides a desirable, low-cost alternative to using natural rubber.

[0303] As discussed further below, the amount of isoprene produced by cells can be greatly increased by introducing a heterologous nucleic acid encoding an isoprene synthase polypeptide (e.g., a plant isoprene synthase polypeptide) into the cells. Isoprene synthase polypeptides convert dimethyl allyl diphosphate (DMAPP) into isoprene. As shown in the Examples, a heterologous Pueraria Montana (kudzu) isoprene synthase polypeptide was expressed in a variety of host cells, such as Escherichia coli, Panteoa citrea, Bacillus subtilis, Yarrowia lipolytica, and Trichoderma reesei. All of these cells produced more isoprene than the corresponding cells without the heterologous isoprene synthase polypeptide. As illustrated in Tables 1 and 2, large amounts of isoprene are produced using the methods described herein. For example, B. subtilis cells with a heterologous isoprene synthase nucleic acid produced approximately 10-fold more isoprene in a 14 liter fermentor than the corresponding control B.
subtilis cells without the heterologous nucleic acid (Table 2). The production of 300 mg of isoprene per liter of broth (mg/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells) by E. coli and 30 mg/L by B. subtilis in fermentors indicates that significant amounts of isoprene can be generated (Table 2). If desired, isoprene can be produced on an even larger scale or other conditions described herein can be used to further increase the amount of isoprene. The vectors listed in Tables 1 and 2 and the experimental conditions are described in further detail below and in the Examples section.

Table 1: Exemplary yields of isoprene from a shake flask using the cell cultures and methods described herein. The assay for measuring isoprene production is described in Example I, part II. For this assay, a sample was removed at one or more time points from the shake flask and cultured for 30 minutes. The amount of isoprene produced in this sample was then measured. The headspace concentration and specific rate of isoprene production are listed in Table 1 and described further herein.

Strain Isoprene Production in a Headspace vial*
Headspace Specific Rate concentration g/Leroth/hr/OD
g/Lgas nmol/ wcm/hr 53.2 E. coli BL21/pTrcKudzu IS 1.40 (781.2) E. coli BL21/ pCL DXS yidi Kudzu 7.61 289.1 IS (4.25 x 10) E. coli BL21/MCM127 with kudzu 874.1 23.0 IS and entire MVA pathway (1.28 x 104) 56.6 E. coli BL21/ pET N-HisKudzu IS 1.49 (831.1) 25.1 Pantoea citrea /pTrcKudzu IS 0.66 (368.6) E. coli w/ Poplar IS 5.6 [Miller (2001)] (82.2) Bacillis licheniformis Fall US 4.2 5849970 (61.4) Yarrowia lipolytica with kudzu -2 -0.05 tg/L
isoprene synthase (-30) Trichoderma reesei with kudzu -2 - 0.05 tg/L
isoprene synthase (-30) E. coli BL21/ pTrcKKDyIkIS with 85.9 3.2 x 103 Strain Isoprene Production in a Headspace vial*
kudzu IS and lower MVA pathway (4.8 x 104) *Normalized to 1 mL of 1 OD600, cultured for 1 hour in a sealed headspace vial with a liquid to headspace volume ratio of 1:19.

Table 2: Exemplary yields of isoprene in a fermentor using the cell cultures and methods described herein. The assay for measuring isoprene production is described in Example I, part II. For this assay, a sample of the off-gas of the fermentor was taken and analyzed for the amount of isoprene. The peak headspace concentration (which is the highest headspace concentration during the fermentation), titer (which is the cumulative, total amount of isoprene produced per liter of broth), and peak specific rate of isoprene production (which is the highest specific rate during the fermentation) are listed in Table 2 and described further herein.

Strain Isoprene Production in Fermentors Peak Headspace Titer Peak Specific rate concentration** (mg/Lbroth) 1g/Lbroth/hr/OD
(Ag/Lgas) nmol/ wcm/hr E. coli BL21 /pTrcKudzu 37 with Kudzu IS 52 41.2 (543.3) E. coli FM5/pTrcKudzu 21.4 IS 3 3.5 (308.1) E. coli BL21/ triple strain 285 300 240 (DXS, yidi, IS) (3.52 x 103) E. coli FM5/ triple strain 180.8 (DXS, yidi, IS) 50.8 29 (2.65 x 103 E. coli/MCM127 with 992.5 Kudzu IS and entire MVA 3815 3044 (1.46 x 104) pathway E. coli BL21/pCLPtrc Upper Pathway gil.2 2418 1640 1248 integrated lower pathway (1.83 x 104) pTrcKudzu E. coli BL21/MCM401 13991 23805 3733 with 4 x 50 M IPTG (5.49 x 104) E. coli BL21/MCM401 22375 19541 5839.5 with 2 x 1000 M IPTG 8.59 x 104) Strain Isoprene Production in Fermentors E. coli BL21/pCLPtrc 1088 UpperPathwayHGS2 - 3500 3300 pTrcKKDyIkIS
(1.60 x 104) 0.8 Bacillus subtilis wild-type 1.5 2.5 (11.7) Bacillus pBS Kudzu IS 16.6 over 100 hrs) (73.4) Bacillus Marburg 6051 24.5 [Wagner and Fall (1999)] 2.04 0.61 (359.8) Bacillus Marburg 6051 6.8 Fall US 5849970 0.7 0.15 (100) E. coli BL21/pCLPtrcUpperPath 5.9 x 103 2.03 x 104 3.22 x 104 way and gil.2KKDyI and (8.66 x 104) pTrcAlba-mMVK

E. coli BL21/pCLPtrcUpper 4 1.28 x 10 Pathway and gil.2KKDyI 3.22 x 104 6.05 x 104 and pTrcAlba-mMVK (1.88 x 105) plus pBBRCMPGI1.5pgl **Normalized to an off-gas flow rate of 1 vvm (1 volume off-gas per 1 Lbroth per minute).

[0304] Additionally, isoprene production by cells that contain a heterologous isoprene synthase nucleic acid can be enhanced by increasing the amount of a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptide and/or an isopentenyl diphosphate isomerase (IDI) polypeptide expressed by the cells. For example, a DXS nucleic acid and/or an IDI nucleic acid can be introduced into the cells. The DXS nucleic acid may be a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid. Similarly, the IDI nucleic acid may be a heterologous nucleic acid or a duplicate copy of an endogenous nucleic acid.
In some embodiments, the amount of DXS and/or IDI polypeptide is increased by replacing the endogenous DXS and/or IDI promoters or regulatory regions with other promoters and/or regulatory regions that result in greater transcription of the DXS and/or IDI
nucleic acids. In some embodiments, the cells contain both a heterologous nucleic acid encoding an isoprene synthase polypeptide (e.g., a plant isoprene synthase nucleic acid) and a duplicate copy of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

[0305] The encoded DXS and IDI polypeptides are part of the DXP pathway for the biosynthesis of isoprene (Figure 19A). DXS polypeptides convert pyruvate and D-glyceraldehyde-3 -phosphate into 1-deoxy-D-xylulose-5-phosphate. While not intending to be bound by any particular theory, it is believed that increasing the amount of DXS polypeptide increases the flow of carbon through the DXP pathway, leading to greater isoprene production.
IDI polypeptides catalyze the interconversion of isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP). While not intending to be bound by any particular theory, it is believed that increasing the amount of IDI polypeptide in cells increases the amount (and conversion rate) of IPP that is converted into DMAPP, which in turn is converted into isoprene.

[0306] For example, fermentation of E. coli cells with a kudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acids was used to produce isoprene. The levels of isoprene varied from 50 to 300 g/L over a time period of 15 hours (Example 7, part VII).

[0307] In some embodiments, the presence of heterologous or extra endogenous isoprene synthase, IDI, and DXS nucleic acids causes cells to grow more reproducibly or remain viable for longer compared to the corresponding cell with only one or two of these heterologous or extra endogenous nucleic acids. For example, cells containing heterologous isoprene synthase, IDI, and DXS nucleic acids grew better than cells with only heterologous isoprene synthase and DXS nucleic acids or with only a heterologous isoprene synthase nucleic acid.
Also, heterologous isoprene synthase, IDI, and DXS nucleic acids were successfully operably linked to a strong promoter on a high copy plasmid that was maintained by E. coli cells, suggesting that large amounts of these polypeptides could be expressed in the cells without causing an excessive amount of toxicity to the cells. While not intending to be bound to a particular theory, it is believed that the presence of heterologous or extra endogenous isoprene synthase and IDI
nucleic acids may reduce the amount of one or more potentially toxic intermediates that would otherwise accumulate if only a heterologous or extra endogenous DXS nucleic acid was present in the cells.

[0308] In some embodiments, the production of isoprene by cells that contain a heterologous isoprene synthase nucleic acid is augmented by increasing the amount of a MVA
polypeptide expressed by the cells (Figures 19A and 19B). Exemplary MVA pathways polypeptides include any of the following polypeptides: acetyl-CoA acetyltransferase (AA-CoA
thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK) polypeptides, phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonate decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDI
polypeptides, and polypeptides (e.g., fusion polypeptides) having an activity of two or more MVA
pathway polypeptides. For example, one or more MVA pathway nucleic acids can be introduced into the cells. In some embodiments, the cells contain the upper MVA pathway, which includes AA-CoA
thiolase, HMG-CoA synthase, and HMG-CoA reductase nucleic acids. In some embodiments, the cells contain the lower MVA pathway, which includes MVK, PMK, MVD, and IDI
nucleic acids. In some embodiments, the cells contain an entire MVA pathway that includes AA-CoA
thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK, PMK, MVD, and IDI nucleic acids.
In some embodiments, the cells contain an entire MVA pathway that includes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK, PMDC, IPK, and IDI nucleic acids.
The MVA pathway nucleic acids may be heterologous nucleic acids or duplicate copies of endogenous nucleic acids. In some embodiments, the amount of one or more MVA
pathway polypeptides is increased by replacing the endogenous promoters or regulatory regions for the MVA pathway nucleic acids with other promoters and/or regulatory regions that result in greater transcription of the MVA pathway nucleic acids. In some embodiments, the cells contain both a heterologous nucleic acid encoding an isoprene synthase polypeptide (e.g., a plant isoprene synthase nucleic acid) and a duplicate copy of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

[0309] For example, E. coli cells containing a nucleic acid encoding a kudzu isoprene synthase polypeptide and nucleic acids encoding Saccharomyces cerevisiae MVK, PMK, MVD, and IDI
polypeptides generated isoprene at a rate of 6.67 x 10-4 mol/Lbrofh/OD600/hr (see Example 8).
Additionally, a 14 liter fermentation of E. coli cells with nucleic acids encoding Enterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase polypeptides produced 22 grams of mevalonic acid (an intermediate of the MVA pathway). A
shake flask of these cells produced 2-4 grams of mevalonic acid per liter. These results indicate that heterologous MVA pathways nucleic acids are active in E. coli. E. coli cells that contain nucleic acids for both the upper MVA pathway and the lower MVA pathway as well as a kudzu isoprene synthase (strain MCM 127) produced significantly more isoprene (874 g/L) compared to E.

coli cells with nucleic acids for only the lower MVA pathway and the kudzu isoprene synthase (strain MCM 131) (see Table 3 and Example 8, part VIII).

[0310] In some embodiments, at least a portion of the cells maintain the heterologous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects described herein, the nucleic acid comprising the heterologous or duplicate copy of an endogenous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid also comprises a selective marker, such as a kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol antibiotic resistance nucleic acid.

[0311] As indicated in Example 7, part VI, the amount of isoprene produced can be further increased by adding yeast extract to the cell culture medium. In this example, the amount of isoprene produced was linearly proportional to the amount of yeast extract in the cell medium for the concentrations tested (Figure 48C). Additionally, approximately 0.11 grams of isoprene per liter of broth was produced from a cell medium with yeast extract and glucose (Example 7, part VIII). Both of these experiments used E. coli cells with kudzu isoprene synthase, S.
cerevisia IDI, and E. coli DXS nucleic acids to produce isoprene. Increasing the amount of yeast extract in the presence of glucose resulted in more isoprene being produced than increasing the amount of glucose in the presence of yeast extract. Also, increasing the amount of yeast extract allowed the cells to produce a high level of isoprene for a longer length of time and improved the health of the cells.

[0312] Isoprene production was also demonstrated using three types of hydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as the carbon source (Figures 46A-C). E. coli cells with kudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acids produced as much isoprene from these hydrolyzed biomass carbon sources as from the equivalent amount of glucose (e.g., 1% glucose, w/v). If desired, any other biomass carbon source can be used in the compositions and methods described herein. Biomass carbon sources are desirable because they are cheaper than many conventional cell mediums, thereby facilitating the economical production of isoprene.

[0313] Additionally, invert sugar was shown to function as a carbon source for the generation of isoprene (Figures 47C and 96-98). For example, 2.4 g/L of isoprene was produced from cells expressing MVA pathway polypeptides and a Kudzu isoprene synthase (Example 8, part XV).
Glycerol was as also used as a carbon source for the generation of 2.2 mg/L of isoprene from cells expressing a Kudzu isoprene synthase (Example 8, part XIV). Expressing a DXS nucleic acid, an IDI nucleic acid, and/or one or more MVA pathway nucleic acids (such as nucleic acids encoding the entire MVA pathway) in addition to an isoprene synthase nucleic acid may increase the production of isoprene from glycerol.

[0314] In some embodiments, an oil is included in the cell medium. For example, B. subtilis cells containing a kudzu isoprene synthase nucleic acid produced isoprene when cultured in a cell medium containing an oil and a source of glucose (Example 4, part III).
As another example, E. coli fadR atoC mutant cells containing the upper and lower MVA
pathway plus kudzu isoprene synthase produced isoprene when cultured in a cell medium containing palm oil and a source of glucose (Example 27, part II). In some embodiments, more than one oil (such as 2, 3, 4, 5, or more oils) is included in the cell medium. While not intending to be bound to any particular theory, it is believed that (i) the oil may increase the amount of carbon in the cells that is available for conversion to isoprene, (ii) the oil may increase the amount of acetyl-CoA in the cells, thereby increasing the carbon flow through the MVA pathway, and/or (ii) the oil may provide extra nutrients to the cells, which is desirable since much of the carbon in the cells is converted to isoprene rather than other products. In some embodiments, cells that are cultured in a cell medium containing oil naturally use the MVA pathway to produce isoprene or are genetically modified to contain nucleic acids for the entire MVA pathway. In some embodiments, the oil is partially or completely hydrolyzed before being added to the cell culture medium to facilitate the use of the oil by the host cells.

[0315] One of the major hurdles to commercial production of small molecules such as isoprene in cells (e.g., bacteria) is the decoupling of production of the molecule from growth of the cells. In some embodiments for the commercially viable production of isoprene, a significant amount of the carbon from the feedstock is converted to isoprene, rather than to the growth and maintenance of the cells ("carbon efficiency"). In various embodiments, the cells convert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture medium into isoprene. In particular embodiments, a significant portion of the carbon from the feedstock that is converted to downstream products is converted to isoprene. As described further in Example 11, E. coli cells expressing MVA pathway and kudzu isoprene synthase nucleic acids exhibited decoupling of the production of isoprene or the intermediate mevalonic acid from growth, resulting in high carbon efficiency. In particular, mevalonic acid was formed from cells expressing the upper MVA pathway from Enterococcusfaecalis.

Isoprene was formed from cells expressing the upper MVA pathway from Enterococcus faecalis, the lower MVA pathway from Saccharomyces cerevisiae, and the isoprene synthase from Pueraria montana (Kudzu). This decoupling of isoprene or mevalonic acid production from growth was demonstrated in four different strains of E. coli: BL21(LDE3), BL21(LDE3) Tuner, FMS, and MG1655. The first two E. coli strains are B strains, and the latter two are K12 strains. Decoupling of production from growth was also demonstrated in a variant of MG 1655 with ack andpta genes deleted. This variant also demonstrated less production of acetate.
Exemplary Polypeptides and Nucleic Acids

[0316] Various isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptides and nucleic acids can be used in the compositions and methods described herein.

[0317] In some embodiments, the fusion polypeptide includes part or all of a first polypeptide (e.g., an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide or catalytically active fragment thereof) and may optionally include part or all of a second polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In some embodiments, the fusion polypeptide has an activity of two or more MVA pathway polypeptides (such as AA-CoA thiolase and HMG-CoA reductase polypeptides). In some embodiments, the polypeptide is a naturally-occurring polypeptide (such as the polypeptide encoded by an Enterococcus faecalis mvaE
nucleic acid) that has an activity of two or more MVA pathway polypeptides.

[0318] In various embodiments, a polypeptide has at least or about 50, 100, 150, 175, 200, 250, 300, 350, 400, or more amino acids. In some embodiments, the polypeptide fragment contains at least or about 25, 50, 75, 100, 150, 200, 300, or more contiguous amino acids from a full-length polypeptide and has at least or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of a corresponding full-length polypeptide. In particular embodiments, the polypeptide includes a segment of or the entire amino acid sequence of any naturally-occurring isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide. In some embodiments, the polypeptide has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide.

[0319] In some embodiments, the polypeptide is an isolated polypeptide. In some embodiments, the polypeptide is a heterologous polypeptide.

[0320] In some embodiments, the nucleic acid is a recombinant nucleic acid. In some embodiments, an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid is operably linked to another nucleic acid encoding all or a portion of another polypeptide such that the recombinant nucleic acid encodes a fusion polypeptide that includes an isoprene synthase, DXS, IDI, MVA
pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide and all or part of another polypeptide (e.g., a peptide that facilitates purification or detection of the fusion polypeptide, such as a His-tag). In some embodiments, part or all of a recombinant nucleic acid is chemically synthesized.
It is to be understood that mutations, including single nucleotide mutations, can occur within a nucleic acid as defined herein.

[0321] In some embodiments, the nucleic acid is a heterologous nucleic acid.
In particular embodiments, the nucleic acid includes a segment of or the entire nucleic acid sequence of any naturally-occurring isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid. In some embodiments, the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, or more contiguous nucleotides from a naturally-occurring isoprene synthase nucleic acid DXS, IDI, MVA
pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid. In some embodiments, the nucleic acid has one or more mutations compared to the sequence of a wild-type (i.e., a sequence occurring in nature) isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid. In some embodiments, the nucleic acid has one or more mutations (e.g., a silent mutation) that increase the transcription or translation of isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, or transcription factor nucleic acid. In some embodiments, the nucleic acid is a degenerate variant of any nucleic acid encoding an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide.

[0322] The accession numbers of exemplary isoprene synthase, DXS, IDI, and/or MVA
pathway polypeptides and nucleic acids are listed in Appendix 1 (the accession numbers of Appendix 1 and their corresponding sequences are herein incorporated by reference in their entireties, particularly with respect to the amino acid and nucleic acid sequences of isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids). The Kegg database also contains the amino acid and nucleic acid sequences of numerous exemplary isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids (see, for example, the world-wide web at "genome.jp/kegg/pathway/map/map00100.html" and the sequences therein, which are each hereby incorporated by reference in their entireties, particularly with respect to the amino acid and nucleic acid sequences of isoprene synthase, DXS, IDI, and/or MVA
pathway polypeptides and nucleic acids). In some embodiments, one or more of the isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and/or nucleic acids have a sequence identical to a sequence publicly available on December 12, 2007 or September 14, 2008 such as any of the sequences that correspond to any of the accession numbers in Appendix 1 or any of the sequences present in the Kegg database. Additional exemplary isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids are described further below.

Exemplary Isoprene Synthase Polypeptides and Nucleic Acids

[0323] As noted above, isoprene synthase polypeptides convert dimethyl allyl diphosphate (DMAPP) into isoprene. Exemplary isoprene synthase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an isoprene synthase polypeptide. Standard methods can be used to determine whether a polypeptide has isoprene synthase polypeptide activity by measuring the ability of the polypeptide to convert DMAPP into isoprene in vitro, in a cell extract, or in vivo. In an exemplary assay, cell extracts are prepared by growing a strain (e.g., the E.
coli/pTrcKudzu strain described herein) in the shake flask method as described in Example 1.
After induction is complete, approximately 10 mL of cells are pelleted by centrifugation at 7000 x g for 10 minutes and re-suspended in 5 ml of PEB without glycerol. The cells are lysed using a French Pressure cell using standard procedures. Alternatively the cells are treated with lysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thaw at -80 C.

[0324] Isoprene synthase polypeptide activity in the cell extract can be measured, for example, as described in Silver et al., J. Biol. Chem. 270:13010-13016, 1995 and references therein, which are each hereby incorporated by reference in their entireties, particularly with respect to assays for isoprene synthase polypeptide activity. DMAPP (Sigma) is evaporated to dryness under a stream of nitrogen and re-hydrated to a concentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 and stored at -20 C. To perform the assay, a solution of 5 L of 1M
MgCl2, 1 mM (250 g/ml) DMAPP, 65 L of Plant Extract Buffer (PEB) (50 mM Tris-HCI, pH
8.0, 20 mM MgCl2, 5% glycerol, and 2 mM DTT) is added to 25 L of cell extract in a 20 ml Headspace vial with a metal screw cap and teflon coated silicon septum (Agilent Technologies) and cultured at 37 C for 15 minutes with shaking. The reaction is quenched by adding 200 L
of 250 mM EDTA and quantified by GC/MS as described in Example 1, part II.

[0325] Exemplary isoprene synthase nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an isoprene synthase polypeptide. Exemplary isoprene synthase polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

[0326] In some embodiments, the isoprene synthase polypeptide or nucleic acid is from the family Fabaceae, such as the Faboideae subfamily. In some embodiments, the isoprene synthase polypeptide or nucleic acid is a polypeptide or nucleic acid from Pueraria montana (kudzu) (Sharkey et al., Plant Physiology 137: 700-712, 2005), Pueraria lobata, poplar (such as Populus alba, Populus nigra, Populus trichocarpa, or Populus alba x tremula (CAC35696) Miller et al., Planta 213: 483-487, 2001) aspen (such as Populus tremuloides) Silver et al., JBC 270(22):
13010-1316, 1995), or English Oak (Quercus robur) (Zimmer et al., WO
98/02550), which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene synthase nucleic acids and the expression of isoprene synthase polypeptides.
Suitable isoprene synthases include, but are not limited to, those identified by Genbank Accession Nos.
AY341431, AY316691, AY279379, AJ457070, and AY182241, which are each hereby incorporated by reference in their entireties, particularly with respect to sequences of isoprene synthase nucleic acids and polypeptides. In some embodiments, the isoprene synthase polypeptide or nucleic acid is not a naturally-occurring polypeptide or nucleic acid from Quercus robur (i.e., the isoprene synthase polypeptide or nucleic acid is an isoprene synthase polypeptide or nucleic acid other than a naturally-occurring polypeptide or nucleic acid from Quercus robur). In some embodiments, the isoprene synthase nucleic acid or polypeptide is a naturally-occurring polypeptide or nucleic acid from poplar. In some embodiments, the isoprene synthase nucleic acid or polypeptide is not a naturally-occurring polypeptide or nucleic acid from poplar.

Exemplary DXS Polypeptides and Nucleic Acids

[0327] As noted above, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptides convert pyruvate and D-glyceraldehyde-3 -phosphate into 1-deoxy-D-xylulose-5-phosphate.
Exemplary DXS polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of a DXS polypeptide.
Standard methods (such as those described herein) can be used to determine whether a polypeptide has DXS
polypeptide activity by measuring the ability of the polypeptide to convert pyruvate and D-glyceraldehyde-3 -phosphate into 1-deoxy-D-xylulose-5-phosphate in vitro, in a cell extract, or in vivo. Exemplary DXS nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of a DXS polypeptide.
Exemplary DXS polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

Exemplary IDI Polypeptides and Nucleic Acids

[0328] Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphate delta-isomerase or IDI) catalyses the interconversion of isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP) (e.g., converting IPP into DMAPP and/or converting DMAPP into IPP). Exemplary IDI polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an IDI
polypeptide. Standard methods (such as those described herein) can be used to determine whether a polypeptide has IDI
polypeptide activity by measuring the ability of the polypeptide to interconvert IPP and DMAPP
in vitro, in a cell extract, or in vivo. Exemplary IDI nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an IDI polypeptide. Exemplary IDI polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

Exemplary MVA Pathway Polypeptides and Nucleic Acids

[0329] Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase (AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA
synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK) polypeptides, phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonate decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDI
polypeptides, and polypeptides (e.g., fusion polypeptides) having an activity of two or more MVA
pathway polypeptides. In particular, MVA pathway polypeptides include polypeptides, fragments of polypeptides, peptides, and fusions polypeptides that have at least one activity of an MVA
pathway polypeptide. Exemplary MVA pathway nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of an MVA pathway polypeptide. Exemplary MVA pathway polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

[0330] In particular, acetyl-CoA acetyltransferase polypeptides (AA-CoA
thiolase or AACT) convert two molecules of acetyl-CoA into acetoacetyl-CoA. Standard methods (such as those described herein) can be used to determine whether a polypeptide has AA-CoA
thiolase polypeptide activity by measuring the ability of the polypeptide to convert two molecules of acetyl-CoA into acetoacetyl-CoA in vitro, in a cell extract, or in vivo.

[0331] 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase or HMGS) polypeptides convert acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA.
Standard methods (such as those described herein) can be used to determine whether a polypeptide has HMG-CoA
synthase polypeptide activity by measuring the ability of the polypeptide to convert acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl-CoA in vitro, in a cell extract, or in vivo.

[0332] 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR) polypeptides convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate. Standard methods (such as those described herein) can be used to determine whether a polypeptide has HMG-CoA
reductase polypeptide activity by measuring the ability of the polypeptide to convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate in vitro, in a cell extract, or in vivo.

[0333] Mevalonate kinase (MVK) polypeptides phosphorylates mevalonate to form mevalonate-5-phosphate. Standard methods (such as those described herein) can be used to determine whether a polypeptide has MVK polypeptide activity by measuring the ability of the polypeptide to convert mevalonate into mevalonate-5-phosphate in vitro, in a cell extract, or in vivo.

[0334] Phosphomevalonate kinase (PMK) polypeptides phosphorylates mevalonate-5-phosphate to form mevalonate-5-diphosphate. Standard methods (such as those described herein) can be used to determine whether a polypeptide has PMK polypeptide activity by measuring the ability of the polypeptide to convert mevalonate-5 -phosphate into mevalonate-5-diphosphate in vitro, in a cell extract, or in vivo.

[0335] Diphosphomevalonate decarboxylase (MVD or DPMDC) polypeptides convert mevalonate-5-diphosphate into isopentenyl diphosphate (IPP). Standard methods (such as those described herein) can be used to determine whether a polypeptide has MVD
polypeptide activity by measuring the ability of the polypeptide to convert mevalonate-5-diphosphate into IPP in vitro, in a cell extract, or in vivo.

[0336] Phosphomevalonate decarboxylase (PMDC) polypeptides convert mevalonate-phosphate into isopentenyl phosphate (IP). Standard methods (such as those described herein) can be used to determine whether a polypeptide has PMDC polypeptide activity by measuring the ability of the polypeptide to convert mevalonate-5-phosphate into IP in vitro, in a cell extract, or in vivo.

[0337] Isopentenyl phosphate kinase (IPK) polypeptides phosphorylate isopentyl phosphate (IP) to form isopentenyl diphosphate (IPP). Standard methods (such as those described herein) can be used to determine whether a polypeptide has IPK polypeptide activity by measuring the ability of the polypeptide to convert IP into IPP in vitro, in a cell extract, or in vivo.

[0338] Exemplary IDI polypeptides and nucleic acids are described above.
Exemplary Hydrogenase Polypeptides and Nucleic Acids

[0339] Hydrogenase polypeptides catalyze the reaction: 2H+ + 2e H H2. In vitro that reaction is reversible, but certain hydrogenases may work in only one direction in vivo, either oxidizing H2 or reducing H+. Hydrogenase polypeptides can be oxygen-sensitive, contain complex metal cofactors as part of their catalytic center and sometimes consist of multiple subunits, with hydrogenase gene expression sometimes involving additional accessory polypeptides, such as `maturation' factors or transcription regulatory factors (i.e., activators or repressors). Hydrogenases are classified into at least three broad groups based upon the type of metal cofactor in their catalytic center: (1) nickel-iron ("NiFe") hydrogenases have a nickel/iron cofactor; (2) iron-iron hydrogenases ("FeFe") have an iron/iron cofactor; and (3) iron/sulfur-free ("Fe") hydrogenases, which lack the 4Fe4S clusters found in groups (1) and (2), have an iron cofactor and a methenyl-tetrahydromethanopterin electron carrier. See, e.g., Chung-Jung Chou et al., "Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels,"
Metabol. Eng. 10:394-404 (2008), and Goniil Vardar-Schara et al., "Metabolically engineered bacteria for producing hydrogen via fermentation," Microbial Biotechnol.
1(2):107-125 (2008), both of which are incorporated herein by reference in their entireties, particularly with respect to the various types and classes of hydrogenases. Although many organisms contain multiple hydrogenases, few contain genes for both NiFe and FeFe hydrogenases.

[0340] The catalytic center of NiFe hydrogenases consists of a nickel atom and an iron atom, each with two carbon monoxide (CO) and two cyanide (CN) ligands. The NiFe hydrogenases all comprise at least a second subunit containing multiple iron-sulfur (Fe-S) centers for the transfer of electrons to and from the catalytic center. The NiFe hydrogenases can be subdivided into four main classes: (1) respiratory enzymes, which are part of multi-enzyme systems that couple the oxidation of H2 to reduction of terminal electron acceptors such as S042 or N03-under anaerobic conditions, or to 02 in aerobic microorganisms; (2) H2 sensors, which activate expression of the metabolically active NiFe hydrogenases; (3) cytoplasmic hydrogenases, containing multiple subunits able to utilize NADP+, which are readily reversible in vitro, but in vivo may only oxidize H2; and (4) membrane-bound, energy-conserving multi-enzyme complexes also found in bacteria and Archaea. Chung-Jung Chou et al., "Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels," Metabol. Eng.
10:394-404 (2008).

[0341] The catalytic center of FeFe hydrogenases contains a catalytic "H
cluster" which coordinates a binuclear (FeFe) site bridged to a [4Fe-4S] center by a single protein (cysteine) ligand. The two iron atoms of the binuclear center each have two carbon monoxide (CO) and two cyanide (CN) ligands, and are also bridged by two sulfur atoms which are part of a small organic molecule. Most FeFe hydrogenases are monomeric enzymes of about 50 kilodaltons (kDa), and appear to function in vivo primarily to dispose of excess reducing equivalents by reducing protons to hydrogen gas. Chung-Jung Chou et al., "Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels," Metabol. Eng. 10:394-404 (2008).

[0342] The catalytic center of Fe hydrogenases was originally thought to have an active site based on an organic cofactor with no metals involved, but was later shown to contain a mononuclear Fe atom. Despite the phylogenetic differences between the three types of hydrogenase, in addition to at least one iron atom, all three groups of hydrogenases also contain at least one carbon monoxide (CO) ligand to the iron atom in their active sites, which facilitates the catalytic oxidation of H2 and the reduction of protons. Chung-Jung Chou et al., "Hydrogenesis in hyperthermophilic microorganisms: implications for biofuels,"
Metabol. Eng.
10:394-404 (2008).

[0343] Exemplary hydrogenase polypeptides include, but are not limited to, the E. coli hydrogenase-1 (Hyd-1) polypeptides, E. coli hydrogenase-2 (Hyd-2) polypeptides, E. coli hydrogenase-3 (Hyd-3) polypeptides, E. coli hydrogenase-4 (Hyd-4) polypeptides, E. coli formate hydrogen lyase (FHL) complex, which produces hydrogen gas from formate and CO2 under anaerobic conditions at acidic pH (see, e.g., Akihito Yoshida et al., "Efficient induction of formate hydrogen lyase of aerobically grown Escherichia coli in a three-step biohydrogen production process," Appl. Microbiol. Biotechnol. 74:754-760 (2007), which is incorporated herein by reference in its entirety, particularly with respect to the induction of expression of formate hydrogen lyase in E. coli), Ralstonia eutropha H16 hydrogenase (R.
eutropha HoxH) Rhodococcus opacus MR] I hydrogenase (R. opacus HoxH) polypeptides, Synechosystis sp. PCC
6803 hydrogenase (Syn. PCC 6803 HoxH) polypeptides, Desulfovibrio gigas hydrogenase (D.
gigas) polypeptides, and Desulfovibrio desulfuricans ATCC 7757 hydrogenase (D.
desulfuricans) polypeptides (see, e.g., Goniil Vardar-Schara et al., "Metabolically engineered bacteria for producing hydrogen via fermentation," Microbial Biotechnol.
1(2):107-125 (2008), which is incorporated herein by reference in its entirety, particularly with respect to the various types and classes of hydrogenases) and polypeptides (e.g., fusion polypeptides) having an activity of two or more hydrogenase polypeptides. In particular, hydrogenase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusion polypeptides that have at least one activity of a hydrogenase polypeptide. Exemplary hydrogenase nucleic acids include nucleic acids that encode a polypeptide, fragment of a polypeptide, peptide, or fusion polypeptide that has at least one activity of a hydrogenase polypeptide, or at least one activity necessary for expression, processing, or maturation of a hydrogenase polypeptide. Exemplary hydrogenase polypeptides and nucleic acids include naturally-occurring polypeptides and nucleic acids from any of the source organisms described herein as well as mutant polypeptides and nucleic acids derived from any of the source organisms described herein.

[0344] E. coli Hyd-3, which is part of the anaerobic formate hydrogen lyase (FHL) complex, is encoded by the hyc operon (comprising the hycA, hycB, hycC, hycD, hycE, hycF, hycG, hycH, and hycl genes). E. coli Hyd-4 is encoded by the hyf operon (comprising the hyfA, hyfB, hyfC, hy)D, hyjE, hyfF, hyfG, hyfH, hyfl, hyfJ, and hyfR genes). E. coli FHL is encoded by six genes from the hyc operon (hycB, hycC, hycD, hycE, hycF and hycG) and the fdhF gene (encoding formate dehydrogenase H (Fdh-H)). Expression of the FHL complex can further involve expression of pyruvate formate lyase (pfl), FhIA, a transcription factor that activates transcription offdhF and the hyc operon, or deletion/inactivation of HycA, a transcription factor encoded by the hycA gene that negatively regulates transcription of FHL. Co-production of isoprene and hydrogen can be improved by expression or inactivation/deletion of additional proteins involved in the regulation of gene expression for hydrogenases and other enzymes, such as, for example, iron-sulfur complex transcriptional regulator (iscR) (Kalim-Akhtar et al., "Deletion of iscR stimulates recombinant Clostridial Fe/Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3)," App/. Microbiol. Biotechnol.
78:853-862 (2008), which is incorporated herein by reference in its entirety, particularly with reference to stimulation of Clostridial Fe/Fe hydrogenase activity and hydrogen accumulation in E. coli by deleting the iscR gene).

[0345] Exemplary ferredoxin-dependent hydrogenase polypeptides include, but are not limited to, Clostridium acetobutulicum hydrogenase A (HydA) (see, e.g., P.W. King et al., "Functional studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system," J.
Bacteriol. 188(6):163-172 (2006), which is incorporated herein by reference in its entirety, particularly with respect to production of hydrogen by HydA and three HydA-associated maturation enzymes (HydE, HydG, and HydF), which may be expressed alone or in conjunction with one or more of. (1) Bacillus subtilis NADPH ferredoxin oxidoreductase (NFOR) (see, e.g., Viet et al., (2008)), which is incorporated herein by reference in its entirety, particularly with respect to production of hydrogen by NFOR; see also PCT Publication No.
WO/2007/089901, which is incorporated herein by reference in its entirety, particularly with respect to optimization of E. coli strains for production of hydrogen), Clostridium kluyveri NADH
ferredoxin oxidoreductase (RnfCDGEAB) (Henning Seedorf et al., "The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features," Proc. Nat'l Acad. Sci. U.S.A.
105(6):2128-2133 (2008), which is incorporated herein by reference in its entirety, particular with reference to NADH ferredoxin oxidoreductase, and with reference to components of the anaerobic ethanol-acetate fermentation pathway), or Clostridium pasteuranium ferredoxin oxidoreductase (Fdx);
(2) glyceraldehyde-6-phosphate ferredoxin oxidoreductase ("GAPOR"); or (3) pyruvate ferredoxin oxidoreductase ("POR"), and polypeptides (e.g., fusion polypeptides) having an activity of two or more hydrogenase polypeptides or of one or more hydrogenase polypeptides and an activity of one or more ferredoxin-dependent oxidoreductases. In particular, ferredoxin-dependent hydrogenase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusion polypeptides that have at least one activity of a ferredoxin-dependent hydrogenase polypeptide.

[0346] Exemplary NADPH-dependent hydrogenase polypeptides include, but are not limited to thermophilic hydrogenase polypeptides such as Pyrococcusfuriosus hydrogenase (see, e.g., J.
Woodward et al., "Enzymatic production of biohydrogen," Nature 405(6790):1014-(2000)), and polypeptides (e.g., fusion polypeptides) having an activity of two or more NADPH-dependent hydrogenase polypeptides. In particular, NADPH-dependent hydrogenase polypeptides include polypeptides, fragments of polypeptides, peptides, and fusion polypeptides that have at least one activity of a NADPH-dependent hydrogenase polypeptide.

[0347] Exemplary oxygen-tolerant or oxygen-insensitive hydrogenases include, but are not limited to, Rubrivivax gelatinosus hydrogenase (see, e.g., P.C. Maness et al., "Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus," Appl. Environ. Microbiol. 68(6):2633-2636 (2002), which is incorporated herein by reference in its entirety, particularly with respect to R. gelatinosus hydrogenase), and Ralstonia eutropha hydrogenase polypeptides (see, e.g., T.
Burgdorf et al., "[NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation," J. Mol. Microbiol. Biotechnol. 10(2-4):181-196 (2005), which is incorporated herein by reference in its entirety, particularly with respect to R. eutropha hydrogenase polypeptides). Alternatively, heterologous nucleic acids encoding hydrogenase polypeptides can be mutagenized and screened for 02-tolerance or 02-insensitivity using standard methods and assays (see, e.g., L.E. Nagy et al., "Application of gene-shuffling for the rapid generation of novel [FeFe]-hydrogenase libraries," Biotechnol. Letts.
29(3)421-430 (2007), which is incorporated herein by reference, particularly with respect to mutagenesis and screening for oxygen tolerant hydrogenase polypeptides).

[0348] Standard methods (such as those described herein) can be used to determine whether a polypeptide has hydrogenase activity by measuring the ability of the polypeptide to produce hydrogen gas in vitro, in a cell extract, or in vivo.

Exemplary Polypeptides and Nucleic Acids for Genes Related to Production of Fermentation Side Products

[0349] In addition to expressing or over-expressing heterologous or native hydrogenases in E.
coli, co-production of isoprene and hydrogen can be improved by inactivation of anaerobic biosynthetic pathways, thereby blocking the carbon flow to a variety of metabolites (i.e., fermentation side products) produced under oxygen-limited or anaerobic conditions, including, but not limited to, lactate, acetate, pyruvate, ethanol, succinate, and glycerol. Exemplary polypeptides involved in the production of fermentation side products include formate dehydrogenase N, alpha subunit (fdnG), formate dehydrogenase 0, large subunit (fdoG), nitrate reductase (narG), formate transporter A (focA), formate transporter B (focB), pyruvate oxidase (poxB), pyruvate dehydrogenase El component ackA/pta (aceE), alcohol dehydrogenase (adhE), fumarate reductase membrane protein (frdC), and lactate dehydrogenase (ldhA).
See, e.g., Toshinori Maeda et al., "Enhanced hydrogen production from glucose by metabolically engineered Escherichia coli," Appl. Microbiol. Biotechnol. 77(4):879-890 (2007), which is incorporated by reference in its entirety, particularly with respect to production of E. coli strains with modified glucose metabolism. Exemplary polypeptides involved in the regulation or expression of genes involved in the production of fermentation side products that may also be inactivated to improve co-production of isoprene and hydrogen include, but are not limited to, repressor of formate hydrogen lyase (hycA), fumarate reductase regulator (fnr), acetyl-coenzyme A synthetase (acs), and formate dehydrogenase regulatory protein (hycA), which regulates expression of the transcriptional regulator fhlA (formate hydrogen lyase transcriptional activator).

Exemplary Polypeptides and Nucleic Acids for Genes Related to Hydrogen Re-uptake

[0350] Exemplary polypeptides involved in hydrogen re-uptake that may also be inactivated to improve co-production of isoprene and hydrogen include, but are not limited to, E. coli hydrogenase-1 (Hyd-1) (hya operon) and E. coli hydrogenase-2 (Hyd-2) (hyb operon). E. coli Hyd-1 is encoded by the hya operon (comprising the hyaA, hyaB, hyaC, hyaD, hyaE, and hyaF
genes). E. coli Hyd-2 is encoded by the hyb operon (comprising the hybA, hybB, hybC, hybD, hybE, hybF, hybG, and hybO genes).

Exemplary Methods for Isolating Nucleic Acids

[0351] Isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids can be isolated using standard methods. Methods of obtaining desired nucleic acids from a source organism of interest (such as a bacterial genome) are common and well known in the art of molecular biology (see, for example, WO
2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to the isolation of nucleic acids of interest). For example, if the sequence of the nucleic acid is known (such as any of the known nucleic acids described herein), suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired nucleic acid sequence.
Once the sequence is isolated, the DNA may be amplified using standard primer directed amplification methods such as polymerase chain reaction (PCR) (U.S. Patent No.
4,683,202, which is incorporated by reference in its entirety, particularly with respect to PCR methods) to obtain amounts of DNA suitable for transformation using appropriate vectors.

[0352] Alternatively, isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids (such as any isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids with a known nucleic acid sequence) can be chemically synthesized using standard methods.

[0353] Additional isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides and nucleic acids which may be suitable for use in the compositions and methods described herein can be identified using standard methods.
For example, cosmid libraries of the chromosomal DNA of organisms known to produce isoprene naturally can be constructed in organisms such as E. coli, and then screened for isoprene production. In particular, cosmid libraries may be created where large segments of genomic DNA (35-45 kb) are packaged into vectors and used to transform appropriate hosts.
Cosmid vectors are unique in being able to accommodate large quantities of DNA. Generally cosmid vectors have at least one copy of the cos DNA sequence which is needed for packaging and subsequent circularization of the heterologous DNA. In addition to the cos sequence, these vectors also contain an origin of replication such as Co1EI and drug resistance markers such as a nucleic acid resistant to ampicillin or neomycin. Methods of using cosmid vectors for the transformation of suitable bacterial hosts are well described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods.

[0354] Typically to clone cosmids, heterologous DNA is isolated using the appropriate restriction endonucleases and ligated adjacent to the cos region of the cosmid vector using the appropriate ligases. Cosmid vectors containing the linearized heterologous DNA
are then reacted with a DNA packaging vehicle such as bacteriophage. During the packaging process, the cos sites are cleaved and the heterologous DNA is packaged into the head portion of the bacterial viral particle. These particles are then used to transfect suitable host cells such as E. coli. Once injected into the cell, the heterologous DNA circularizes under the influence of the cos sticky ends. In this manner, large segments of heterologous DNA can be introduced and expressed in host cells.

[0355] Additional methods for obtaining isoprene synthase, DXS, IDI, MVA
pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids include screening a metagenomic library by assay (such as the headspace assay described herein) or by PCR using primers directed against nucleotides encoding for a length of conserved amino acids (for example, at least 3 conserved amino acids). Conserved amino acids can be identified by aligning amino acid sequences of known isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides. Conserved amino acids for isoprene synthase polypeptides can be identified based on aligned sequences of known isoprene synthase polypeptides. An organism found to produce isoprene naturally can be subjected to standard protein purification methods (which are well known in the art) and the resulting purified polypeptide can be sequenced using standard methods. Other methods are found in the literature (see, for example, Julsing et al., Applied. Microbiol. Biotechnol.
75: 1377-84, 2007;
Withers et al., Appl Environ Microbiol. 73(19):6277-83, 2007, which are each hereby incorporated by reference in their entireties, particularly with respect to identification of nucleic acids involved in the synthesis of isoprene).

[0356] Additionally, standard sequence alignment and/or structure prediction programs can be used to identify additional DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides and nucleic acids based on the similarity of their primary and/or predicted polypeptide secondary structure with that of known DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides and nucleic acids.
Standard databases such as the swissprot-trembl database (world-wide web at "expasy.org", Swiss Institute of Bioinformatics Swiss-Prot group CMU - 1 rue Michel Servet Geneva 4, Switzerland) can also be used to identify isoprene synthase, DXS, IDI, MVA
pathway, hydrogenase, hydrogenase maturation and/or transcription regulatory polypeptides and nucleic acids. The secondary and/or tertiary structure of an isoprene synthase, DXS, IDI, MVA
pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptide can be predicted using the default settings of standard structure prediction programs, such as PredictProtein (630 West, 168 Street, 1313217, New York, N.Y. 10032, USA).
Alternatively, the actual secondary and/or tertiary structure of an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptide can be determined using standard methods. Additional isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids can also be identified by hybridization to probes generated from known isoprene synthase, DXS, IDI, MVA
pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids.

Exemplary Promoters and Vectors

[0357] Any of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids described herein can be included in one or more vectors. Accordingly, also described herein are vectors with one more nucleic acids encoding any of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides that are described herein.
In some embodiments, the vector contains a nucleic acid under the control of an expression control sequence.

[0358] In some embodiments, the vector contains a selective marker or selectable marker.
Markers useful in vector systems for transformation of Trichoderma are known in the art (see, e.g., Finkelstein, Chapter 6 in Biotechnology of Filamentous Fungi, Finkelstein et al., Eds.
Butterworth-Heinemann, Boston, MA, Chap. 6., 1992; and Kinghorn et al., Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London, 1992, which are each hereby incorporated by reference in their entireties, particularly with respect to selective markers). In some embodiments, the selective marker is the amdS
nucleic acid, which encodes the enzyme acetamidase, allowing transformed cells to grow on acetamide as a nitrogen source. The use of an A. nidulans amdS nucleic acid as a selective marker is described in Kelley et al., EMBO J. 4:475 - 479, 1985 and Penttila et al., Gene 61:155-164, 1987 (which are each hereby incorporated by reference in their entireties, particularly with respect to selective markers). In some embodiments, an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation, or transcription regulatory nucleic acid integrates into a chromosome of the cells without a selective marker.

[0359] Suitable vectors are those which are compatible with the host cell employed. Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast, or a plant. Protocols for obtaining and using such vectors are known to those in the art (see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to the use of vectors).

[0360] Promoters are well known in the art. Any promoter that functions in the host cell can be used for expression of an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid in the host cell. Initiation control regions or promoters, which are useful to drive expression of isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids in various host cells are numerous and familiar to those skilled in the art (see, for example, WO

2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors for the expression of nucleic acids of interest). Virtually any promoter capable of driving these nucleic acids can be used including, but not limited to, CYC1, HIS3, GALL, GAL10, ADH1, PGK, PHO5, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, XPL, XPR, T7, tac, and trc (useful for expression in E. coli).

[0361] In some embodiments, a glucose isomerase promoter is used (see, for example, U.S.
Patent No. 7,132,527 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect promoters and plasmid systems for expressing polypeptides of interest). Reported glucose isomerase promoter mutants can be used to vary the level of expression of the polypeptide encoded by a nucleic acid operably linked to the glucose isomerase promoter (U.S. Patent No. 7,132,527). In various embodiments, the glucose isomerase promoter is contained in a low, medium, or high copy plasmid (U.S. Patent No. 7,132,527).

[0362] In various embodiments, an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is contained in a low copy plasmid (e.g., a plasmid that is maintained at about 1 to about 4 copies per cell), medium copy plasmid (e.g., a plasmid that is maintained at about 10 to about 15 copies per cell), or high copy plasmid (e.g., a plasmid that is maintained at about 50 or more copies per cell). In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA
pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to a T7 promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid operably linked to a T7 promoter is contained in a medium or high copy plasmid. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to a Trc promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid operably linked to a Trc promoter is contained in a medium or high copy plasmid. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to a Lac promoter.
In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA

pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid operably linked to a Lac promoter is contained in a low copy plasmid. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to an endogenous promoter, such as an endogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces, or Trichoderma promoter or an endogenous alkaline serine protease, isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor promoter. In some embodiments, the heterologous or extra endogenous isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid operably linked to an endogenous promoter is contained in a high copy plasmid. In some embodiments, the vector is a replicating plasmid that does not integrate into a chromosome in the cells. In some embodiments, part or all of the vector integrates into a chromosome in the cells.

[0363] In some embodiments, the vector is any vector which when introduced into a fungal host cell is integrated into the host cell genome and is replicated. Reference is made to the Fungal Genetics Stock Center Catalogue of Strains (FGSC, the world-wide web at "fgsc.net"
and the references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors) for a list of vectors.
Additional examples of suitable expression and/or integration vectors are provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987, Supplement 30, section 7.7.18); van den Hondel et al. in Bennett and Lasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp. 396-428, 1991; and U.S. Patent No. 5,874,276, which are each hereby incorporated by reference in their entireties, particularly with respect to vectors.
Particularly useful vectors include pFB6, pBR322, PUC18, pUC 100, and pENTR/D.

[0364] In some embodiments, an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is operably linked to a suitable promoter that shows transcriptional activity in a fungal host cell. The promoter may be derived from one or more nucleic acids encoding a polypeptide that is either endogenous or heterologous to the host cell. In some embodiments, the promoter is useful in a Trichoderma host. Suitable non-limiting examples of promoters include cbh 1, cbh2, egll, egl2, pepA, hfb1, hfb2, xyn 1, and amy. In some embodiments, the promoter is one that is native to the host cell.
For example, in some embodiments when T. reesei is the host, the promoter is a native T.
reesei promoter. In some embodiments, the promoter is T. reesei cbhl, which is an inducible promoter and has been deposited in GenBank under Accession No. D86235, which is incorporated by reference in its entirety, particularly with respect to promoters. In some embodiments, the promoter is one that is heterologous to the fungal host cell. Other examples of useful promoters include promoters from the genes of A. awamori and A. niger glucoamylase (glaA) (Nunberg et al., Mol.
Cell Biol.
4:2306-2315, 1984 and Boel et al., EMBO J. 3:1581-1585, 1984, which are each hereby incorporated by reference in their entireties, particularly with respect to promoters); Aspergillus niger alpha amylases, Aspergillus oryzae TAKA amylase, T. reesei xln1, and the T. reesei cellobiohydrolase 1 (EP 137280, which is incorporated by reference in its entirety, particularly with respect to promoters).

[0365] In some embodiments, the expression vector also includes a termination sequence.
Termination control regions may also be derived from various genes native to the host cell. In some embodiments, the termination sequence and the promoter sequence are derived from the same source. In another embodiment, the termination sequence is endogenous to the host cell. A
particularly suitable terminator sequence is cbh1 derived from a Trichoderma strain (such as T.
reesei). Other useful fungal terminators include the terminator from an A.
niger or A. awamori glucoamylase nucleic acid (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boel et al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated by reference in their entireties, particularly with respect to fungal terminators). Optionally, a termination site may be included.
For effective expression of the polypeptides, DNA encoding the polypeptide are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger RNA.

[0366] In some embodiments, the promoter, coding, region, and terminator all originate from the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid to be expressed. In some embodiments, the coding region for an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid is inserted into a general-purpose expression vector such that it is under the transcriptional control of the expression construct promoter and terminator sequences. In some embodiments, genes or part thereof are inserted downstream of the strong cbh 1 promoter.

[0367] An isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid can be incorporated into a vector, such as an expression vector, using standard techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is hereby incorporated by reference in its entirety, particularly with respect to the screening of appropriate DNA sequences and the construction of vectors). Methods used to ligate the DNA construct comprising a nucleic acid of interest (such as an isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid), a promoter, a terminator, and other sequences and to insert them into a suitable vector are well known in the art. For example, restriction enzymes can be used to cleave the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid and the vector. Then, the compatible ends of the cleaved isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid and the cleaved vector can be ligated. Linking is generally accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide linkers are used in accordance with conventional practice (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, and Bennett and Lasure, More Gene Manipulations in Fungi, Academic Press, San Diego, pp 70-76, 1991, which are each hereby incorporated by reference in their entireties, particularly with respect to oligonucleotide linkers). Additionally, vectors can be constructed using known recombination techniques (e.g., Invitrogen Life Technologies, Gateway Technology).

[0368] In some embodiments, it may be desirable to over-express isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids at levels far higher than currently found in naturally-occurring cells.
This result may be accomplished by the selective cloning of the nucleic acids encoding those polypeptides into multicopy plasmids or placing those nucleic acids under a strong inducible or constitutive promoter. Methods for over-expressing desired polypeptides are common and well known in the art of molecular biology and examples may be found in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to cloning techniques.

[0369] In some embodiments, it may be desirable to under-express (e.g., mutate, inactivate, or delete) isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation, or transcription factor polypeptide-encoding nucleic acids at levels far below that those currently found in naturally-occurring cells. This result may be accomplished by the mutation or inactivation of transcriptional regulatory proteins required for expression of isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids, by deletion of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids, or by placing those nucleic acids under the control of a strong repressible promoter. Methods for mutating, inactivating, or deleting desired polypeptides are common and well known in the art of molecular biology and examples may be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to cloning and mutagenesis techniques.

[0370] The following resources include descriptions of additional general methodology useful in accordance with the compositions and methods described herein: Kreigler, Gene Transfer and Expression; A Laboratory Manual, 1990 and Ausubel et al., Eds. Current Protocols in Molecular Biology, 1994, which are each hereby incorporated by reference in their entireties, particularly with respect to molecular biology and cloning techniques.

Exemplary Source Organisms

[0371] Isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids (and their encoded polypeptides) can be obtained from any organism that naturally contains isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids. As noted above, isoprene is formed naturally by a variety of organisms, such as bacteria, yeast, plants, and animals.
Organisms contain the MVA pathway, DXP pathway, or both the MVA and DXP
pathways for producing isoprene (Figures 19A and 19B). Thus, DXS nucleic acids can be obtained, e.g., from any organism that contains the DXP pathway or contains both the MVA and DXP
pathways.
IDI and isoprene synthase nucleic acids can be obtained, e.g., from any organism that contains the MVA pathway, DXP pathway, or both the MVA and DXP pathways. MVA pathway nucleic acids can be obtained, e.g., from any organism that contains the MVA pathway or contains both the MVA and DXP pathways. Hydrogenase nucleic acids can be obtained, e.g., from any organism that oxidizes hydrogen or reduces hydrogen ions. Fermentation side product genes can be obtained or identified, e.g., from any organism that undergoes oxygen-limited or anaerobic respiration, such as glycolysis.

[0372] In some embodiments, the nucleic acid sequence of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic is identical to the sequence of a nucleic acid that is produced by any of the following organisms in nature. In some embodiments, the amino acid sequence of the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptide is identical to the sequence of a polypeptide that is produced by any of the following organisms in nature. In some embodiments, the isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acid or polypeptide is a mutant nucleic acid or polypeptide derived from any of the organisms described herein. As used herein, "derived from" refers to the source of the nucleic acid or polypeptide into which one or more mutations is introduced. For example, a polypeptide that is "derived from a plant polypeptide"
refers to polypeptide of interest that results from introducing one or more mutations into the sequence of a wild-type (i.e., a sequence occurring in nature) plant polypeptide.

[0373] In some embodiments, the source organism is a fungus, examples of which are species of Aspergillus such as A. oryzae and A. niger, species of Saccharomyces such as S. cerevisiae, species of Schizosaccharomyces such as S. pombe, and species of Trichoderma such as T. reesei.
In some embodiments, the source organism is a filamentous fungal cell. The term "filamentous fungi" refers to all filamentous forms of the subdivision Eumycotina (see, Alexopoulos, C. J.
(1962), Introductory Mycology, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic. The filamentous fungal parent cell may be a cell of a species of, but not limited to, Trichoderma, (e.g., Trichoderma reesei, the asexual morph of Hypocrea jecorina, previously classified as T. longibrachiatum, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol.
Biotechnol 20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp., Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea); Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillus sp. (e.g., A. oryzae, A. niger, A sojae, A. japonicus, A.
nidulans, or A. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993 and Goedegebuur et al., Genet 41:
89-98, 2002), Fusarium sp., (e.g., F. roseum, F. graminum F. cerealis, F.
oxysporuim, or F.
venenatum), Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M. miehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci. 228: 21-26, 1985). The term "Trichoderma" or "Trichoderma sp." or "Trichoderma spp." refer to any fungal genus previously or currently classified as Trichoderma.

[0374] In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F. oxysporum, or F. solani.
Aspergillus strains are disclosed in Ward et al., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur et al., Curr Gene 41:89-98, 2002, which are each hereby incorporated by reference in their entireties, particularly with respect to fungi. In particular embodiments, the fungus is a strain of Trichoderma, such as a strain of T. reesei. Strains of T. reesei are known and non-limiting examples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCC No.
56765, ATCC No. 56767, and NRRL 15709, which are each hereby incorporated by reference in their entireties, particularly with respect to strains of T. reesei. In some embodiments, the host strain is a derivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl.
Microbiol.
Biotechnology 20:46-53, 1984, which is hereby incorporated by reference in its entirety, particularly with respect to strains of T. reesei.

[0375] In some embodiments, the source organism is a yeast, such as Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp. In some embodiments, the Saccharomyces sp. is Saccharomyces cerevisiae.

[0376] In some embodiments, the source organism is a bacterium, such as strains of Bacillus such as B. lichenformis or B. subtilis, strains of Pantoea such as P. citrea, strains of Pseudomonas such as P. alcaligenes, P. putida, or P. fluorescens, strains of Streptomyces such as S. lividans or S. rubiginosus, strains of Corynebacterium sp. such as Corynebacterium glutamicum, strains of Rhodopseudomonas sp. such as Rhodopseudomonas palustris, or strains of Escherichia such as E. coli.

[0377] As used herein, "the genus Bacillus" includes all species within the genus "Bacillus,"
as known to those of skill in the art, including but not limited to B.
subtilis, B. licheniformis, B.
lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.
amyloliquefaciens, B. clausii, B.
halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B.
thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named "Geobacillus stearothermophilus." The production of resistant endospores in the presence of oxygen is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0378] In some embodiments, the source organism is a gram-positive bacterium.
Non-limiting examples include strains of Streptomyces (e.g., S. lividans, S. coelicolor, or S. griseus) and Bacillus. In some embodiments, the source organism is a gram-negative bacterium, such as E.

coli., Rhodopseudomonas sp. such as Rhodopseudomonas palustris, or Pseudomonas sp., such as P. alcaligenes, P. putida, or P. fluorescens.

[0379] In some embodiments, the source organism is a plant, such as a plant from the family Fabaceae, such as the Faboideae subfamily. In some embodiments, the source organism is kudzu, poplar (such as Populus alba x tremula CAC35696), aspen (such as Populus tremuloides), or Quercus robur.

[0380] In some embodiments, the source organism is an algae, such as a green algae, red algae, glaucophytes, chlorarachniophytes, euglenids, chromista, or dinoflagellates.

[0381] In some embodiments, the source organism is a cyanobacteria, such as cyanobacteria classified into any of the following groups based on morphology:
Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, or Stigonematales.

[0382] In some embodiments, the source organism is an anaerobic organism.
Anaerobic organisms can include, but are not limited to, obligate anaerobes, facultatitive anaerobes, and aerotolerant anaerobes. Such organisms can be any of the organisms listed above, bacteria, yeast, etc. In one embodiment, the obligate anaerobes can be any one or combination selected from the group consisting of Clostridium jungdahlii, Clostridium autoethanogenum, Eurobacterium limosum, Clostridium carboxydivorans, Peptostreptococcus productus, and Butyribacterium methylotrophicum. It is to be understood that any combination of any of the source organisms described herein can be used for other embodiments of the invention.
Exemplary Host Cells

[0383] A variety of host cells can be used to express isoprene synthase, DXS, IDI, MVA
pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptides and to co-produce isoprene and hydrogen in the methods described herein. Exemplary host cells include cells from any of the organisms listed in the prior section under the heading "Exemplary Source Organisms." The host cell may be a cell that naturally produces isoprene or a cell that does not naturally produce isoprene. In some embodiments, the host cell naturally produces isoprene using the DXP pathway, and an isoprene synthase, DXS, and/or IDI
nucleic acid is added to enhance production of isoprene using this pathway. In some embodiments, the host cell naturally produces isoprene using the MVA pathway, and an isoprene synthase and/or one or more MVA pathway nucleic acids are added to enhance production of isoprene using this pathway. In some embodiments, the host cell naturally produces isoprene using the DXP
pathway and one or more MVA pathway nucleic acids are added to produce isoprene using part or all of the MVA pathway as well as the DXP pathway. In some embodiments, the host cell naturally produces isoprene using both the DXP and MVA pathways and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways.

[0384] In some embodiments, the host cell naturally produces isoprene using both the DXP
and MVA pathways, and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways, one or more hydrogenase nucleic acids are added to enhance hydrogen production and one or more fermentation side product-producing genes are inactivated or deleted to limit production of fermentation side products. In some embodiments, the host cell naturally co-produces isoprene and hydrogen using both the DXP and MVA pathways and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways, one or more hydrogenase nucleic acids are added to enhance hydrogen production, one or more fermentation side product-producing genes are inactivated or deleted to limit production of fermentation side products, and one or more hydrogen reuptake genes are inactivated or deleted to increase hydrogen production. In some embodiments, the host cell naturally co-produces isoprene and hydrogen using both the DXP and MVA
pathways and a hydrogenase, and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways, one or more hydrogenase nucleic acids are added to enhance hydrogen production, one or more hydrogenase maturation nucleic acids are added to enhance hydrogen production, one or more fermentation side product-producing genes are inactivated or deleted to limit production of fermentation side products, and one or more hydrogen reuptake genes are inactivated or deleted to increase hydrogen production. In some embodiments, the host cell naturally co-produces isoprene and hydrogen using both the DXP and MVA pathways and one or more isoprene synthase, DXS, IDI, or MVA pathway nucleic acids are added to enhance production of isoprene by one or both of these pathways, one or more hydrogenase nucleic acids are added to enhance hydrogen production, one or more hydrogenase maturation nucleic acids are added to enhance hydrogen production, one or more transcription factor nucleic acids are added or inactivated or deleted to enhance hydrogenase production, one or more fermentation side product-producing genes are inactivated or deleted to limit production of fermentation side products, and one or more hydrogen reuptake genes are inactivated or deleted to increase hydrogen production.

Exemplary Transformation Methods

[0385] Isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor nucleic acids or vectors containing them can be inserted into a host cell (e.g., a plant cell, a fungal cell, a yeast cell, or a bacterial cell described herein) using standard techniques for expression of the encoded isoprene synthase, DXS, IDI, MVA pathway, hydrogenase, hydrogenase maturation and/or transcription factor polypeptide.
Introduction of a DNA construct or vector into a host cell can be performed using techniques such as transformation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated or DEAE-Dextrin mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are known in the art (see, e.g., Current Protocols in Molecular Biology (F. M.
Ausubel et al. (eds) Chapter 9, 1987; Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989; and Campbell et al., Curr. Genet.
16:53-56, 1989, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods). The expression of heterologous polypeptide in Trichoderma is described in U.S. Patent No. 6,022,725; U.S. Patent No. 6,268,328; U.S. Patent No.
7,262,041;WO 2005/001036; Harkki et al., Enzyme Microb. Technol. 13:227-233, 1991; Harkki et al., Bio Technol. 7:596-603, 1989; EP 244,234; EP 215,594; and Nevalainen et al., "The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes," in Molecular Industrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY pp. 129 - 148, 1992, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation and expression methods). Reference is also made to Cao et al., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al., Proceedings. Natl.
Acad. Sci. USA 81:1470-1474, 1984 (which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods) for transformation of Aspergillus strains. The introduced nucleic acids may be integrated into chromosomal DNA
or maintained as extrachromosomal replicating sequences.

[0386] Any method known in the art may be used to select transformants. In one non-limiting example, stable transformants including an amdS marker are distinguished from unstable transformants by their faster growth rate and the formation of circular colonies with a smooth, rather than ragged outline on solid culture medium containing acetamide.
Additionally, in some cases a further test of stability is conducted by growing the transformants on a solid non-selective medium (e.g., a medium that lacks acetamide), harvesting spores from this culture medium, and determining the percentage of these spores which subsequently germinate and grow on selective medium containing acetamide.

[0387] In some embodiments, fungal cells are transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a known manner. In one specific embodiment, the preparation of Trichoderma sp.
for transformation involves the preparation of protoplasts from fungal mycelia (see, Campbell et al., Curr. Genet. 16:53-56, 1989, which is incorporated by reference in its entirety, particularly with respect to transformation methods). In some embodiments, the mycelia are obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall resulting in protoplasts. The protoplasts are then protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually the concentration of these stabilizers varies between 0.8 M and 1.2 M. It is desirable to use about a 1.2 M solution of sorbitol in the suspension medium.

[0388] Uptake of DNA into the host Trichoderma sp. strain is dependent upon the calcium ion concentration. Generally, between about 10 mM CaCl2 and 50 mM CaC12 is used in an uptake solution. In addition to the calcium ion in the uptake solution, other compounds generally included are a buffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 MM
EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG).
While not intending to be bound to any particular theory, it is believed that the polyethylene glycol acts to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoderma sp. strain and the plasmid DNA
to be transferred to the nucleus. This fusion frequently leaves multiple copies of the plasmid DNA
integrated into the host chromosome.

[0389] Usually a suspension containing the Trichoderma sp. protoplasts or cells that have been subjected to a permeability treatment at a density of 105 to 107/mL (such as 2 x 106/mL) are used in the transformation. A volume of 100 L of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaC12) are mixed with the desired DNA.
Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. In some embodiments, about 0.25 volumes are added to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, and the like may also be added to the uptake solution and aid in transformation. Similar procedures are available for other fungal host cells (see, e.g., U.S. Patent Nos. 6,022,725 and 6,268,328, which are each hereby incorporated by reference in their entireties, particularly with respect to transformation methods).

[0390] Generally, the mixture is then cultured at approximately 0 C for a period of between 10 to 30 minutes. Additional PEG is then added to the mixture to further enhance the uptake of the desired nucleic acid sequence. The 25% PEG 4000 is generally added in volumes of 5 to 15 times the volume of the transformation mixture; however, greater and lesser volumes may be suitable. The 25% PEG 4000 is desirably about 10 times the volume of the transformation mixture. After the PEG is added, the transformation mixture is then cultured either at room temperature or on ice before the addition of a sorbitol and CaC12 solution.
The protoplast suspension is then further added to molten aliquots of a growth medium. When the growth medium includes a growth selection (e.g., acetamide or an antibiotic) it permits the growth of transformants only.

[0391] The transformation of bacterial cells maybe performed according to conventional methods, e.g., as described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor, 1982, which is hereby incorporated by reference in its entirety, particularly with respect to transformation methods.

Exemplary Cell Culture Media

[0392] Also described herein is a cell or a population of cells in culture that co-produce isoprene and hydrogen. By "cells in culture" is meant two or more cells in a solution (e.g., a cell growth medium) that allows the cells to undergo one or more cell divisions.
"Cells in culture" do not include plant cells that are part of a living, multicellular plant containing cells that have differentiated into plant tissues. In various embodiments, the cell culture includes at least or about 10, 20, 50, 100, 200, 500, 1,000, 5,000, 10,000 or more cells.

[0393] By "cells in oxygen-limited culture" is meant two or more cells in a solution (e.g., a cell growth medium) that allows the cell to under go one or more cell divisions, wherein the solution contains a limiting amount of oxygen. The term "oxygen-limited culture" means that the culture is either anoxic or contains less than the required amount of oxygen to support respiration via the biological transfer of reducing equivalents to oxygen, and also encompasses anaerobic cultures. Under oxygen-limited culture conditions, some electrons derived from carbon metabolism cannot be accepted because oxygen concentrations are too low, causing cells to switch to hydrogen production if they comprise the appropriate metabolic pathways for doing so. Oxygen-limited culture conditions occur when the oxygen transfer rate ("OTR") is less than the oxygen uptake rate ("OUR") indicated by dissolved oxygen concentrations of close to zero in culture medium.

[0394] Any carbon source can be used to cultivate the host cells. The term "carbon source"
refers to one or more carbon-containing compounds capable of being metabolized by a host cell or organism. For example, the cell medium used to cultivate the host cells may include any carbon source suitable for maintaining the viability or growing the host cells.

[0395] In some embodiments, the carbon source is a carbohydrate (such as monosaccharide, disaccharide, oligosaccharide, or polysaccharides), invert sugar (e.g., enzymatically treated sucrose syrup), glycerol, glycerine (e.g., a glycerine byproduct of a biodiesel or soap-making process), dihydroxyacetone, one-carbon source, oil (e.g., a plant or vegetable oil such as corn, palm, or soybean oil), animal fat, animal oil, fatty acid (e.g., a saturated fatty acid, unsaturated fatty acid, or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbial or plant protein or peptide), renewable carbon source (e.g., a biomass carbon source such as a hydrolyzed biomass carbon source), yeast extract, component from a yeast extract, polymer, acid, alcohol, aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, or any combination of two or more of the foregoing. In some embodiments, the carbon source is a product of photosynthesis, including, but not limited to, glucose.

[0396] Exemplary monosaccharides include glucose and fructose; exemplary oligosaccharides include lactose and sucrose, and exemplary polysaccharides include starch and cellulose.
Exemplary carbohydrates include C6 sugars (e.g., fructose, mannose, galactose, or glucose) and C5 sugars (e.g., xylose or arabinose). In some embodiments, the cell medium includes a carbohydrate as well as a carbon source other than a carbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, or a component from a yeast extract). In some embodiments, the cell medium includes a carbohydrate as well as a polypeptide (e.g., a microbial or plant protein or peptide). In some embodiments, the microbial polypeptide is a polypeptide from yeast or bacteria. In some embodiments, the plant polypeptide is a polypeptide from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

[0397] In some embodiments, the concentration of the carbohydrate is at least or about 5 grams per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the carbohydrate is between about 50 and about 400 g/L, such as between about 100 and about 360 g/L, between about 120 and about 360 g/L, or between about 200 and about 300 g/L. In some embodiments, this concentration of carbohydrate includes the total amount of carbohydrate that is added before and/or during the culturing of the host cells.

[0398] In some embodiments, the cells are cultured under limited glucose conditions. By "limited glucose conditions" is meant that the amount of glucose that is added is less than or about 105% (such as about 100%) of the amount of glucose that is consumed by the cells. In particular embodiments, the amount of glucose that is added to the culture medium is approximately the same as the amount of glucose that is consumed by the cells during a specific period of time. In some embodiments, the rate of cell growth is controlled by limiting the amount of added glucose such that the cells grow at the rate that can be supported by the amount of glucose in the cell medium. In some embodiments, glucose does not accumulate during the time the cells are cultured. In various embodiments, the cells are cultured under limited glucose conditions for greater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours. In various embodiments, the cells are cultured under limited glucose conditions for greater than or about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time the cells are cultured. While not intending to be bound by any particular theory, it is believed that limited glucose conditions may allow more favorable regulation of the cells.

[0399] In some embodiments, the cells are cultured in the presence of an excess of glucose. In particular embodiments, the amount of glucose that is added is greater than about 105% (such as about or greater than 110, 120, 150, 175, 200, 250, 300, 400, or 500%) or more of the amount of glucose that is consumed by the cells during a specific period of time. In some embodiments, glucose accumulates during the time the cells are cultured.

[0400] Exemplary lipids are any substance containing one or more fatty acids that are C4 and above fatty acids that are saturated, unsaturated, or branched.

[0401] Exemplary oils are lipids that are liquid at room temperature. In some embodiments, the lipid contains one or more C4 or above fatty acids (e.g., contains one or more saturated, unsaturated, or branched fatty acid with four or more carbons). In some embodiments, the oil is obtained from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, linseed, oleagineous microbial cells, Chinese tallow, or any combination of two or more of the foregoing.

[0402] Exemplary fatty acids include compounds of the formula RCOOH, where "R"
is a hydrocarbon. Exemplary unsaturated fatty acids include compounds where "R"
includes at least one carbon-carbon double bond. Exemplary unsaturated fatty acids include, but are not limited to, oleic acid, vaccenic acid, linoleic acid, palmitoleic acid, and arachidonic acid. Exemplary polyunsaturated fatty acids include compounds where "R" includes a plurality of carbon-carbon double bonds. Exemplary saturated fatty acids include compounds where "R" is a saturated aliphatic group. In some embodiments, the carbon source includes one or more C12-C22 fatty acids, such as a C12 saturated fatty acid, a C14 saturated fatty acid, a C16 saturated fatty acid, a C18 saturated fatty acid, a C20 saturated fatty acid, or a C22 saturated fatty acid. In an exemplary embodiment, the fatty acid is palmitic acid. In some embodiments, the carbon source is a salt of a fatty acid (e.g., an unsaturated fatty acid), a derivative of a fatty acid (e.g., an unsaturated fatty acid), or a salt of a derivative of fatty acid (e.g., an unsaturated fatty acid). Suitable salts include, but are not limited to, lithium salts, potassium salts, sodium salts, and the like. Di- and triglycerols are fatty acid esters of glycerol.

[0403] In some embodiments, the concentration of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is at least or about 1 gram per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such as at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments, the concentration of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 10 and about 400 g/L, such as between about 25 and about 300 g/L, between about 60 and about 180 g/L, or between about 75 and about 150 g/L. In some embodiments, the concentration includes the total amount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both (i) a lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride and (ii) a carbohydrate, such as glucose. In some embodiments, the ratio of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e., one carbon in the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride per carbohydrate carbon). In particular embodiments, the amount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride is between about 60 and 180 g/L, and the amount of the carbohydrate is between about 120 and 360 g/L.

[0404] Exemplary microbial polypeptide carbon sources include one or more polypeptides from yeast or bacteria. Exemplary plant polypeptide carbon sources include one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

[0405] Exemplary renewable carbon sources include cheese whey permeate, cornsteep liquor, sugar beet molasses, barley malt, and components from any of the foregoing.
Exemplary renewable carbon sources also include glucose, hexose, pentose and xylose present in biomass, such as corn, switchgrass, sugar cane, cell waste of fermentation processes, and protein by-product from the milling of soy, corn, or wheat. In some embodiments, the biomass carbon source is a lignocellulosic, hemicellulosic, or cellulosic material such as, but are not limited to, a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft wood pulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, corn stover, switch grass, rice hull product, or a by-product from wet or dry milling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat, and/or distillers grains). Exemplary cellulosic materials include wood, paper and pulp waste, herbaceous plants, and fruit pulp. In some embodiments, the carbon source includes any plant part, such as stems, grains, roots, or tubers. In some embodiments, all or part of any of the following plants are used as a carbon source: corn, wheat, rye, sorghum, triticate, rice, millet, barley, cassava, legumes, such as beans and peas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. In some embodiments, the carbon source is a biomass hydrolysate, such as a biomass hydrolysate that includes both xylose and glucose or that includes both sucrose and glucose.

[0406] In some embodiments, the renewable carbon source (such as biomass) is pretreated before it is added to the cell culture medium. In some embodiments, the pretreatment includes enzymatic pretreatment, chemical pretreatment, or a combination of both enzymatic and chemical pretreatment (see, for example, Farzaneh et al., Bioresource Technology 96 (18): 2014-2018, 2005; U.S. Patent No. 6,176,176; U.S. Patent No. 6,106,888; which are each hereby incorporated by reference in their entireties, particularly with respect to the pretreatment of renewable carbon sources). In some embodiments, the renewable carbon source is partially or completely hydrolyzed before it is added to the cell culture medium.

[0407] In some embodiments, the renewable carbon source (such as corn stover) undergoes ammonia fiber expansion (AFEX) pretreatment before it is added to the cell culture medium (see, for example, Farzaneh et al., Bioresource Technology 96 (18): 2014-2018, 2005). During AFEX
pretreatment, a renewable carbon source is treated with liquid anhydrous ammonia at moderate temperatures (such as about 60 to about 100 C) and high pressure (such as about 250 to about 300 psi) for about 5 minutes. Then, the pressure is rapidly released. In this process, the combined chemical and physical effects of lignin solubilization, hemicellulose hydrolysis, cellulose decrystallization, and increased surface area enables near complete enzymatic conversion of cellulose and hemicellulose to fermentable sugars. AFEX pretreatment has the advantage that nearly all of the ammonia can be recovered and reused, while the remaining serves as nitrogen source for microbes in downstream processes. Also, a wash stream is not required for AFEX
pretreatment. Thus, dry matter recovery following the AFEX treatment is essentially 100%. AFEX
is basically a dry to dry process. The treated renewable carbon source is stable for long periods and can be fed at very high solid loadings in enzymatic hydrolysis or fermentation processes.
Cellulose and hemicellulose are well preserved in the AFEX process, with little or no degradation.
There is no need for neutralization prior to the enzymatic hydrolysis of a renewable carbon source that has undergone AFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sources produces clean sugar streams for subsequent fermentation use.

[0408] In some embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to at least or about 0.1, 0.5, 1, 1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50%
glucose (w/v). The equivalent amount of glucose can be determined by using standard HPLC
methods with glucose as a reference to measure the amount of glucose generated from the carbon source. In some embodiments, the concentration of the carbon source (e.g., a renewable carbon source) is equivalent to between about 0.1 and about 20% glucose, such as between about 0.1 and about 10% glucose, between about 0.5 and about 10% glucose, between about 1 and about 10% glucose, between about 1 and about 5% glucose, or between about 1 and about 2%
glucose.

[0409] In some embodiments, the carbon source includes yeast extract or one or more components of yeast extract. In some embodiments, the concentration of yeast extract is at least 1 gram of yeast extract per liter of broth (g/L, wherein the volume of broth includes both the volume of the cell medium and the volume of the cells), such at least or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In some embodiments, the concentration of yeast extract is between about 1 and about 300 g/L, such as between about 1 and about 200 g/L, between about 5 and about 200 g/L, between about 5 and about 100 g/L, or between about 5 and about 60 g/L. In some embodiments, the concentration includes the total amount of yeast extract that is added before and/or during the culturing of the host cells. In some embodiments, the carbon source includes both yeast extract (or one or more components thereof) and another carbon source, such as glucose. In some embodiments, the ratio of yeast extract to the other carbon source is about 1:5, about 1:10, or about 1:20 (w/w).

[0410] Additionally the carbon source may also be one-carbon substrates such as carbon dioxide, or methanol. Glycerol production from single carbon sources (e.g., methanol, formaldehyde, or formate) has been reported in methylotrophic yeasts (Yamada et al., Agric.
Biol. Chem., 53(2) 541-543, 1989, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources) and in bacteria (Hunter et. al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). These organisms can assimilate single carbon compounds, ranging in oxidation state from methane to formate, and produce glycerol. The pathway of carbon assimilation can be through ribulose monophosphate, through serine, or through xylulose-momophosphate (Gottschalk, Bacterial Metabolism, Second Edition, Springer-Verlag: New York, 1986, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). The ribulose monophosphate pathway involves the condensation of formate with ribulose-5 -phosphate to form a six carbon sugar that becomes fructose and eventually the three carbon product glyceraldehyde-3-phosphate. Likewise, the serine pathway assimilates the one-carbon compound into the glycolytic pathway via methylenetetrahydrofolate.

[0411] In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7th ed., 415-32. Editors: Murrell et al., Publisher:
Intercept, Andover, UK, 1993, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources). Similarly, various species of Candida metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153(5), 485-9, 1990, which is hereby incorporated by reference in its entirety, particularly with respect to carbon sources).

[0412] In some embodiments, cells are cultured in a standard medium containing physiological salts and nutrients (see, e.g., Pourquie, J. et al., Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al., Academic Press, pp. 71-86, 1988 and Ilmen et al., App/. Environ. Microbiol. 63:1298-1306, 1997, which are each hereby incorporated by reference in their entireties, particularly with respect to cell medias). Exemplary growth media are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of particular host cells are known by someone skilled in the art of microbiology or fermentation science.

[0413] In addition to an appropriate carbon source, the cell medium desirably contains suitable minerals, salts, cofactors, buffers, and other components known to those skilled in the art suitable for the growth of the cultures or the enhancement of isoprene production (see, for example, WO 2004/033646 and references cited therein and WO 96/35796 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect cell medias and cell culture conditions). In some embodiments where an isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acid is under the control of an inducible promoter, the inducing agent (e.g., a sugar, metal salt or antimicrobial), is desirably added to the medium at a concentration effective to induce expression of an isoprene synthase, DXS, IDI, and/or MVA pathway polypeptide. In some embodiments, cell medium has an antibiotic (such as kanamycin) that corresponds to the antibiotic resistance nucleic acid (such as a kanamycin resistance nucleic acid) on a vector that has one or more DXS, IDI, or MVA
pathway nucleic acids.

Exemplary Cell Culture Conditions

[0414] Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Exemplary techniques may be found in Manual of Methods for General Bacteriology Gerhardt et al., eds), American Society for Microbiology, Washington, D.C.
(1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA, which are each hereby incorporated by reference in their entireties, particularly with respect to cell culture techniques. In some embodiments, the cells are cultured in a culture medium under conditions permitting the expression of one or more isoprene synthase, DXS, IDI, or MVA pathway polypeptides encoded by a nucleic acid inserted into the host cells.

[0415] Standard cell culture conditions can be used to culture the cells (see, for example, WO
2004/033646 and references cited therein, which are each hereby incorporated by reference in their entireties, particularly with respect to cell culture and fermentation conditions). Cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as at about 20 C
to about 37 C, at about 6% to about 84% C02, and at a pH between about 5 to about 9). In some embodiments, cells are grown at 35 C in an appropriate cell medium. In some embodiments, e.g., cultures are cultured at approximately 28 C in appropriate medium in shake cultures or fermentors until the desired amount of isoprene and hydrogen co-production is achieved. In some embodiments, the pH ranges for fermentation are between about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5 to about 7.0). Reactions may be performed under aerobic, anoxic, or anaerobic conditions based on the requirements of the host cells. In some embodiments, the cells are cultured under oxygen-limited conditions. In some embodiments, the cells are cultured in the presence of oxygen under conditions where 0.5 moles of oxygen are taken up per mole of isoprene produced. In some embodiments, the cells are cultured under anaerobic conditions. Exemplary culture conditions for a given filamentous fungus are known in the art and may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection and Fungal Genetics Stock Center.

[0416] In various embodiments, the cells are grown using any known mode of fermentation, such as batch, fed-batch, or continuous processes. In some embodiments, a batch method of fermentation is used. Classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the cell medium is inoculated with the desired host cells and fermentation is permitted to occur adding nothing to the system.
Typically, however, "batch" fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly until the time the fermentation is stopped. Within batch cultures, cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. In some embodiments, cells in log phase are responsible for the bulk of the isoprene production. In some embodiments, cells in stationary phase produce isoprene.

[0417] In some embodiments, a variation on the standard batch system is used, such as the Fed-Batch system. Fed-Batch fermentation processes comprise a typical batch system with the exception that the carbon source is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of carbon source in the cell medium. Fed-batch fermentations may be performed with the carbon source (e.g., glucose) in a limited or excess amount. Measurement of the actual carbon source concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen, and the partial pressure of waste gases such as CO2. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., which is hereby incorporated by reference in its entirety, particularly with respect to cell culture and fermentation conditions.

[0418] In some embodiments, continuous fermentation methods are used.
Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.

[0419] Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or isoprene production. For example, one method maintains a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allows all other parameters to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration (e.g., the concentration measured by media turbidity) is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, the cell loss due to media being drawn off is balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, Biotechnology:
A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., which is hereby incorporated by reference in its entirety, particularly with respect to cell culture and fermentation conditions.

[0420] In some embodiments, cells are immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isoprene production.

[0421] In some embodiments, bottles of liquid culture are placed in shakers in order to introduce oxygen to the liquid and maintain the uniformity of the culture. In some embodiments, an incubator is used to control the temperature, humidity, shake speed, and/or other conditions in which a culture is grown. The simplest incubators are insulated boxes with an adjustable heater, typically going up to -65 C. More elaborate incubators can also include the ability to lower the temperature (via refrigeration), or the ability to control humidity or CO2 levels. Most incubators include a timer; some can also be programmed to cycle through different temperatures, humidity levels, etc. Incubators can vary in size from tabletop to units the size of small rooms.

[0422] If desired, a portion or all of the cell medium can be changed to replenish nutrients and/or avoid the build up of potentially harmful metabolic byproducts and dead cells. In the case of suspension cultures, cells can be separated from the media by centrifuging or filtering the suspension culture and then resuspending the cells in fresh media. In the case of adherent cultures, the media can be removed directly by aspiration and replaced. In some embodiments, the cell medium allows at least a portion of the cells to divide for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution).

[0423] In some embodiments, a constitutive or leaky promoter (such as a Trc promoter) is used and a compound (such as IPTG) is not added to induce expression of the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid(s) operably linked to the promoter. In some embodiments, a compound (such as IPTG) is added to induce expression of the isoprene synthase, DXS, IDI, or MVA pathway nucleic acid(s) operably linked to the promoter.
Exemplary Methods for Decoupling Isoprene Production from Cell Growth

[0424] Desirably, carbon from the feedstock is converted to isoprene rather than to the growth and maintenance of the cells. In some embodiments, the cells are grown to a low to medium OD600, then production of isoprene is started or increased. This strategy permits a large portion of the carbon to be converted to isoprene.

[0425] In some embodiments, cells reach an optical density such that they no longer divide or divide extremely slowly, but continue to make isoprene for several hours (such as about 2, 4, 6, 8, 10, 15, 20, 25, 30, or more hours). For example, Figs. 60A-67C illustrate that cells may continue to produce a substantial amount of mevalonic acid or isoprene after the cells reach an optical density such that they no longer divide or divide extremely slowly. In some cases, the optical density at 550 nm decreases over time (such as a decrease in the optical density after the cells are no longer in an exponential growth phase due to cell lysis), and the cells continue to produce a substantial amount of mevalonic acid or isoprene. In some embodiments, the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cells produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/gwcm/hr) during this time period. In some embodiments, the amount of isoprene is between about 2 to about 5,000 nmole/gwcm/hr, such as between about 2 to about nmole/gwcm/hr, about 100 to about 500 nmole/gwcm/hr, about 150 to about 500 nmole/gwcm /hr, about 500 to about 1,000 nmole/gwcm/hr, about 1,000 to about 2,000 nmole/gwcm/hr, or about 2,000 to about 5,000 nmole/gwcm/hr. In some embodiments, the amount of isoprene is between about 20 to about 5,000 nmole/gwcm/hr, about 100 to about 5,000 nmole/gwcm/hr, about 200 to about 2,000 nmole/gwcm/hr, about 200 to about 1,000 nmole/gwcm/hr, about 300 to about 1,000 nmole/gwcm/hr, or about 400 to about 1,000 nmole/gwcm/hr.

[0426] In some embodiments, the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cells produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/Lbroth, wherein the volume of broth includes the volume of the cells and the cell medium) during this time period. In some embodiments, the amount of isoprene is between about 2 to about 5,000 mg/Lbroth, such as between about 2 to about 100 mg/Lbrofh, about 100 to about 500 mg/Lbroth, about 500 to about 1,000 mg/Lbroth, about 1,000 to about 2,000 mg/Lbroth, or about 2,000 to about 5,000 mg/Lbroth. In some embodiments, the amount of isoprene is between about 20 to about 5,000 mg/Lbroth, about 100 to about 5,000 mg/Lbroth, about 200 to about 2,000 mg/Lbroth, about 200 to about 1,000 mg/Lbroth, about 300 to about 1,000 mg/Lbroth, or about 400 to about 1,000 mg/Lbroth.

[0427] In some embodiments, the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cells convert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture medium into isoprene during this time period. In some embodiments, the percent conversion of carbon into isoprene is between such as about 0.002 to about 4.0%, about 0.002 to about 3.0%, about 0.002 to about 2.0%, about 0.002 to about 1.6%, about 0.002 to about 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about 1.0%, or about 1.0 to about 1.6%. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 0.4%, 0.002 to about 0.16%, 0.04 to about 0.16%, about 0.005 to about 0.3%, about 0.01 to about 0.3%, or about 0.05 to about 0.3%.

[0428] In some embodiments, isoprene is only produced in stationary phase. In some embodiments, isoprene is produced in both the growth phase and stationary phase. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD600) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene produced during the growth phase for the same length of time. In various embodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of the total amount of isoprene that is produced (such as the production of isoprene during a fermentation for a certain amount of time, such as 20 hours) is produced while the cells are in stationary phase. In various embodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of the total amount of isoprene that is produced (such as the production of isoprene during a fermentation for a certain amount of time, such as 20 hours) is produced while the cells divide slowly or not at all such that the optical density at 550 nm of the cells increases by less than or about 50% (such as by less than or about 40, 30, 20, 10, 5, or 0%). In some embodiments, isoprene is only produced in the growth phase.

[0429] In some embodiments, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids are placed under the control of a promoter or factor that is more active in stationary phase than in the growth phase. For example, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids may be placed under control of a stationary phase sigma factor, such as RpoS. In some embodiments, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids are placed under control of a promoter inducible in stationary phase, such as a promoter inducible by a response regulator active in stationary phase.

Production of Isoprene within Safe Operating Ranges

[0430] The production of isoprene within safe operating levels according to its flammability characteristics simplifies the design and construction of commercial facilities, vastly improves the ability to operate safely, and limits the potential for fires to occur. In particular, the optimal ranges for the production of isoprene are within the safe zone, i.e., the nonflammable range of isoprene concentrations. In one such aspect, described herein is a method for the production of isoprene within the nonflammable range of isoprene concentrations (outside the flammability envelope of isoprene).

[0431] Thus, computer modeling and experimental testing were used to determine the flammability limits of isoprene (such as isoprene in the presence of 02, N2, C02, or any combination of two or more of the foregoing gases) in order to ensure process safety. The flammability envelope is characterized by the lower flammability limit (LFL), the upper flammability limit (UFL), the limiting oxygen concentration (LOC), and the limiting temperature. For a system to be flammable, a minimum amount of fuel (such as isoprene) must be in the presence of a minimum amount of oxidant, typically oxygen. The LFL
is the minimum amount of isoprene that must be present to sustain burning, while the UFL is the maximum amount of isoprene that can be present. Above this limit, the mixture is fuel rich and the fraction of oxygen is too low to have a flammable mixture. The LOC indicates the minimum fraction of oxygen that must also be present to have a flammable mixture. The limiting temperature is based on the flash point of isoprene and is that lowest temperature at which combustion of isoprene can propagate. These limits are specific to the concentration of isoprene, type and concentration of oxidant, inerts present in the system, temperature, and pressure of the system. Compositions that fall within the limits of the flammability envelope propagate combustion and require additional safety precautions in both the design and operation of process equipment.

[0432] The following conditions were tested using computer simulation and mathematical analysis and experimental testing. If desired, other conditions (such as other temperature, pressure, and permanent gas compositions) may be tested using the methods described herein to determine the LFL, UFL, and LOC concentrations.

(1) Computer simulation and mathematical analysis Test Suite 1:
isoprene: 0 wt% - 14 wt%
02:6 wt% - 21 wt%
N2: 79 wt% - 94 wt%
Test Suite 2:
isoprene: 0 wt% - 14 wt%
02:6 wt% - 21 wt%
N2: 79 wt% - 94 wt%
Saturated with H2O
Test Suite 3:
isoprene: 0 wt% - 14 wt%
02:6 wt% - 21 wt%

N2: 79 wt% - 94 wt%
C02: 5 wt% - 30 wt%

(2) Experimental testing for final determination of flammability limits Test Suite 1:
isoprene: 0 wt% - 14 wt%
02:6 wt% - 21 wt%
N2: 79 wt% - 94 wt%
Test Suite 2:
isoprene: 0 wt% - 14 wt%
02:6 wt% - 21 wt%
N2: 79 wt% - 94 wt%
Saturated with H2O

[0433] Simulation software was used to give an estimate of the flammability characteristics of the system for several different testing conditions. CO2 showed no significant affect on the system's flammability limits. Test suites 1 and 2 were confirmed by experimental testing. The modeling results were in-line with the experimental test results. Only slight variations were found with the addition of water.

[0434] The LOC was determined to be 9.5 vol% for an isoprene, 02, N2, and CO2 mixture at 40 C and 1 atmosphere. The addition of up to 30% CO2 did not significantly affect the flammability characteristics of an isoprene, 02, and N2 mixture. Only slight variations in flammability characteristics were shown between a dry and water saturated isoprene, 02, and N2 system. The limiting temperature is about -54 C. Temperatures below about -54 C are too low to propagate combustion of isoprene.

[0435] In some embodiments, the LFL of isoprene ranges from about 1.5 vol.% to about 2.0 vol%, and the UFL of isoprene ranges from about 2.0 vol.% to about 12.0 vol.%, depending on the amount of oxygen in the system. In some embodiments, the LOC is about 9.5 vol% oxygen.
In some embodiments, the LFL of isoprene is between about 1.5 vol.% to about 2.0 vol%, the UFL of isoprene is between about 2.0 vol.% to about 12.0 vol.%, and the LOC is about 9.5 vol%

oxygen when the temperature is between about 25 C to about 55 C (such as about 40 C) and the pressure is between about 1 atmosphere and 3 atmospheres.

[0436] In some embodiments, isoprene is produced in the presence of less than about 9.5 vol%
oxygen (that is, below the LOC required to have a flammable mixture of isoprene). In some embodiments in which isoprene is produced in the presence of greater than or about 9.5 vol%
oxygen, the isoprene concentration is below the LFL (such as below about 1.5 vol.%). For example, the amount of isoprene can be kept below the LFL by diluting the isoprene composition with an inert gas (e.g., by continuously or periodically adding an inert gas such as nitrogen to keep the isoprene composition below the LFL). In some embodiments in which isoprene is produced in the presence of greater than or about 9.5 vol% oxygen, the isoprene concentration is above the UFL (such as above about 12 vol.%). For example, the amount of isoprene can be kept above the UFL by using a system (such as any of the cell culture systems described herein) that produces isoprene at a concentration above the UFL. If desired, a relatively low level of oxygen can be used so that the UFL is also relatively low. In this case, a lower isoprene concentration is needed to remain above the UFL.

[0437] In some embodiments in which isoprene is produced in the presence of greater than or about 9.5 vol% oxygen, the isoprene concentration is within the flammability envelope (such as between the LFL and the UFL). In some embodiments when the isoprene concentration may fall within the flammability envelope, one or more steps are performed to reduce the probability of a fire or explosion. For example, one or more sources of ignition (such as any materials that may generate a spark) can be avoided. In some embodiments, one or more steps are performed to reduce the amount of time that the concentration of isoprene remains within the flammability envelope. In some embodiments, a sensor is used to detect when the concentration of isoprene is close to or within the flammability envelope. If desired, the concentration of isoprene can be measured at one or more time points during the culturing of cells, and the cell culture conditions and/or the amount of inert gas can be adjusted using standard methods if the concentration of isoprene is close to or within the flammability envelope. In particular embodiments, the cell culture conditions (such as fermentation conditions) are adjusted to either decrease the concentration of isoprene below the LFL or increase the concentration of isoprene above the UFL. In some embodiments, the amount of isoprene is kept below the LFL by diluting the isoprene composition with an inert gas (such as by continuously or periodically adding an inert gas to keep the isoprene composition below the LFL).

[0438] In some embodiments, the amount of flammable volatiles other than isoprene (such as one or more sugars) is at least about 2, 5, 10, 50, 75, or 100-fold less than the amount of isoprene produced. In some embodiments, the portion of the gas phase other than isoprene gas comprises between about 0% to about 100% (volume) oxygen, such as between about 0% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 90% to about 90%, or about 90% to about 100% (volume) oxygen. In some embodiments, the portion of the gas phase other than isoprene gas comprises between about 0% to about 99%
(volume) nitrogen, such as between about 0% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 90% to about 90%, or about 90% to about 99% (volume) nitrogen.

[0439] In some embodiments, the portion of the gas phase other than isoprene gas comprises between about 1% to about 50% (volume) C02, such as between about 1% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, or about 40%
to about 50% (volume)C02-

[0440] In some embodiments, an isoprene composition also contains ethanol. For example, ethanol may be used for extractive distillation of isoprene, resulting in compositions (such as intermediate product streams) that include both ethanol and isoprene.
Desirably, the amount of ethanol is outside the flammability envelope for ethanol. The LOC of ethanol is about 8.7 vol%, and the LFL for ethanol is about 3.3 vol% at standard conditions, such as about 1 atmosphere and about 60 F (NFPA 69 Standard on Explosion Prevention Systems, 2008 edition, which is hereby incorporated by reference in its entirety, particularly with respect to LOC, LFL, and UFL
values). In some embodiments, compositions that include isoprene and ethanol are produced in the presence of less than the LOC required to have a flammable mixture of ethanol (such as less than about 8.7% vol%). In some embodiments in which compositions that include isoprene and ethanol are produced in the presence of greater than or about the LOC required to have a flammable mixture of ethanol, the ethanol concentration is below the LFL (such as less than about 3.3 vol.%).

[0441] In various embodiments, the amount of oxidant (such as oxygen) is below the LOC of any fuel in the system (such as isoprene or ethanol). In various embodiments, the amount of oxidant (such as oxygen) is less than about 60, 40, 30, 20, 10, or 5% of the LOC of isoprene or ethanol. In various embodiments, the amount of oxidant (such as oxygen) is less than the LOC

of isoprene or ethanol by at least 2, 4, 5, or more absolute percentage points (vol %). In particular embodiments, the amount of oxygen is at least 2 absolute percentage points (vol %) less than the LOC of isoprene or ethanol (such as an oxygen concentration of less than 7.5 vol%
when the LOC of isoprene is 9.5 vol%). In various embodiments, the amount of fuel (such as isoprene or ethanol) is less than or about 25, 20, 15, 10, or 5% of the LFL
for that fuel.

High Efficiency Production and Recovery of Isoprene, a Volatile Hydrocarbon, by Fermentation

[0442] Methods are provided herein of producing isoprene comprising a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the liquid phase concentration of isoprene is less than about 200 mg/L. In some embodiments, the liquid phase concentration of isoprene in the culture is less than about any of 175 mg/L, 150 mg/L, 125 mg/L, 100 mg/L, 75 mg/L, 50 mg/L, 25 mg/L, 20 mg/L, 15 mg/L, 10 mg/L, 5 mg/L, or 2.5 mg/L. In some embodiments, the liquid phase concentration of isoprene in culture is between about any of 0.1 mg/L to 200 mg/L, 1 mg/L to 200 mg/L, 1 mg/L to 150 mg/L, 1 mg/L to 100 mg/L, 1 mg/L to 50 mg/L, 1 mg/L to 25 mg/L, 1 mg/L to 20 mg/L, or 10 mg/L to 20 mg/L. In some embodiments, the isoprene produced is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene." In some embodiments, the liquid phase concentration is below the solubility limit of isoprene.

[0443] In some embodiments of the methods, the cells produce greater than about 400 nmole/gwcm/hour of isoprene. In some embodiments, the amount of isoprene is between about .m/hour, 400 nmole/gwcm/hour to 1 mmole/gwcm/hour, any of 400 nmole/gwcm/hour to 1 mole/gw, 400 nmole/gwcm/hour to 40 mmole/gwcm/hour, 400 nmole/gwcm/hour to 4 mmole/gwcm/hour, 1 mmole/gwcm/hour to 1.5 mmole/gwcm/hour, 1.5 mmole/gwcm/hour to 3 mmole/gwcm/hour, 3 mmole/gwcm/hour to 5 mmole/gwcm/hour, 5 mmole/gwcm/hour to 25 mmole/gwcm/hour, mmole/gwcm/hour to 100 mmole/gwcm/hour, 100 mmole/gwcm/hour to 500 mmole/gwcm/hour, or 500 mmole/gwcm/hour to 1000 mmole/gwcm/hour. In some embodiments, the amount of isoprene is about any of 1 mmole/gwcm/hour, 1.5 mmole/gwcm/hour, 2 mmole/gwcm/hour, 3 mmole/gwcm/hour, 4 mmole/gwcm/hour, or 5 mmole/gwcm/hour.

[0444] The low value for Henry's coefficient (in M atmi units) means that isoprene can be recovered from fermentation broth by gas stripping at low sparging rates, for example 0.01 vvm to 2 vvm. In some embodiments, the gas sparging rate is between about any of 0.1 vvm to 1 vvm, 0.01 vvm to 0.5 vvm, 0.2 vvm to 1 vvm, or 0.5 vvm to 1 vvm. In some embodiments, the gas sparging rate is about any of 0.1 vvm, 0.25 vvm, 0.5 vvm, 0.75 vvm, 1 vvm, 1.25 vvm, 1.5 vvm, 1.75 vvm, or 2 vvm. In some embodiments, the low sparging rates are maintained for the entire course of the fermentation run, during growth phase, or during stationary phase. In some embodiments, the low sparging rates are maintained for between about any of 1 hour to 5 hours, hours to 10 hours, 10 hours to 20 hours, 20 hours to 30 hours, 30 hours to 40 hours, 40 hours to 50 hours, or 50 hours to 60 hours. The lower desirable gas sparge limit is defined by the point at which the aqueous phase becomes saturated with isoprene and a liquid organic phase forms.
This can only occur below the boiling point of isoprene (34.1 C at 1 atm), above which a liquid isoprene phase will never form. At temperatures below the boiling point of isoprene, the formation of a liquid phase is determined by the aqueous solubility of isoprene, which is approximately 650 mg/L at 25 C. While it is highly desirable to avoid the formation of a liquid isoprene phase, it is not absolutely required provided that the cells can tolerate the presence of liquid isoprene without toxic effects.

[0445] In some embodiments, the oxygen, C02, and isoprene are any of the amounts or concentrations discussed in the section entitled "Production of Isoprene with Safe Operating Ranges." In some embodiments, all the oxygen is consumed by the cells while maintaining fully aerobic metabolism. In some embodiments, an excess of oxygen is used in order to satisfy the oxygen demands of the cells. Desirable ranges of oxygen in the off-gas are less than 20%, or less than 15% or less than 10% (v/v). Levels of oxygen below the limiting oxygen concentration required for combustion of isoprene (9.5% v/v at 1 atm) are particularly desirable. In some embodiments, oxygen-enriched air is utilized with the purpose of allowing minimal gas sweep rates while satisfying the cellular oxygen demand. In some embodiments, the portion of the gas phase of the gas sweep comprises between about 0.1 % to about 10%, about 10%
to about 20%, or about 20% to about 30% (volume) oxygen. In some embodiments, isoprene fermentations are performed under high pressure in order minimize the amount of excess oxygen required to maintain the required dissolved oxygen levels in the liquid phase.

[0446] In some embodiments, the reduction of the gas sweep rate through the fermentor is advantageous for an integrated isoprene production process in that such conditions enrich the off-gas isoprene levels up to about 30,000 g/L (about 1% v/v) without adversely affecting the physiology of the cells.

[0447] In some embodiments, reduced gas-sparge rates do not significantly adversely affect the physiology of the cells. In some embodiments, the carbon dioxide evolution rate of cells in culture with reduced gas-sparge rates is between about any of 1 x 10-18 mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour. In some embodiments, cell viability with reduced gas-sparge rates is reduced by less than about any of 1.75-fold, 1.5-fold, 1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold.
In some embodiments, cell viability with reduced gas-sparge rates is reduced by about 2-fold. In some embodiments, cell viability with reduced gas-sparge rates of a cell expressing a MVA
pathway and/or DXP
pathway RNA and/or protein from one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid is compared to a control cell lacking one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP
pathway nucleic acid with reduced gas-sparge rates. In some embodiments, cell viability with reduced gas-sparge rates of a cell expressing a MVA pathway and/or DXP pathway RNA and/or protein from one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP
pathway nucleic acid under the control of an inducible promoter, wherein the promotor is induced, is compared to a control cell containing one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid under the control of an inducible promoter, wherein the promotor is not induced (uninduced) with reduced gas-sparge rates. In some embodiments, the inducible promoter is a beta-galactosidase promotor.

[0448] In some embodiments, the fermentation of a genetically modified host organism that converts at least 5% of the total carbon consumed by the organism into a volatile, unsaturated hydrocarbon. In some embodiments, the production of an unsaturated hydrocarbon at such a rate as to be present in the fermentation off-gas at a level of at least about any of 100 g/L, 500 g/L, 1000 g/L, 2, 500 g/L, 5,000 g/L, 7,500 g/L, or 10,000 g/L.

[0449] In some embodiments, the unsaturated hydrocarbon is recovered from the off-gas stream in a manner that is suited to high-rates of production, which correspond to concentrations in the off-gas of at least about any of 100 g/L, 500 g/L, 1000 g/L, 2,500 g/L, 5,000 g/L, 7,500 g/L, or 10,000 g/L. In some embodiments, the continuous extraction and recovery of an unsaturated hydrocarbon from the fermentation off-gas in particular at low gas sweep rates such that the resulting off-gas is enriched in the volatile component of interest.
In some embodiments, recovery of the volatile hydrocarbon by methods that depend on elevated concentrations of the volatile. For example, efficient capture of isoprene in fermentation off-gas through the use of compression/condensation or extractive distillation technologies. Also contemplated is the use of activated carbon cartridges in addition to silica gel adsorbants, desorption and concentration of isoprene from carbon cartridges, and/or construction and fermentation of host organisms such as E. coli strains that can convert about 5% or more of the glucose substrate to isoprene and result in off-gas concentrations of greater than about 15,000 g/L isoprene. Recovery methods include any of the methods described herein.

[0450] Also provided herein are methods of producing a compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry's law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L.
In some embodiments, the method comprises: a) culturing cells under suitable conditions for production of the compound, wherein gas is added (such as the addition of gas to a system such as a fermentation system) at a gas sparging rate between about 0.01 vvm to about 2 vvm; and b) producing the compound.

[0451] In some embodiments, the amount of the compound that partitions into the cell mass is not included in the liquid phase solubility values. In some embodiments, the liquid phase concentration is below the solubility limit of compound.

[0452] In some embodiments, the compounds can be continuously recovered from fermentation broth by gas stripping at moderate to low gas sparging rates, in particular those compounds with Henry's law coefficients of about any of less than 250 M/atm, 200 M/atm, 150 M/atm, 100 M/atm, 75 M/atm, 50 M/atm, 25 M/atm, 10 M/atm, 5 M/atm, or 1 M/atm.
Examples include aldehydes such as acetaldehyde (15 M/atm), ketones such as acetone (30 M/atm) or 2-butanone (20 M/atm), or alcohols including methanol (220 M/atm), ethanol (200 M/atm), 1-butanol (120 M/atm) or C5 alcohols including 3-methyl-3-buten-l-ol, and 3-methyl-2-buten-l-ol (50- 100 M/atm). Esters of alcohols generally have lower Henry's constants than the respective alcohols, for example ethyl acetate (6-9 M/atm) or the acetyl esters of C5 alcohols (<5 M/atm).Compounds with Henry's law coefficients of less than 1 M/atm are particularly desirable. Examples include hemiterpenes, monoterpenes, or sesquiterpenes, in addition to other hydrocarbons such as Cl to C5 hydrocarbons (e.g., methane, ethane, ethylene, or propylene). In some embodiments, the hydrocarbons such as Cl to C5 hydrocarbons are saturated, unsaturated, or branched.

[0453] In general, there is a correlation between Henry's law coefficient and water solubility in that compounds with very low coefficients are sparingly soluble in water (substantially water insoluble). Although volatiles with infinite solubilities in water (e.g.
acetone or ethanol) can be removed by gas stripping, desirable solubility limits are less than about any of 100 g/L, 75 g/L, 50 g/L, 25 g/L, 10 g/L, 5 g/L, or lg/L.

[0454] In some embodiments of any of the methods of producing any of the compounds described above, the gas sparging rate is between about any of 0.1 vvm to 1 vvm, 0.2 vvm to 1 vvm, or 0.5 vvm to 1 vvm. In some embodiments, the gas sparging rate is about any of 0.1 vvm, 0.25 vvm, 0.5 vvm, 0.75 vvm, 1 vvm, 1.25 vvm, 1.5 vvm, 1.75 vvm, or 2 vvm. In some embodiments, the low sparging rates are maintained for the entire course of the fermentation run, during growth phase, or during stationary phase. In some embodiments, the low sparging rates are maintained for between about any of 1 hour to 5 hours, 5 hours to 10 hours, 10 hours to 20 hours, 20 hours to 30 hours, 30 hours to 40 hours, 40 hours to 50 hours, or 50 hours to 60 hours.

[0455] Any of the systems described herein can be used in the methods of producing a compound described above. Standard methods would be used to purify such as those described in the section entitled "Exemplary Purification Methods." Separation can be performed post-recovery for example, by distillation or selective adsorption techniques.

Exemplary Production of Biolsoprene

[0456] In some embodiments, the cells are cultured in a culture medium under conditions permitting the production of isoprene by the cells.

[0457] By "peak absolute productivity" is meant the maximum absolute amount of isoprene in the off-gas during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By "peak absolute productivity time point" is meant the time point during a fermentation run when the absolute amount of isoprene in the off-gas is at a maximum during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). In some embodiments, the isoprene amount is measured at the peak absolute productivity time point. In some embodiments, the peak absolute productivity for the cells is about any of the isoprene amounts disclosed herein.

[0458] By "peak specific productivity" is meant the maximum amount of isoprene produced per cell during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By "peak specific productivity time point" is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per cell is at a maximum. The peak specific productivity is determined by dividing the total productivity by the amount of cells, as determined by optical density at 600nm (OD600). In some embodiments, the isoprene amount is measured at the peak specific productivity time point. In some embodiments, the peak specific productivity for the cells is about any of the isoprene amounts per cell disclosed herein.

[0459] By "peak volumetric productivity" is meant the maximum amount of isoprene produced per volume of broth (including the volume of the cells and the cell medium) during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By "peak specific volumetric productivity time point" is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per volume of broth is at a maximum. The peak specific volumetric productivity is determined by dividing the total productivity by the volume of broth and amount of time. In some embodiments, the isoprene amount is measured at the peak specific volumetric productivity time point. In some embodiments, the peak specific volumetric productivity for the cells is about any of the isoprene amounts per volume per time disclosed herein.

[0460] By "peak concentration" is meant the maximum amount of isoprene produced during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). By "peak concentration time point" is meant the time point during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run) when the amount of isoprene produced per cell is at a maximum. In some embodiments, the isoprene amount is measured at the peak concentration time point. In some embodiments, the peak concentration for the cells is about any of the isoprene amounts disclosed herein.

[0461] By "average volumetric productivity" is meant the average amount of isoprene produced per volume of broth (including the volume of the cells and the cell medium) during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). The average volumetric productivity is determined by dividing the total productivity by the volume of broth and amount of time. In some embodiments, the average specific volumetric productivity for the cells is about any of the isoprene amounts per volume per time disclosed herein.

[0462] By "cumulative total productivity" is meant the cumulative, total amount of isoprene produced during the culturing of cells for a particular period of time (e.g., the culturing of cells during a particular fermentation run). In some embodiments, the cumulative, total amount of isoprene is measured. In some embodiments, the cumulative total productivity for the cells is about any of the isoprene amounts disclosed herein.

[0463] As used herein, "relative detector response" refers to the ratio between the detector response (such as the GC/MS area) for one compound (such as isoprene) to the detector response (such as the GC/MS area) of one or more compounds (such as all C5 hydrocarbons). The detector response may be measured as described herein, such as the GC/MS
analysis performed with an Agilent 6890 GC/MS system fitted with an Agilent HP-5MS GC/MS column (30 m x 250 m; 0.25 m film thickness). If desired, the relative detector response can be converted to a weight percentage using the response factors for each of the compounds. This response factor is a measure of how much signal is generated for a given amount of a particular compound (that is, how sensitive the detector is to a particular compound). This response factor can be used as a correction factor to convert the relative detector response to a weight percentage when the detector has different sensitivities to the compounds being compared.
Alternatively, the weight percentage can be approximated by assuming that the response factors are the same for the compounds being compared. Thus, the weight percentage can be assumed to be approximately the same as the relative detector response.

[0464] In some embodiments, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 12,500, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 125,000, 150,000, 188,000, or more nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/gwcm/hr). In some embodiments, the amount of isoprene is between about 2 to about 200,000 nmole/gwcm/hr, such as between about 2 to about 100 nmole/gwcm/hr, about 100 to about 500 nmole/gwcm/hr, about 150 to about 500 nmole/gwcm /hr, about 500 to about 1,000 nmole/gwcm/hr, about 1,000 to about 2,000 nmole/gwcm/hr, or about 2,000 to about 5,000 nmole/gwcm/hr, about 5,000 to about 10,000 nmole/gwcm/hr, about 10,000 to about 50,000 nmole/gwcm/hr, about 50,000 to about 100,000 nmole/gwcm/hr, about 100,000 to about 150,000 nmole/gwcm/hr, or about 150,000 to about 200,000 nmole/gwcm/hr. In some embodiments, the amount of isoprene is between about 20 to about 5,000 nmole/gwcm/hr, about 100 to about 5,000 nmole/gwcm/hr, about 200 to about 2,000 nmole/gwcm/hr, about 200 to about 1,000 nmole/gwcm/hr, about 300 to about 1,000 nmole/gwcm/hr, or about 400 to about 1,000 nmole/gwcm/hr, about 1,000 to about 5,000 nmole/gwcm/hr, about 2,000 to about 20,000 nmole/gwcm/hr, about 5,000 to about 50,000 nmole/gwcm/hr, about 10,000 to about 100,000 nmole/gwcm/hr, about 20,000 to about 150,000 nmole/gwcm/hr, or about 20,000 to about 200,000 nmole/gwcm/hr.

[0465] The amount of isoprene in units of nmole/gwcm/hr can be measured as disclosed in U.S.
Patent No. 5,849,970, which is hereby incorporated by reference in its entirety, particularly with respect to the measurement of isoprene production. For example, two mL of headspace (e.g., headspace from a culture such as 2 mL of culture cultured in sealed vials at 32 C with shaking at 200 rpm for approximately 3 hours) are analyzed for isoprene using a standard gas chromatography system, such as a system operated isothermally (85 C) with an n-octane/porasil C column (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2 mercuric oxide reduction gas detector (Trace Analytical, Menlo Park, CA) (see, for example, Greenberg et al, Atmos. Environ. 27A: 2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, which are each hereby incorporated by reference in their entireties, particularly with respect to the measurement of isoprene production). The gas chromatography area units are converted to nmol isoprene via a standard isoprene concentration calibration curve. In some embodiments, the value for the grams of cells for the wet weight of the cells is calculated by obtaining the A600 value for a sample of the cell culture, and then converting the A600 value to grams of cells based on a calibration curve of wet weights for cell cultures with a known A600 value. In some embodiments, the grams of the cells is estimated by assuming that one liter of broth (including cell medium and cells) with an A600 value of 1 has a wet cell weight of 1 gram. The value is also divided by the number of hours the culture has been incubating for, such as three hours.

[0466] In some embodiments, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wet weight of the cells/hr (ng/gwcm/h). In some embodiments, the amount of isoprene is between about 2 to about 5,000 ng/gwcm/h, such as between about 2 to about 100 ng/gwcm/h, about 100 to about 500 ng/gwcm/h, about 500 to about 1,000 ng/gwcm/h, about 1,000 to about 2,000 ng/gwcm/h, or about 2,000 to about 5,000 ng/gwcm/h. In some embodiments, the amount of isoprene is between about 20 to about 5,000 ng/gwcm/h, about 100 to about 5,000 ng/gwcm/h, about 200 to about 2,000 ng/gwcm/h, about 200 to about 1,000 ng/gwcm/h, about 300 to about 1,000 ng/gwcm/h, or about 400 to about 1,000 ng/gwcm/h. The amount of isoprene in ng/gwcm/h can be calculated by multiplying the value for isoprene production in the units of nmole/gwcm/hr discussed above by 68.1 (as described in Equation 5 below).

[0467] In some embodiments, the cells in culture produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/Lbroth, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the amount of isoprene is between about 2 to about 5,000 mg/Lbroth, such as between about 2 to about 100 mg/Lbroth, about 100 to about 500 mg/Lbro , about 500 to about 1,000 mg/Lbro , about 1,000 to about 2,000 mg/Lbro , or about 2,000 to about 5,000 mg/Lbroth. In some embodiments, the amount of isoprene is between about 20 to about 5,000 mg/Lbroth, about 100 to about 5,000 mg/Lbroth, about 200 to about 2,000 mg/Lbroth, about 200 to about 1,000 mg/Lbroth, about 300 to about 1,000 mg/Lbrofh, or about 400 to about 1,000 mg/Lbrofh.

[0468] The specific productivity of isoprene in mg of isoprene/L of headspace from shake flask or similar cultures can be measured by taking a 1 ml sample from the cell culture at an OD600 value of approximately 1.0, putting it in a 20 mL vial, incubating for 30 minutes, and then measuring the amount of isoprene in the headspace (as described, for example, in Example I, part II). If the OD600 value is not 1.0, then the measurement can be normalized to an OD600 value of 1.0 by dividing by the OD600 value. The value of mg isoprene/L headspace can be converted to mg/Lbroth/hr/OD600 of culture broth by multiplying by a factor of 38. The value in units of mg/Lbroth/hr/OD600 can be multiplied by the number of hours and the OD600 value to obtain the cumulative titer in units of mg of isoprene/L of broth.

[0469] In some embodiments, the cells in culture have an average volumetric productivity of isoprene at greater than or about 0.1, 1.0, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, or more mg of isoprene/L of broth/hr (mg/Lbrofh/hr, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the average volumetric productivity of isoprene is between about 0.1 to about 3,500 mg/Lbrofh/hr, such as between about 0.1 to about 100 mg/Lbrofh/hr, about 100 to about 500 mg/Lbroth/hr, about 500 to about 1,000 mg/Lbroth/hr, about 1,000 to about 1,500 mg/Lbroth/hr, about 1,500 to about 2,000 mg/Lbrofh/hr, about 2,000 to about 2,500 mg/Lbrofh/hr, about 2,500 to about 3,000 mg/Lbroth/hr, or about 3,000 to about 3,500 mg/Lbroth/hr. In some embodiments, the average volumetric productivity of isoprene is between about 10 to about 3,500 mg/Lbroth/hr, about 100 to about 3,500 mg/Lbroth/hr, about 200 to about 1,000 mg/Lbroth/hr, about 200 to about 1,500 mg/Lbroth/hr, about 1,000 to about 3,000 mg/Lbrofh/hr, or about 1,500 to about 3,000 mg/Lbrofh/hr.

[0470] In some embodiments, the cells in culture have a peak volumetric productivity of isoprene at greater than or about 0.5, 1.0, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000, 5,250, 5,500, 5,750, 6,000, 6,250, 6,500, 6,750, 7,000, 7,250, 7,500, 7,750, 8,000, 8,250, 8,500, 8,750, 9,000, 9,250, 9,500, 9,750, 10,000, 12,500, 15,000, or more mg of isoprene/L of broth/hr (mg/Lbroth/hr, wherein the volume of broth includes the volume of the cells and the cell medium). In some embodiments, the peak volumetric productivity of isoprene is between about 0.5 to about 15,000 mg/Lbrofh/hr, such as between about 0.5 to about 10 mg/Lbroth/hr, about 1.0 to about 100 mg/Lbrofh/hr, about 100 to about 500 mg/Lbroth/hr, about 500 to about 1,000 mg/Lbroth/hr, about 1,000 to about 1,500 mg/Lbroth/hr, about 1,500 to about 2,000 mg/Lbrofh/hr, about 2,000 to about 2,500 mg/Lbroth/hr, about 2,500 to about 3,000 mg/Lbrofh/hr, about 3,000 to about 3,500 mg/Lbroth/hr, about 3,500 to about 5,000 mg/Lbroeh/hr, about 5,000 to about 7,500 mg/Lbroeh/hr, about 7,500 to about 10,000 mg/Lbroth/hr, about 10,000 to about 12,500 mg/Lbrofh/h, or about 12,500 to about 15,000 mg/Lbroth/hr. In some embodiments, the peak volumetric productivity of isoprene is between about 10 to about 15,000 mg/Lbroeh/hr, about 100 to about 2,500 mg/Lbroeh/hr, about 1,000 to about 5,000 mg/Lbroeh/hr, about 2,500 to about 7,500 mg/Lbrofh/hr, about 5,000 to about 10,000 mg/Lbroth/hr, about 7,500 to about 12,500 mg/Lbrofh/hr, or about 10,000 to about 15,000 mg/Lbrofh/hr.

[0471] The instantaneous isoprene production rate in mg/Lbrofh/hr in a fermentor can be measured by taking a sample of the fermentor off-gas, analyzing it for the amount of isoprene (in units such as mg of isoprene per Lgas) as described, for example, in Example I, part II and multiplying this value by the rate at which off-gas is passed though each liter of broth (e.g., at 1 vvm (volume of air/volume of broth/minute) this is 60 Lgas per hour). Thus, an off-gas level of 1 mg/Lgas corresponds to an instantaneous production rate of 60 mg/Lbroth/hr at air flow of 1 vvm.
If desired, the value in the units mg/Lbroeh/hr can be divided by the OD600 value to obtain the specific rate in units of mg/Lbroth/hr/OD. The average value of mg isoprene/Lgas can be converted to the total product productivity (grams of isoprene per liter of fermentation broth, mg/Lbroth) by multiplying this average off-gas isoprene concentration by the total amount of off-gas sparged per liter of fermentation broth during the fermentation. Thus, an average off-gas isoprene concentration of 0.5 mg/Lbroth/hr over 10 hours at 1 vvm corresponds to a total product concentration of 300 mg isoprene/Lbroth.

[0472] In some embodiments, the cells in culture convert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 23.2, 23.4, 23.6, 23.8, 24.0, 25.0, 30.0, 31.0, 32.0, 33.0, 35.0, 37.5, 40.0, 45.0, 47.5, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, or 90.0 molar % of the carbon in the cell culture medium into isoprene. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 90.0 molar %, such as about 0.002 to about 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about 1.0%, about 1.0 to about 1.6%, about 1.6 to about 3.0%, about 3.0 to about 5.0%, about 5.0 to about 8.0%, about 8.0 to about 10.0%, about 10.0 to about 15.0%, about 15.0 to about 20.0%, about 20.0 to about 25.0%, about 25.0% to 30.0%, about 30.0% to 35.0%, about 35.0% to 40.0%, about 45.0% to 50.0%, about 50.0% to 55.0%, about 55.0% to 60.0%, about 60.0% to 65.0%, about 65.0% to 70.0%, about 75.0% to 80.0%, about 80.0% to 85.0%, or about 85.0% to 90.0%. In some embodiments, the percent conversion of carbon into isoprene is between about 0.002 to about 0.4 molar %, 0.002 to about 0.16 molar %, 0.04 to about 0.16 molar %, about 0.005 to about 0.3 molar %, about 0.01 to about 0.3 molar %, about 0.05 to about 0.3 molar %, about 0.1 to 0.3 molar %, about 0.3 to about 1.0 molar %, about 1.0 to about 5.0 molar %, about 2 to about 5.0 molar %, about 5.0 to about 10.0 molar %, about 7 to about 10.0 molar %, about 10.0 to about 20.0 molar %, about 12 to about 20.0 molar %, about 16 to about 20.0 molar %, about 18 to about 20.0 molar %, about 18 to 23.2 molar %, about 18 to 23.6 molar %, about 18 to about 23.8 molar %, about 18 to about 24.0 molar %, about 18 to about 25.0 molar %, about 20 to about 30.0 molar %, about 30 to about 40.0 molar %, about 30 to about 50.0 molar %, about 30 to about 60.0 molar %, about 30 to about 70.0 molar %, about 30 to about 80.0 molar %, or about 30 to about 90.0 molar %.

[0473] The percent conversion of carbon into isoprene (also referred to as "%
carbon yield") can be measured by dividing the moles carbon in the isoprene produced by the moles carbon in the carbon source (such as the moles of carbon in batched and fed glucose and yeast extract).
This number is multiplied by 100% to give a percentage value (as indicated in Equation 1).
Equation 1 % Carbon Yield = (moles carbon in isoprene produced)/(moles carbon in carbon source) * 100

[0474] For this calculation, yeast extract can be assumed to contain 50% w/w carbon. As an example, for the 500 liter described in Example 7, part VIII, the percent conversion of carbon into isoprene can be calculated as shown in Equation 2.

Equation 2 % Carbon Yield = (39.1 g isoprene * 1/68.lmol/g * 5 C/mol)/[(181221 g glucose * 1/180 mol/g * 6 C/mol) + (17780 g yeast extract * 0.5 * 1/12 mol/g)] * 100 = 0.042%

[0475] For the two 500 liter fermentations described herein (Example 7, parts VII and VIII), the percent conversion of carbon into isoprene was between 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14 liter systems as described herein. Example 11, part V describes the 1.53% conversion of carbon to isoprene using the methods described herein.

[0476] One skilled in the art can readily convert the rates of isoprene production or amount of isoprene produced into any other units. Exemplary equations are listed below for interconverting between units.

Units for Rate of Isoprene production (total and specific) Equation 3 1 g isoprene/Lbroth/hr = 14.7 mmol isoprene/Lbroth/hr (total volumetric rate) Equation 4 1 nmol isoprene /gw, .m/hr = 1 nmol isoprene /Lbroth/hr/OD600 (This conversion assumes that one liter of broth with an OD600 value of 1 has a wet cell weight of 1 gram.) Equation 5 1 nmol isoprene/gwcm/hr = 68.1 ng isoprene/gwcm/hr (given the molecular weight of isoprene) Equation 6 1 nmol isoprene/Lgas 02/hr = 90 nmol isoprene/Lbrofh/hr (at an 02 flow rate of 90 L/hr per L of culture broth) Equation 7 1 g isoprene/Lgas isoprene in off-gas = 60 g isoprene/Lbroth/hr at a flow rate of 60 Lgas per Lbroth (1 vvm) Units for Titer (total and specific) Equation 8 1 nmol isoprene/mg cell protein = 150 nmol isoprene/Lbroth/OD600 (This conversion assumes that one liter of broth with an OD600 value of 1 has a total cell protein of approximately 150 mg) (specific productivity) Equation 9 1 g isoprene/Lbroth = 14.7 mmol isoprene/Lbroth (total titer)

[0477] If desired, Equation 10 can be used to convert any of the units that include the wet weight of the cells into the corresponding units that include the dry weight of the cells.
Equation 10 Dry weight of cells = (wet weight of cells)/3.3

[0478] If desired, Equation 11 can be used to convert between units of ppm and g/L. In particular, "ppm" means parts per million defined in terms of g/g (w/w).
Concentrations of gases can also be expressed on a volumetric basis using "ppmv" (parts per million by volume), defined in terms of L/L (vol/vol). Conversion of g/L to ppm (e.g., g of analyte per g of gas) can be performed by determining the mass per L of off-gas (i.e., the density of the gas). For example, a liter of air at standard temperature and pressure (STP; 101.3 kPa (1 bar) and 273.15K). has a density of approximately 1.29 g/L. Thus, a concentration of 1 ppm ( g/g) equals 1.29 g/L at STP (equation 11). The conversion of ppm ( g/g) to g/L is a function of both pressure, temperature, and overall composition of the off-gas.

Equation 11 1 ppm ( g/g) equals 1.29 g/L at standard temperature and pressure (STP; 101.3 kPa (1 bar) and 273.15K).

[0479] Conversion of g/L to ppmv (e.g., L of analyte per L of gas) can be performed using the Universal Gas Law (equation 12). For example, an off-gas concentration of 1000 g/Lgas corresponds to 14.7 mol/Lgas. The universal gas constant is 0.082057 L.atm K71mol-1, so using equation 12, the volume occupied by 14.7 mol of HG at STP is equal to 0.329 mL. Therefore, the concentration of 1000 g/L HG is equal to 329 ppmv or 0.0329% (v/v) at STP.

Equation 12

[0480] PV = nRT, where "P" is pressure, "V" is volume, "n" is moles of gas, "R" is the Universal gas constant, and "T" is temperature in Kelvin.

[0481] The amount of impurities in isoprene compositions are typically measured herein on a weight per volume (w/v) basis in units such as g/L. If desired, measurements in units of g/L
can be converted to units of mg/m3 using equation 13.

Equation 13 1 g/L = 1 mg/m3

[0482] In some embodiments described herein, a cell comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide produces an amount of isoprene that is at least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of isoprene produced from a corresponding cell grown under essentially the same conditions without the heterologous nucleic acid encoding the isoprene synthase polypeptide.

[0483] In some embodiments described herein, a cell comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide and one or more heterologous nucleic acids encoding a DXS, IDI, and/or MVA pathway polypeptide produces an amount of isoprene that is at least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of isoprene produced from a corresponding cell grown under essentially the same conditions without the heterologous nucleic acids.

[0484] In some embodiments, the isoprene composition comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has a relative detector response of greater than or about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.99, or 100% for isoprene compared to the detector response for all C5 hydrocarbons in the composition. In some embodiments, the isoprene composition comprises between about 99.90 to about 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96, about 99.96 to about 99.98, about 99.98 to 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.

[0485] In some embodiments, the isoprene composition comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-l-yne, trans-pent-3-ene-l-yne, or cis-pent-3-ene-l-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has a relative detector response of less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for C5 hydrocarbons other than isoprene compared to the detector response for all C5 hydrocarbons in the composition. In some embodiments, the composition has a relative detector response of less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-l-yne compared to the detector response for all C5 hydrocarbons in the composition. In some embodiments, the isoprene composition comprises between about 0.02 to about 0.04%, about 0.04 to about 0.06%, about 0.06 to 0.08%, about 0.08 to 0.10%, or about 0.10 to about 0.12% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3 -methyl-l-butyne, pent-4-ene-l-yne, trans-pent-3-ene-l-yne, or cis-pent-3-ene-l-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition.

[0486] In some embodiments, the isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In some embodiments, the isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 g/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In some embodiments, the isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a hydrocarbon other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne). In some embodiments, the isoprene composition comprises between about 0.005 to about 50, such as about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005 g/L of a hydrocarbon other than isoprene. In some embodiments, the isoprene composition comprises less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a protein or fatty acid (such as a protein or fatty acid that is naturally associated with natural rubber).

[0487] In some embodiments, the isoprene composition comprises less than or about 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of alpha acetylenes, piperylenes, acetonitrile, or 1,3-cyclopentadiene. In some embodiments, the isoprene composition comprises less than or about 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of sulfur or allenes. In some embodiments, the isoprene composition comprises less than or about 30, 20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes (such as 1-pentyne, 2-pentyne, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne). In some embodiments, the isoprene composition comprises less than or about 2000, 1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of isoprene dimers, such as cyclic isoprene dimmers (e.g., cyclic C10 compounds derived from the dimerization of two isoprene units).

[0488] In some embodiments, the isoprene composition includes ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), or any two or more of the foregoing. In particular embodiments, the isoprene composition comprises greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 g/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), or any two or more of the foregoing. In some embodiments, the isoprene composition comprises between about 0.005 to about 120, such as about 0.01 to about 80, about 0.01 to about 60, about 0.01 to about 40, about 0.01 to about 30, about 0.01 to about 20, about 0.01 to about 10, about 0.1 to about 80, about 0.1 to about 60, about 0.1 to about 40, about 5 to about 80, about 5 to about 60, or about 5 to about 40 g/L of ethanol, acetone, a C5 prenyl alcohol, or any two or more of the foregoing.

[0489] In some embodiments, the isoprene composition includes one or more of the following components: 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-l-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). In various embodiments, the amount of one of these components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w). In some embodiments, the relative detector response for the second compound compared to the detector response for isoprene is greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%. In various embodiments, the amount of one of these components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is between about 0.01 to about 105 % (w/w), such as about 0.01 to about 90, about 0.01 to about 80, about 0.01 to about 50, about 0.01 to about 20, about 0.01 to about 10, about 0.02 to about 50, about 0.05 to about 50, about 0.1 to about 50, or 0.1 to about 20% (w/w).

[0490] In some embodiments, the isoprene composition includes one or more of the following:
an alcohol, an aldehyde, or a ketone (such as any of the alcohols, aldehydes, or ketones described herein). In some embodiments, the isoprene composition includes (i) an alcohol and an aldehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) an alcohol, an aldehyde, and a ketone.

[0491] In some embodiments, the isoprene composition contains one or more of the following:
methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole. In some embodiments, the isoprene composition contains 1 ppm or more of one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole. In some embodiments, the concentration of more of one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole, is between about 1 to about 10,000 ppm in an isoprene composition (such as off-gas before it is purified).
In some embodiments, the isoprene composition (such as off-gas after it has undergone one or more purification steps) includes one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole, at a concentration between about 1 to about 100 ppm, such as about 1 to about 10 ppm, about 10 to about 20 ppm, about 20 to about 30 ppm, about 30 to about 40 ppm, about 40 to about 50 ppm, about 50 to about 60 ppm, about 60 to about 70 ppm, about 70 to about 80 ppm, about 80 to about 90 ppm, or about 90 to about 100 ppm. Volatile organic compounds from cell cultures (such as volatile organic compounds in the headspace of cell cultures) can be analyzed using standard methods such as those described herein or other standard methods such as proton transfer reaction-mass spectrometry (see, for example, Bunge et al., Applied and Environmental Microbiology, 74(7):2179-2186, 2008 which is hereby incorporated by reference in its entirety, particular with respect to the analysis of volatile organic compounds).

[0492] In some embodiments, the composition comprises greater than about 2 mg of isoprene, such as greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. In some embodiments, the amount of isoprene in the composition is between about 2 to about 5,000 mg, such as between about 2 to about 100 mg, about 100 to about 500 mg, about 500 to about 1,000 mg, about 1,000 to about 2,000 mg, or about 2,000 to about 5,000 mg. In some embodiments, the amount of isoprene in the composition is between about 20 to about 5,000 mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg, about 200 to about 1,000 mg, about 300 to about 1,000 mg, or about 400 to about 1,000 mg. In some embodiments, greater than or about 20, 25, 30, 40, 50, 60, 70, 80, 90, or 95% by weight of the volatile organic fraction of the composition is isoprene.

[0493] In some embodiments, the composition includes ethanol. In some embodiments, the composition includes between about 75 to about 90% by weight of ethanol, such as between about 75 to about 80%, about 80 to about 85%, or about 85 to about 90% by weight of ethanol.
In some embodiments in which the composition includes ethanol, the composition also includes between about 4 to about 15% by weight of isoprene, such as between about 4 to about 8%, about 8 to about 12%, or about 12 to about 15% by weight of isoprene.

[0494] In some embodiments described herein, a cell comprising one or more heterologous nucleic acids encoding an isoprene synthase polypeptide, DXS polypeptide, IDI
polypeptide, and/or MVA pathway polypeptide produces an amount of an isoprenoid compound (such as a compound with 10 or more carbon atoms that is formed from the reaction of one or more IPP
molecules with one or more DMAPP molecules) that is greater than or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of the isoprenoid compound produced from a corresponding cell grown under essentially the same conditions without the one or more heterologous nucleic acids. In some embodiments described herein, a cell comprising one or more heterologous nucleic acids encoding an isoprene synthase polypeptide, DXS polypeptide, IDI polypeptide, and/or MVA pathway polypeptide produces an amount of a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-1-ol) that is greater than or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount of the C5 prenyl alcohol produced from a corresponding cell grown under essentially the same conditions without the one or more heterologous nucleic acids.

Exemplary Co-Production of Biolsoprene and Hydrogen

[0495] In some embodiments, any of the isoprene-producing cells described herein that comprise one or more heterologous nucleic acids encoding an isoprene synthase polypeptide, a DXS polypeptide, an IDI polypeptide, and/or an MVA pathway polypeptide operably linked to a promoter further comprise a heterologous nucleic acid also operably linked to a promoter encoding one or more hydrogenase polypeptides or one or more polypeptides involved in the regulation or expression of hydrogenase polypeptides (e.g., hydrogenase maturation proteins or transcription factors). In some embodiments, any of the isoprene-producing cells described herein that comprise one or more heterologous nucleic acids encoding an isoprene synthase polypeptide, a DXS polypeptide, an IDI polypeptide, an MVA pathway polypeptide, one or more hydrogenase polypeptides or one or more polypeptides involved in the regulation or expression of hydrogenase polypeptides operably linked to a promoter further comprise a mutation or deletion inactivating one or more polypeptides involved in the production of fermentation side products, one or more polypeptides involved in the regulation or expression of genes for the production of fermentation side products, or one or more polypeptides involved in hydrogen reuptake. Such cells can co-produce isoprene and hydrogen.

[0496] In some embodiments of any of the aspects described herein, the cells are bacterial cells, such as gram-positive bacterial cells (e.g., Bacillus cells such as Bacillus subtilis cells or Streptomyces cells such as Streptomyces lividans, Streptomyces coelicolor, or Streptomyces griseus cells). In some embodiments of any of the aspects described herein, the cells are gram-negative bacterial cells (e.g., Escherichia cells such as Escherichia coli cells, Rhodopseudomonas sp. such as Rhodopseudomonas palustris cells, Pseudomonas sp.
such as Pseudomonasfluorescens cells or Pseudomonas putida cells, or Pantoea cells such as Pantoea citrea cells). In some embodiments of any of the aspects described herein, the cells are fungal, cells such as filamentous fungal cells (e.g., Trichoderma cells such as Trichoderma reesei cells or Aspergillus cells such as Aspergillus oryzae and Aspergillus niger) or yeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells or Sacchraomyces cells such as Saccaromyces cerevisiae).

[0497] In some embodiments of any of the aspects described herein, the isoprene synthase polypeptide is a polypeptide from a plant such as Pueraria (e.g., Pueraria montana or Pueraria lobata) or Populus (e.g., Populus tremuloides, Populus alba, Populus nigra, Populus trichocarpa, or the hybrid, Populus alba x Populus tremula).

[0498] In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding a DXS
polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding a DXS polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise one or more nucleic acids encoding an IDI polypeptide and a DXS polypeptide. In some embodiments of any of the aspects described herein, one nucleic acid encodes the isoprene synthase polypeptide, IDI
polypeptide, and DXS polypeptide. In some embodiments of any of the aspects described herein, one vector encodes the isoprene synthase polypeptide, IDI polypeptide, and DXS
polypeptide. In some embodiments, the vector comprises a selective marker or a selectable marker, such as an antibiotic resistance nucleic acid.

[0499] In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding an MVA pathway polypeptide (such as an MVA pathway polypeptide from Saccharomyces cerevisia or Enterococcus faecalis). In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an MVA pathway polypeptide (such as an MVA
pathway polypeptide from Saccharomyces cerevisia or Enterococcus faecalis). In some embodiments of any of the aspects described herein, the cells comprise an isoprene synthase, DXS, and MVA
pathway nucleic acid. In some embodiments of any of the aspects described herein, the cells comprise an isoprene synthase nucleic acid, a DXS nucleic acid, an IDI nucleic acid, and a MVA
pathway nucleic acid.

[0500] In some embodiments, the isoprene-producing cells described herein further comprise a heterologous nucleic acid encoding a hydrogenase polypeptide operably linked to a promoter. In some embodiments, the hydrogenase polypeptide comprises E. coli hydrogenase-1 (Hyd-1), E.
coli hydrogenase-2 (Hyd-2), E. coli hydrogenase-3 (Hyd-3), E. coli hydrogenase-4 (Hyd-4), E.
coli formate hydrogen lyase (FHL) complex, which produces hydrogen gas from formate and CO2 under anaerobic conditions at acidic pH, Rhodococcus opacus MRII
hydrogenase (R.
opacus HoxH), Synechosystis sp. PCC 6803 hydrogenase (Syn. PCC 6803 HoxH), Desulfovibrio gigas hydrogenase (D. gigas), and Desulfovibrio desulfuricans ATCC 7757 hydrogenase (D.
desulfuricans). In some embodiments, the isoprene-producing cells furthercomprising a heterologous nucleic acid encoding a hydrogenase polypeptide operably linked to a promoter further comprise E. coli hydrogenase-3 (Hyd-3), E. coli pyruvate formate lyase (pfl), and E. coli formate hydrogen lyase (FHL) complex.

[0501] In some embodiments, the hydrogenase polypeptide encodes a ferredoxin-dependent hydrogenase polypeptide. In some embodiments, the ferredoxin-dependent hydrogenase polypeptide comprises Clostridium acetobutulicum hydrogenase A (HydA), which can be expressed in conjunction with one or more of. (1) Bacillus subtilis NADPH
ferredoxin oxidoreductase (NFOR) or Clostridium kluyveri NADH ferredoxin oxidoreductase (RnfCDGEAB), Clostridium pasteuranium ferredoxin oxidoreductase (Fdx); (2) glyceraldehyde-6-phosphate ferredoxin oxidoreductase (GAPOR); or (3) pyruvate ferredoxin oxidoreductase (POR). In some embodiments, the ferredoxin-dependent hydrogenase polypeptide Clostridium acetobutulicum hydrogenase A (HydA) is expressed with three HydA-associated maturation enzymes (HydE, HydG, and HydF), and further in conjunction with one or more of: (1) Bacillus subtilis NADPH ferredoxin oxidoreductase (NFOR) or Clostridium kluyveri NADH ferredoxin oxidoreductase (RnfCDGEAB), Clostridium pasteuranium ferredoxin oxidoreductase (Fdx); (2) glyceraldehyde-6-phosphate ferredoxin oxidoreductase (GAPOR); or (3) pyruvate ferredoxin oxidoreductase (POR).

[0502] In some embodiments, the hydrogenase polypeptide encodes an NADPH-dependent hydrogenase polypeptide. In some embodiments, the NADPH-dependent hydrogenase polypeptide comprises Pyrococcusfuriosus hydrogenase. In some embodiments, the hydrogenase polypeptide encodes an oxygen-tolerant hydrogenase. In some embodiments, the oxygen-tolerant hydrogenase comprises Rubrivivax gelatinosus hydrogenase, and Ralstonia eutropha hydrogenase.

[0503] In some embodiments, the isoprene-producing cells described herein further comprise a mutation or deletion inactivating a gene involved in regulation of hydrogenase activity, such as iron-sulfur complex transcriptional regulator (iscR) (Kalim-Akhtar et al., "Deletion of iscR
stimulates recombinant Clostridial Fe/Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3)," Appl. Microbiol. Biotechnol. 78:853-862 (2008), which is incorporated herein by reference in its entirety, particularly with reference to stimulation of Clostridial Fe/Fe hydrogenase activity and hydrogen accumulation in E. coli by deleting the iscR
gene).

[0504] In some embodiments, the isoprene-producing cells described herein further comprise a mutation or deletion inactivating a gene encoding one or more cellular polypeptides involved in production of fermentation side products, such as lactate, acetate, pyruvate, ethanol, succinate, and glycerol. In some embodiments, the inactivated polypeptides involved in production of fermentation side products comprise one or more polypeptides encoding formate dehydrogenase N, alpha subunit (fdnG), formate dehydrogenase 0, large subunit (fdoG), nitrate reductase (narG), formate transporter A (focA), formate transporter B (focB), pyruvate oxidase (poxB), pyruvate dehydrogenase El component ackA/pta (aceE), alcohol dehydrogenase (adhE), fumarate reductase membrane protein (frdC), or lactate dehydrogenase (ldhA).

[0505] In some embodiments, the isoprene-producing cells described herein further comprise a mutation or deletion inactivating a gene encoding one or more cellular polypeptides involved in the regulation or expression of genes involved in production of fermentation side products. In some embodiments, the inactivated polypeptides involved in the regulation or expression of genes involved in production of fermentation side products comprise repressor of formate hydrogen lyase (hycA), fumarate reductase regulator (fnr), acetyl-coenzyme A
synthetase (acs), and formate dehydrogenase regulatory protein (hycA).

[0506] In some embodiments, the isoprene-producing cells described herein further comprise a mutation or deletion inactivating a gene encoding one or more cellular polypeptides involved in hydrogen re-uptake. In some embodiments, the inactivated polypeptides involved in hydrogen re-uptake comprise E. coli hydrogenase-1 (Hyd-1) (hya operon) and E. coli hydrogenase-2 (Hyd-2) (hyb operon).

[0507] In some embodiments of any of the aspects described herein, the heterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor polypeptide or nucleic acid is operably linked to a T7 promoter, such as a T7 promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase, DXS
polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid is operably linked to a Trc promoter, such as a Trc promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase, DXS polypeptide, IDI
polypeptide, MVA
pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid is operably linked to a Lac promoter, such as a Lac promoter contained in a low copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase, DXS
polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid is operably linked to an endogenous promoter, such as an endogenous alkaline serine protease promoter. In some embodiments, the heterologous isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid integrates into a chromosome of the cells without a selective marker or without a selectable marker.

[0508] In some embodiments, one or more MVA pathway, IDI, DXS, isoprene synthase, hydrogenase, hydrogenase maturation or transcription factor nucleic acids are placed under the control of a promoter or factor that is more active in stationary phase than in the growth phase.
For example, one or more MVA pathway, IDI, DXS, isoprene synthase, hydrogenase, hydrogenase maturation or transcription factor nucleic acids may be placed under control of a stationary phase sigma factor, such as RpoS. In some embodiments, one or more MVA pathway, IDI, DXS, isoprene synthase, hydrogenase, hydrogenase maturation or transcription factor nucleic acids are placed under control of a promoter inducible in stationary phase, such as a promoter inducible by a response regulator active in stationary phase.

[0509] In some embodiments of any of the aspects described herein, at least a portion of the cells maintain the heterologous isoprene synthase, DXS polypeptide, IDI
polypeptide, MVA
pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects described herein, the nucleic acid comprising the isoprene synthase, DXS polypeptide, IDI polypeptide, MVA pathway, hydrogenase, hydrogenase maturation or transcription factor nucleic acid also comprises a selective marker or a selectable marker, such as an antibiotic resistance nucleic acid.

[0510] In some embodiments of any of the aspects described herein, cells that co-produce isoprene and hydrogen are cultured in any of the culture media described herein, under oxygen-limited conditions to facilitate the co-production of isoprene and hydrogen by the cells. In some embodiments, the cells are grown in oxygen-limited culture. In some embodiments, the cells are grown in the presence of 0.5 moles of oxygen per mole of isoprene. In some embodiments, the cells are grown anaerobically, in the absence of oxygen.

[0511] In some embodiments, any of the cells described herein are grown in oxygen-limited culture and co-produce isoprene and hydrogen. In some embodiments, the cells in oxygen-limited culture produce isoprene at a rate greater than about 400 nmole/gwcm/hr, and produce hydrogen at a rate greater than about 125 nmole/gwcm/hr. In some embodiments, the cells in oxygen-limited culture produce isoprene at a rate between about 400 nmole/gwcm/hr to about 2.0 X 105 nmole/gw~m/hr and hydrogen at a rate between about 125 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr. In some embodiments, the cells in oxygen-limited culture produce isoprene at a rate between about 400 nmole/gwcm/hr and about 2.0 x 105 nmole/gwcm/hr, between about 500 nmole/gwcm/hr and about 1.5 x 105 nmole/gwcm/hr, between about 750 nmole/gwcm/hr and about 1 X 105 nmole/gwcm/hr, between about 1000 nmole/gwcm/hr and about 1 x 105 nmole/gwcm/hr, between about 2500 nmole/gwcm/hr and about 1 x 105 nmole/gwcm/hr, between about 5000 nmole/gwcm/hr and about 1 x 105 nmole/gwcm/hr, between about 7500 nmole/gwcm/hr and about 1 X 105 nmole/gwcm/hr, and between about 1 X 104 nmole/gwcm/hr and about 1 X 105 nmole/gwcm/hr.
In some embodiments, the cells in oxygen-limited culture produce greater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/gwcm/hr isoprene. In some embodiments, the cells in oxygen-limited culture produce hydrogen at a rate between about 125 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 250 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 500 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 750 nmole/gwcm/hr to about 1.25 nmole/gwcm/hr, between about 1000 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 1250 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 2500 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 5000 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 7500 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, and between about 1.00 x 104 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr. In some embodiments, the cells in oxygen-limited culture produce greater than about 125, 250, 500, 750, 1000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, 10,000, or more nmole/gwcm/hr hydrogen.

[0512] In some embodiments, any of the cells described herein are grown in oxygen-limited culture and co-produce isoprene and hydrogen. In some embodiments, the cells in oxygen-limited culture have an average volumetric productivity of isoprene greater than about 0.1 mg/Lbroth/hr and an average volumetric productivity of hydrogen greater than about 0.005 mg/Lbroth/hr. In some embodiments, the cells in oxygen-limited culture have a peak volumetric productivity of isoprene greater than about 1000 mg/Lbroth/hr and a peak volumetric productivity of hydrogen greater than about 5 mg/Lbroth/hr. In some embodiments, the cells in oxygen-limited culture have a peak volumetric productivity of isoprene greater than about 3000 mg/Lbroth/hr and a peak volumetric productivity of hydrogen greater than about 5 mg/Lbroth/hr.
In some embodiments, the cells in oxygen-limited culture have a peak volumetric productivity of isoprene greater than about 5000 mg/Lbroth/hr and a peak volumetric productivity of hydrogen greater than about 5 mg/Lbroth/hr. In some embodiments, the cells in oxygen-limited culture have an average volumetric productivity of isoprene between about 0.1 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, and an average volumetric productivity of hydrogen between about 0.005 mg/Lbroth/hr and about 5 mg/Lbroth/hr. In some embodiments, the cells in oxygen-limited culture have an average volumetric productivity of isoprene between about 1 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, between about 5 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, between about 10 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, between about 25 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, between about 50 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, between about 100 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, between about 250 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, between about 500 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, between about 1000 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, and between about 2500 mg/Lbroth/hr and about 5000 mg/Lbroth/hr, and an average volumetric productivity of hydrogen between about 0.01 mg/Lbroth/hr and about 5 mg/Lbroth/hr, between about 0.025 mg/Lbroth/hr and about 5 mg/Lbroth/hr, between about 0.05 mg/Lbroth/hr and about 5 mg/Lbroth/hr, between about 0.1 mg/Lbroth/hr and about 5 mg/Lbroth/hr, between about 0.25 mg/Lbroth/hr and about 5 mg/Lbroth/hr, between about 0.5 mg/Lbroth/hr and about 5 mg/Lbroth/hr, between about 1 mg/Lbroth/hr and about 5 mg/Lbroth/hr, and between about 2.5 mg/Lbroth/hr and about 5 mg/Lbroth/hr.

[0513] In some embodiments, any of the cells described herein are grown in oxygen-limited culture and co-produce isoprene and hydrogen. In some embodiments, the cells in oxygen-limited culture convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and produce hydrogen equivalent to more than about 0.024 molar percent of the carbon that the cells consume from a cell culture medium. In some embodiments, the cells in oxygen-limited culture convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and produce hydrogen equivalent to more than about 400 molar percent of the carbon that the cells consumer from a cell culture medium.

[0514] In some embodiments, any of the cells described herein that co-produce isoprene and hydrogen are grown in oxygen-limited culture. In some embodiments, the cells in oxygen-limited culture co-produce isoprene and hydrogen in a ratio ranging from at least one molar percent of isoprene for every three molar percent of hydrogen to at least one molar percent of isoprene for every four molar percent of hydrogen. In some embodiments, the cells in oxygen-limited culture produce an off-gas containing from 1 to 11 molar percent isoprene and from 3 to 33 molar percent hydrogen. In some embodiments, the cells produce from 1 to 11 molar percent isoprene and from 4 to 44 molar percent hydrogen. In some embodiments, the cells in oxygen-limited culture also produce an off-gas containing oxygen, carbon dioxide, or nitrogen. In some embodiments, the cells in oxygen limited culture produce an off-gas containing from 0 to 21 molar percent oxygen, from 18 to 44 molar percent carbon dioxide, and from 0 to 78 molar percent nitrogen.

[0515] In another aspect, provided herein are cells in oxygen-limited culture that co-produce isoprene and hydrogen, comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the cells: (i) produce isoprene at a rate greater than about 400 nmole/gwcm/hr and produce hydrogen at a rate greater than about 125 nmole/gwcm/hr; (ii) have an average volumetric productivity of isoprene greater than about 0.1 mg/Lbrofh/hr and an average volumetric productivity of hydrogen greater than about 0.005 mg/Lbroth/hr; or (iii) convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and produce hydrogen equivalent to more than about 0.024 molar percent of the carbon that the cells consume from a cell culture medium.

[0516] In some embodiments, the cells in oxygen-limited culture comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein the cells produce greater than about 400 nmole/gwcm/hr of isoprene and greater than about 125 nmole/gwcm/hr of hydrogen. In some embodiments, the cells in oxygen-limited culture comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein the cells have an average volumetric productivity of isoprene greater than about 0.1 mg/Lbroth/hr and an average volumetric productivity of hydrogen greater than about 0.005 mg/Lbroth/hr. In some embodiments, the cells in oxygen-limited culture comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein the cells convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and more than about 0.024 molar percent of the carbon that the cells consume from a cell culture medium into hydrogen. In some embodiments, the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

[0517] In some embodiments, the cells in oxygen-limited culture comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide produce isoprene at a rate between about 400 nmole/gwcm/hr and about 2.0 x 105 nmole/gwcm/hr, between about 500 nmole/gwcm/hr and about 1.5 x 105 nmole/gwcm/hr, between about 750 nmole/gwcm/hr and about 1 x nmole/gwcm/hr, between about 1000 nmole/gwcm/hr and about 1 X 105 nmole/gwcm/hr, between about 2500 nmole/gwcm/hr and about 1 x 105 nmole/gwcm/hr, between about 5000 nmole/gwcm/hr and about 1 X 105 nmole/gwcm/hr, between about 7500 nmole/gwcm/hr and about 1 x 105 nmole/gwcm/hr, and between about 1 X 104 nmole/gwcm/hr and about 1 x 105 nmole/gwcm/hr, and produce hydrogen at a rate between about 125 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 250 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 500 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 750 nmole/gwcm/hr to about 1.25 x 104 nmole/gw~m/hr, between about 1000 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 1250 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 2500 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 5000 nmole/gwcm/hr to about 1.25 x 104 nmole/gw~m/hr, between about 7500 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, and between about 1.00 x 104 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr.

[0518] In some embodiments, provided herein are methods of co-producing isoprene and hydrogen, the methods comprising: (a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and (b) co-producing isoprene and hydrogen, wherein the cells produce greater than about 400 nmole/gwcm/hour of isoprene, and wherein the cells produce greater than about 125 nmole/gwcm/hr of hydrogen.

[0519] In some embodiments, the cells in oxygen-limited culture comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide produce isoprene at a rate between about 400 nmole/gwcm/hr and about 2.0 x 105 nmole/gwcm/hr, between about 500 nmole/gwcm/hr and about 1.5 x 105 nmole/gwcm/hr, between about 750 nmole/gwcm/hr and about 1 x nmole/gwcm/hr, between about 1000 nmole/gwcm/hr and about 1 X 105 nmole/gwcm/hr, between about 2500 nmole/gwcm/hr and about 1 x 105 nmole/gwcm/hr, between about 5000 nmole/gwcm/hr and about 1 X 105 nmole/gwcm/hr, between about 7500 nmole/gwcm/hr and about 1 x 105 nmole/gwcm/hr, and between about 1 X 104 nmole/gwcm/hr and about 1 x 105 nmole/gwcm/hr, and produce hydrogen at a rate between about 125 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 250 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 500 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 750 nmole/gwcm/hr to about 1.25 x 104 nmole/gw~m/hr, between about 1000 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 1250 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 2500 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, between about 5000 nmole/gwcm/hr to about 1.25 x 104 nmole/gw~m/hr, between about 7500 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr, and between about 1.00 x 104 nmole/gwcm/hr to about 1.25 x 104 nmole/gwcm/hr.

[0520] In some embodiments, provided herein are methods of co-producing isoprene and hydrogen, the methods comprising: (a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and (b) co-producing isoprene and hydrogen, wherein the cells have an average volumetric productivity of isoprene greater than about 0.1 mg/Lbrofh/hr and an average volumetric productivity of hydrogen greater than about 0.005 mg/Lbrofh/hr.

[0521] In some embodiments, provided herein are methods of co-producing isoprene and hydrogen, the methods comprising: (a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and (b) co-producing isoprene and hydrogen, wherein the cells convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, and produce hydrogen equivalent to more than about 0.024 molar percent of the carbon that the cells consume from a cell culture medium.

[0522] In some embodiments, provided herein are compositions comprising isoprene and hydrogen in a ratio ranging from at least one molar percent of isoprene for every three molar percent of hydrogen to at least one molar percent of isoprene for every four molar percent of hydrogen, and 0.1 molar percent or less of volatile impurities. In some embodiments, the compositions further comprise from 1 to 11 molar percent isoprene and from 4 to 44 molar percent hydrogen. In some embodiments, the compositions further comprise oxygen, carbon dioxide, or nitrogen. In some embodiments, the compositions further comprise from 0 to 21 molar percent oxygen, from 18 to 44 molar percent carbon dioxide, and from 0 to 78 molar percent nitrogen. In some embodiments, the composition further comprises 1.0 x 10-4 molar percent or less of non-methane volatile impurities. In some embodiments, the non-methane volatile impurities comprise one or more of the following: 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, 2,3-cycloheptenolpyridine, or a linear isoprene polymer (such as a linear isoprene dimer or a linear isoprene trimer derived from the polymerization of multiple isoprene units). In some embodiments, the non-methane volatile impurities comprise one or more of the following: the isoprene composition includes one or more of the following: an alcohol, an aldehyde, or a ketone (such as any of the alcohols, aldehydes, or ketones described herein). In some embodiments, the isoprene composition includes (i) an alcohol and an aldehyde, (ii) an alcohol and a ketone, (iii) an aldehyde and a ketone, or (iv) an alcohol, an aldehyde, and a ketone. In some embodiments, the non-methane volatile impurities comprise one or more of the following: methanol, acetaldehyde, ethanol, methanethiol, 1-butanol, 3-methyl-l-propanol, acetone, acetic acid, 2-butanone, 2-methyl-l-butanol, or indole.

[0523] Also provided herein are methods of co-producing isoprene and hydrogen, the methods comprising: a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and b) co-producing isoprene and hydrogen, wherein the peak concentration of the isoprene produced by the cells in oxygen-limited culture is greater than about 10 ng/Lbroth and the hydrogen evolution rate of the cells is greater than about 0.0025 mmol/Lbroth/hour. In some embodiments of any of these methods, the hydrogen evolution rate is between about any of 0.0025 mmol/Lbroth/hr and about 10 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 5 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 2.5 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 1 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 0.5 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 0.25 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 0.025 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 0.5 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 1 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 2.5 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 5 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 10 mmol/Lbroth/hr, between about 0.25 mmol/Lbroth/hr and 1 mmol/Lbroth/hr, between about 0.25 mmol/Lbroth/hr and 2.5 mmol/Lbroth/hr, between about 0.25 mmol/Lbroth/hr and 2.5 mmol/Lbroth/hr, and between about 0.25 mmol/Lbroth/hr and 10 mmol/Lbroth/hr.

[0524] Provided herein are also methods of co-producing isoprene and hydrogen comprising a) culturing cells under conditions suitable for the co-production of isoprene and hydrogen; and b) co-producing isoprene and hydrogen, wherein the liquid phase concentration of isoprene is less than about 200 mg/L, the cells produce greater than about 400 nmole/gwcm/hour of isoprene, and the hydrogen evolution rate of the cells is greater than about 0.0025 mmol/L/hour. In some embodiments, the liquid phase concentration of isoprene in the culture is less than about any of 175 mg/L, 150 mg/L, 125 mg/L, 100 mg/L, 75 mg/L, 50 mg/L, 25 mg/L, 20 mg/L, 15 mg/L, 10 mg/L, 5 mg/L, or 2.5 mg/L. In some embodiments, the liquid phase concentration of isoprene in culture is between about any of 0.1 mg/L to 200 mg/L, 1 mg/L to 200 mg/L, 1 mg/L to 150 mg/L, 1 mg/L to 100 mg/L, 1 mg/L to 50 mg/L, 1 mg/L to 25 mg/L, 1 mg/L to 20 mg/L, or 10 mg/L to 20 mg/L. In some embodiments of any of these methods, the hydrogen evolution rate is between about any of 0.0025 mmol/Lbroth/hr and about 10 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 5 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 2.5 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 1 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 0.5 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 0.25 mmol/Lbroth/hr, between about 0.0025 mmol/Lbroth/hr and about 0.025 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 0.5 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 1 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 2.5 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 5 mmol/Lbroth/hr, between about 0.025 mmol/Lbroth/hr and about 10 mmol/Lbroth/hr, between about 0.25 mmol/Lbroth/hr and 1 mmol/Lbroth/hr, between about 0.25 mmol/Lbroth/hr and 2.5 mmol/Lbroth/hr, between about 0.25 mmol/Lbroth/hr and 2.5 mmol/Lbroth/hr, and between about 0.25 mmol/Lbroth/hr and 10 mmol/Lbroth/hr.

[0525] In one aspect, provided herein are cells in oxygen-limited culture that co-produce isoprene and hydrogen. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the cells in oxygen-limited culture produce greater than about 400 nmole/gwcm/hr of isoprene and greater than about 125 nmole/gwcm/hr of hydrogen. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

[0526] In some embodiments, provided herein are cells in oxygen-limited culture that convert more than about 0.002% of the carbon in a cell culture medium into isoprene and produce hydrogen equivalent to more than about 0.024 molar percent of the carbon in a cell culture medium. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

[0527] In some embodiments, provided herein are cells in oxygen-limited culture that comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

[0528] In one aspect, provided herein are methods of co-producing isoprene with another compound, such as methods of using any of the cells described herein to co-produce isoprene and hydrogen. In some embodiments, the method involves culturing cells under oxygen-limited conditions sufficient to produce greater than about 400 nmole/gwcm/hr of isoprene and greater than about 125 nmole/gwcm/hr of hydrogen. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the method also includes recovering the isoprene and hydrogen produced by the cells. In some embodiments, the method further includes purifying the isoprene and the hydrogen produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD600) during stationary phase is greater than or about 2 or more times the amount of isoprene produced during the growth phase for the same length of time.

[0529] In some embodiments, the method includes culturing cells under oxygen-limited conditions sufficient to convert more than about 0.002% of the carbon (mol/mol) in a cell culture medium into isoprene and to produce hydrogen equivalent to more than about 0.024 molar percent of the carbon in a cell culture medium. In some embodiments, the oxygen-limited culture is anaerobic. In some embodiments, the method also includes recovering isoprene and hydrogen produced by the cells. In some embodiments, the method further includes purifying isoprene and hydrogen produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing.

[0530] In some embodiments of any of the aspects described herein, the microbial polypeptide carbon source includes one or more polypeptides from yeast or bacteria. In some embodiments of any of the aspects described herein, the plant polypeptide carbon source includes one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

[0531] In some embodiments, isoprene and hydrogen are only co-produced in stationary phase. In some embodiments, isoprene and hydrogen are co-produced in both the growth phase and stationary phase. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD600) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene produced during the growth phase for the same length of time. In various embodiments, the amount of hydrogen produced (such as the total amount of hydrogen produced or the amount of hydrogen produced per liter of broth per hour per OD600) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of hydrogen produced during the growth phase for the same length of time.

[0532] In some embodiments, the compositions provided herein comprise hydrogen and greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-l-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-l-yne by weight compared to the total weight of all C5 hydrocarbons in the composition. In particular embodiments, the composition has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.
In some embodiments, the composition has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In particular embodiments, the composition also comprises greater than about 2 mg of isoprene and greater than about 0.48 mg of hydrogen.

[0533] In some embodiments, the volatile organic fraction of the gas phase has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a compound that inhibits the polymerization of isoprene for any compound in the volatile organic fraction of the gas phase that inhibits the polymerization of isoprene. In particular embodiments, the volatile organic fraction of the gas phase also has greater than about 2 mg of isoprene and greater than about 0.48 mg of hydrogen.

[0534] In some embodiments, the systems include any of the cells and/or compositions described herein. In some embodiments, the system includes a reactor that chamber comprises cells in oxygen-limited culture that produce greater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/gwcm/hr isoprene and greater than about 125, 250, 500, 750, 1000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 7,500, 10,000, or more nmole/gw, .m/hr hydrogen. In some embodiments, the system is not a closed system. In some embodiments, at least a portion of the isoprene is removed from the system. In some embodiments, the system includes a gas phase comprising isoprene and hydrogen. In various embodiments, the gas phase comprises any of the compositions described herein.

[0535] In one aspect, featured herein is a product produced by any of the compositions or methods described herein.

Cell Viability at High Isoprene Titer

[0536] Isoprene is a hydrophobic molecule secreted by many plants, animals, and microbes.
Bacteria, such as Bacillus, produce isoprene at fairly low levels. While there is some evidence that plants secrete isoprene to help with thermoprotection, it has been hypothesized that isoprene may act antagonistically to cyanobacteria or fungi, or as an antimicrobial agent. See, e.g., Ladygina et al., Process Biochemistry 41:1001-1014 (2006), which is incorporated by reference in its entirety, particularly with respect to isoprene acting antagonistically. Since the very low production levels happening in nature are sufficient to be anti-microbial, it was of great concern that the titers and productivity levels of isoprene necessary for commercialization of isoprene would kill the host microbe.

[0537] We have found methods for producing titers and productivity levels of isoprene for commercialization of isoprene while maintaining cell viability and/or metabolic activity as indicated by carbon dioxide evolution rate or total carbon dioxide evolution rate.

[0538] Provided herein are methods of producing isoprene comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein cells produce greater than about 400 nmole/gwcm/hour of isoprene, and the carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. In some embodiments, the isoprene produced is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene." In some embodiments, the amount of isoprene is between about any of nmole/gwcm/hour to 1 mole/gwcm/hour, 400 nmole/gwcm/hour to 1 mmole/gwcm/hour, nmole/gwcm/hour to 40 mmole/gwcm/hour, 400 nmole/gwcm/hour to 4 mmole/gwcm/hour, 1 mmole/gwcm/hour to 1.5 mmole/gwcm/hour, 1.5 mmole/gwcm/hour to 3 mmole/gwcm/hour, 3 mmole/gwcm/hour to 5 mmole/gwcm/hour, 5 mmole/gwcm/hour to 25 mmole/gwcm/hour, mmole/gwcm/hour to 100 mmole/gwcm/hour, 100 mmole/gwcm/hour to 500 mmole/gwcm/hour, or 500 mmole/gwcm/hour to 1000 mmole/gwcm/hour. In some embodiments, the amount of isoprene is about any of 1 mmole/gwcm/hour, 1.5 mmole/gwcm/hour, 2 mmole/gwcm/hour, 3 mmole/gwcm/hour, 4 mmole/gwcm/hour, or 5 mmole/gwcm/hour. In some embodiments, the carbon dioxide evolution rate is between about any of 1 x 10-18 mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour.
In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

[0539] Provided herein are also methods of producing isoprene comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein cells produce greater than about 400 nmole/gwcm/hour of isoprene, and cell viability is reduced by less than about two-fold. In some embodiments, the isoprene produced is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene."
In some embodiments, the amount of isoprene is between about any of 400 nmole/gwcm/hour to 1 mole/gwcm/hour, 400 nmole/gwcm/hour to 1 mmole/gwcm/hour, 400 nmole/gwcm/hour to 40 mmole/gwcm/hour, 400 nmole/gwcm/hour to 4 mmole/gwcm/hour, 1 mmole/gwcm/hour to 1.5 mmole/gwcm/hour, 1.5 mmole/gwcm/hour to 3 mmole/gwcm/hour, 3 mmole/gwcm/hour to 5 mmole/gwcm/hour, 5 mmole/gwcm/hour to 25 mmole/gwcm/hour, 25 mmole/gwcm/hour to 100 mmole/gwcm/hour, 100 mmole/gwcm/hour to 500 mmole/gwcm/hour, or 500 mmole/gwcm/hour to 1000 mmole/gwcm/hour. In some embodiments, the amount of isoprene is about any of 1 mmole/gwcm/hour, 1.5 mmole/gwcm/hour, 2 mmole/gwcm/hour, 3 mmole/gwcm/hour, 4 mmole/gwcm/hour, or 5 mmole/gwcm/hour. In some embodiments, cell viability is reduced by less than about any of 1.75-fold, 1.5-fold, 1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold. In some embodiments, cell viability is reduced by about 2-fold.

[0540] Further provided herein are methods of producing isoprene comprising:
a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/Lbroth/hour and the carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. In some embodiments, the cumulative total productivity of isoprene is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene." In some embodiments, the cumulative total productivity of the isoprene is between about any of 0.2 mg/Lbroth/hour to 5 g/Lbroth/hour, 0.2 mg/Lbroth/hour to 1 g/Lbroth/hour, 1 g/Lbroth/hour to 2.5 g/Lbroth/hour, 2.5 g/Lbroth/hour to 5 g/Lbroth/hour. In some embodiments, the carbon dioxide evolution rate is between about any of 1 x 10-18 mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour.
In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

[0541] Provided herein are methods of producing isoprene comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/Lbroth/hour and cell viability is reduced by less than about two-fold. In some embodiments, the cumulative total productivity of isoprene is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene." In some embodiments, the cumulative total productivity of the isoprene is between about any of 0.2 mg/Lbroth/hour to 5 g/Lbroth/hour, 0.2 mg/Lbroth/hour to 1 g/Lbroth/hour, 1 g/Lbroth/hour to 2.5 g/Lbroth/hour, 2.5 g/Lbroth/hour to 5 g/Lbroth/hour. In some embodiments, cell viability is reduced by less than about any of 1.75-fold, 1.5-fold, 1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold.

[0542] Methods of producing isoprene are also provided herein comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/Lbroth and the carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. In some embodiments, the peak concentration of isoprene is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene."
In some embodiments, the peak concentration of isoprene is between about any of 10 ng/Lbroth to 500 ng/Lbroth, 500 ng/Lbroth to 1 g/Lbroth, 1 g/Lbroth to 5 g/Lbroth, 5 g/Lbroth to 50 g/Lbroth, 5 g/Lbroth to 100 g/Lbroth, 5 g/Lbroth to 250 g/Lbroth, 250 g/Lbroth to 500 g/Lbroth, 500 g/Lbroth to 1 mg/Lbroth, 1 mg/Lbroth to 50 mg/Lbroth, 1 mg/Lbroth to 100 mg/Lbroth, 1 mg/Lbroth to 200 mg/Lbroth, 10 ng/Lbroth to 200 mg/Lbroth, 5 g/Lbroth to 100 mg/Lbroth, or 5 g/Lbroth to 200 mg/Lbroth.
In some embodiments, the peak concentration is any of about 10 ng/Lbroth, 100 ng/Lbroth, 1 g/Lbroth, 5 g/Lbroth, 1 mg/Lbroth, 30 mg/ Lbroth, 100 mg/Lbroth, or 200 mg/Lbroth. In some embodiments, the carbon dioxide evolution rate is between about any of 1 x 10-18 mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

[0543] In addition, methods of producing isoprene are also provided herein comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/Lbroth and cell viability is reduced by less than about two-fold.
In some embodiments, the peak concentration of isoprene is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene." In some embodiments, the peak concentration of isoprene is between about any of 10 ng/Lbroth to 500 ng/Lbroth, 500 ng/Lbroth to 1 g/Lbroth, 1 g/Lbroth to 5 g/Lbroth, 5 g/Lbmth to 50 g/Lbroth, 5 g/Lbmth to 100 g/Lbroth, 5 g/Lbroth to 250 g/Lbroth, 250 g/Lbroth to 500 g/Lbroth, 500 g/Lbroth to 1 mg/Lbroth, 1 mg/Lbroth to 50 mg/Lbroth, 1 mg/Lbroth to 100 mg/Lbroth, 1 mg/Lbroth to 200 mg/Lbroth, 10 ng/Lbroth to 200 mg/Lbroth, 5 g/Lbroth to 100 mg/Lbroth, or 5 g/Lbroth to 200 mg/Lbroth. In some embodiments, the peak concentration is any of about 10 ng/Lbroth, 100 ng/Lbroth, 1 g/Lbroth, 5 g/Lbroth, 1 mg/Lbroth, 30 mg/ Lbroth, 100 mg/Lbroth, or 200 mg/Lbroth. In some embodiments, cell viability is reduced by less than about any of 1.75-fold, 1.5-fold, 1.25-fold, 1-fold, 0.75-fold, 0.5-fold, or 0.25-fold. In some embodiments, cell viability is reduced by about 2-fold.

[0544] Cells in culture are also provided herein comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein the cells produce greater than about nmole/gwcm/hour of isoprene and carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. In some embodiments, the isoprene produced is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene."
In some embodiments, the amount of isoprene is between about any of 400 nmole/gwcm/hour to 1 mole/gwcm/hour, 400 nmole/gw, .m/hour to 1 mmole/gwcm/hour, 400 nmole/gwcm/hour to 40 mmole/gwcm/hour, 400 nmole/gwcm/hour to 4 mmole/gwcm/hour, 1 mmole/gwcm/hour to 1.5 mmole/gwcm/hour, 1.5 mmole/gwcm/hour to 3 mmole/gwcm/hour, 3 mmole/gwcm/hour to 5 mmole/gwcm/hour, 5 mmole/gwcm/hour to 25 mmole/gwcm/hour, 25 mmole/gwcm/hour to 100 mmole/gwcm/hour, 100 mmole/gwcm/hour to 500 mmole/gwcm/hour, or 500 mmole/gwcm/hour to 1000 mmole/gwcm/hour. In some embodiments, the amount of isoprene is about any of 1 mmole/gwcm/hour, 1.5 mmole/gwcm/hour, 2 mmole/gwcm/hour, 3 mmole/gwcm/hour, 4 mmole/gwcm/hour, or 5 mmole/gwcm/hour. In some embodiments, the carbon dioxide evolution rate is between about any of 1 x 10-18 mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

[0545] Provided herein are also cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/Lbroth/hour and carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. In some embodiments, the cumulative total productivity of isoprene is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene." In some embodiments, the cumulative total productivity of the isoprene is between about any of 0.2 mg/Lbroth/hour to 5 g/Lbroth/hour, 0.2 mg/Lbroth/hour to 1 g/Lbroth/hour, 1 g/Lbroth/hour to 2.5 g/Lbroth/hour, 2.5 g/Lbroth/hour to 5 g/Lbroth/hour. In some embodiments, the carbon dioxide evolution rate is between about any of 1 x 10-18 mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

[0546] In addition, provided herein are cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/Lbroth and carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. In some embodiments, the peak concentration of isoprene is any concentration or amount disclosed in the section entitled "Exemplary Production of Isoprene." In some embodiments, the peak concentration of isoprene is between about any of ng/Lbroth to 500 ng/Lbroth, 500 ng/Lbroth to 1 g/Lbroth, 1 g/Lbroth to 5 g/Lbroth, 5 g/Lbroth to 50 g/Lbroth, 5 g/Lbroth to 100 g/Lbroth, 5 g/Lbroth to 250 g/Lbroth, 250 g/Lbroth to 500 g/Lbroth, 500 g/Lbroth to 1 mg/Lbroth, 1 mg/Lbroth to 50 mg/Lbroth, 1 mg/Lbroth to 100 mg/Lbroth, 1 mg/Lbroth to 200 mg/Lbroth, 10 ng/Lbroth to 200 mg/Lbroth, 5 g/Lbroth to 100 mg/Lbroth, or 5 g/Lbroth to 200 mg/Lbroth. In some embodiments, the peak concentration is any of about 10 ng/Lbroth, 100 ng/Lbroth, 1 g/Lbroth, 5 g/Lbroth, 1 mg/Lbroth, 30 mg/ Lbroth, 100 mg/Lbroth, or 200 mg/Lbroth. In some embodiments, the carbon dioxide evolution rate is between about any of 1 x 10-18 mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about any of 50 mmol/L/hour, 100 mmol/L/hour, 150 mmol/L/hour, 200 mmol/L/hour, 250 mmol/L/hour, 300 mmol/L/hour, 350 mmol/L/hour, 400 mmol/L/hour, 450 mmol/L/hour, or 500 mmol/L/hour.

[0547] In some embodiments of any of the methods and cells described herein, carbon dioxide evolution rate and/or cell viability of a cell expressing a MVA pathway and/or DXP pathway RNA and/or protein from one or more of a heterologous and/or duplicate copy of a MVA
pathway and/or DXP pathway nucleic acid is compared to a control cell lacking one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid. In some embodiments, carbon dioxide evolution rate and/or cell viability of a cell expressing a MVA pathway and/or DXP pathway RNA and/or protein from one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid under the control of an inducible promoter, wherein the promotor is induced, is compared to a control cell containing one or more of a heterologous and/or duplicate copy of a MVA pathway and/or DXP pathway nucleic acid under the control of an inducible promoter, wherein the promotor is not induced (uninduced). In some embodiments, the inducible promoter is a beta-galactosidase promoter.

[0548] In some embodiments, the methods of producing isoprene comprise: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein cells produce greater than about 400 nmole/gwcm/hour of isoprene, and the carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. Further provided herein are methods of producing isoprene comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/Lbroth/hour and the carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour.
Methods of producing isoprene are also provided herein comprising: a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/Lbroth and the carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. In some embodiments of any of these methods, the carbon dioxide evolution rate is between about any of 1 x 10-18 mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour. In some embodiments, the carbon dioxide evolution rate is about 50 mmol/L/hour or about 500 mmol/L/hour.

[0549] Further provided herein are cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein the cells produce greater than about nmole/gwcm/hour of isoprene and carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. Provided herein are also cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein cumulative total productivity of the isoprene produced by the cells in culture is greater than about 0.2 mg/Lbrofh/hour and carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. In addition, provided herein are cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein peak concentration of the isoprene produced by the cells in culture is greater than about 10 ng/Lbroth and carbon dioxide evolution rate of the cells is greater than about 1 x 10-18 mmol/L/hour. In some embodiments of any of these cells in culture, the carbon dioxide evolution rate is between about any of 1 x 10-18 mmol/L/hour to about 1 mol/L/hour, 1 mmol/L/hour to 1 mol/L/hour, 25 mmol/L/hour to 750 mmol/L/hour, 25 mmol/L/hour to 75 mmol/L/hour, 250 mmol/L/hour to 750 mmol/L/hour, or 450 mmol/L/hour to 550 mmol/L/hour.
In some embodiments, the carbon dioxide evolution rate is about 50 mmol/L/hour or about 500 mmol/L/hour.

[0550] Provided herein are also methods of producing isoprene comprising a) culturing cells under suitable conditions for production of isoprene; and b) producing isoprene, wherein the liquid phase concentration of isoprene is less than about 200 mg/L and the cells produce greater than about 400 nmole/gw, .m/hour of isoprene. In some embodiments, the liquid phase concentration of isoprene in the culture is less than about any of 175 mg/L, 150 mg/L, 125 mg/L, 100 mg/L, 75 mg/L, 50 mg/L, 25 mg/L, 20 mg/L, 15 mg/L, 10 mg/L, 5 mg/L, or 2.5 mg/L. In some embodiments, the liquid phase concentration of isoprene in culture is between about any of 0.1 mg/L to 200 mg/L, 1 mg/L to 200 mg/L, 1 mg/L to 150 mg/L, 1 mg/L to 100 mg/L, 1 mg/L
to 50 mg/L, 1 mg/L to 25 mg/L, 1 mg/L to 20 mg/L, or 10 mg/L to 20 mg/L.

[0551] Also provided herein are methods of producing a compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry's law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L.

In some embodiments, the method comprises: a) culturing cells under suitable conditions for production of the compound, wherein gas is added (such as the addition of gas to a system such as a fermentation system) at a gas sparging rate between about 0.01 vvm to about 2 vvm; and b) producing the compound. In some embodiments, the Henry's law coefficient of the compound is less than about any of 200 M/atm, 150 M/atm, 100 M/atm, 75 M/atm, 50 M/atm, 25 M/atm, 10 M/atm, 5 M/atm, or 1 M/atm. In some embodiments, the solubility in water of the compound is less than about any of 75 g/L, 50 g/L, 25 g/L, 10 g/L, 5 g/L, or l g/L. In some embodiments, the compound is selected from a group consisting of isoprene, an aldehyde (e.g., acetaldehyde), a ketone (e.g., acetone or 2-butanone), an alcohol (e.g., methanol, ethanol, 1-butanol, or C5 alcohols such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), an ester of an alcohol (e.g., ethyl acetate or acetyl esters of C5 alcohols), a hemiterpene, a monoterpene, a sesquiterpene, and Cl to C5 hydrocarbons (e.g., methane, ethane, ethylene, or propylene). In some embodiments, the Cl to C5 hydrocarbons are saturated, unsaturated, or branched. In particular embodiments, the compound is isoprene. In some embodiments of the methods of producing any of the compounds described above, the gas sparging rate is between about any of 0.1 vvm to 1 vvm, 0.2 vvm to 1 vvm, or 0.5 vvm to 1 vvm.

[0552] In one aspect, cells in culture are used to produce isoprene. In some embodiments, the cells in culture produce greater than about 400 nmole of isoprene/gram of cells for the wet weight of the cells/hour (nmole/gwcm/hr) of isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

[0553] In some embodiments, the cells in culture convert more than about 0.002% of the carbon in a cell culture medium into isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

[0554] In some embodiments, the cells in culture comprise a heterologous nucleic acid encoding an isoprene synthase polypeptide. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

[0555] In one aspect, described herein are methods of producing isoprene, such as methods of using any of the cells described herein to produce isoprene. In some embodiments, the method involves culturing cells under conditions sufficient to produce greater than about 400 nmole/gwcm/hr of isoprene. In some embodiments, the method also includes recovering isoprene produced by the cells. In some embodiments, the method includes purifying isoprene produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD600) during stationary phase is greater than or about 2 or more times the amount of isoprene produced during the growth phase for the same length of time. In some embodiments, the gas phase comprises greater than or about 9.5 % (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit. In particular embodiments, (i) the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit, and (ii) the cells produce greater than about 400 nmole/gwcm/hr of isoprene.

[0556] In some embodiments, the method includes culturing cells under conditions sufficient to convert more than about 0.002% of the carbon (mol/mol) in a cell culture medium into isoprene. In some embodiments, the method also includes recovering isoprene produced by the cells. In some embodiments, the method includes purifying isoprene produced by the cells. In some embodiments, the method includes polymerizing the isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions.

[0557] In some embodiments, isoprene is only produced in stationary phase. In some embodiments, isoprene is produced in both the growth phase and stationary phase. In various embodiments, the amount of isoprene produced (such as the total amount of isoprene produced or the amount of isoprene produced per liter of broth per hour per OD600) during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene produced during the growth phase for the same length of time.

[0558] In one aspect, described herein are compositions and systems that comprise isoprene.
In some embodiments, the composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene(w/w) of the volatile organic fraction of the composition is isoprene.

[0559] In some embodiments, the composition comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3 -methyl-l-butyne, pent-4-ene-l-yne, trans-pent-3-ene-l-yne, or cis-pent-3-ene-l-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the composition has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-l-yne, trans-pent-3-ene-l-yne, or cis-pent-3-ene-l-yne by weight compared to the total weight of all C5 hydrocarbons in the composition. In particular embodiments, the composition has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.

[0560] In some embodiments, the composition has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene.
In particular embodiments, the composition also has greater than about 2 mg of isoprene.

[0561] In some embodiments, the composition has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the composition has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 g/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), or any two or more of the foregoing. In particular embodiments, the composition has greater than about 2 mg of isoprene and has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.

[0562] In some embodiments, the composition includes isoprene and one or more second compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. In various embodiments, the amount of one of these second components relative to the amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w).

[0563] In some embodiments, the composition comprises (i) a gas phase that comprises isoprene and (ii) cells in culture that produce greater than about 400 nmole/gwcm/hr of isoprene.
In some embodiments, the composition comprises a closed system, and the gas phase comprises greater than or about 5. 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g/L of isoprene when normalized to 1 mL of 1 OD600 cultured for 1 hour. In some embodiments, the composition comprises an open system, and the gas phase comprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g/L of isoprene when sparged at a rate of 1 vvm. In some embodiments, the volatile organic fraction of the gas phase comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-l-yne) by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-l-yne, or cis-pent-3-ene-l-yne by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In particular embodiments, the volatile organic fraction of the gas phase has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction.

[0564] In some embodiments, the volatile organic fraction of the gas phase has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a compound that inhibits the polymerization of isoprene for any compound in the volatile organic fraction of the gas phase that inhibits the polymerization of isoprene. In particular embodiments, the volatile organic fraction of the gas phase also has greater than about 2 mg of isoprene.

[0565] In some embodiments, the volatile organic fraction of the gas phase has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the volatile organic fraction of the gas phase has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 g/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), or any two or more of the foregoing. In particular embodiments, the volatile organic fraction of the gas phase has greater than about 2 mg of isoprene and has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.

[0566] In some embodiments, the volatile organic fraction of the gas phase has includes isoprene and one or more second compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. In various embodiments, the amount of one of these second components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w) in the volatile organic fraction of the gas phase.

[0567] In some embodiments of any of the compositions described herein, at least a portion of the isoprene is in a gas phase. In some embodiments, at least a portion of the isoprene is in a liquid phase (such as a condensate). In some embodiments, at least a portion of the isoprene is in a solid phase. In some embodiments, at least a portion of the isoprene is adsorbed to a solid support, such as a support that includes silica and/or activated carbon. In some embodiments, the composition includes ethanol. In some embodiments, the composition includes between about 75 to about 90% by weight of ethanol, such as between about 75 to about 80%, about 80 to about 85%, or about 85 to about 90% by weight of ethanol. In some embodiments, the composition includes between about 4 to about 15% by weight of isoprene, such as between about 4 to about 8%, about 8 to about 12%, or about 12 to about 15% by weight of isoprene.

[0568] In some embodiments, the systems include any of the cells and/or compositions described herein. In some embodiments, the system includes a reactor that chamber comprises cells in culture that produce greater than about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/gwcm/hr isoprene. In some embodiments, the system is not a closed system. In some embodiments, at least a portion of the isoprene is removed from the system. In some embodiments, the system includes a gas phase comprising isoprene. In various embodiments, the gas phase comprises any of the compositions described herein.

[0569] In some embodiments of any of the compositions, systems, and methods described herein, a nonflammable concentration of isoprene in the gas phase is produced.
In some embodiments, the gas phase comprises less than about 9.5 % (volume) oxygen. In some embodiments, the gas phase comprises greater than or about 9.5 % (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit. In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 100% (volume) oxygen, such as between about 10% to about 100% (volume) oxygen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 0% to about 99% (volume) nitrogen. In some embodiments, the portion of the gas phase other than isoprene comprises between about 1% to about 50% (volume) C02-

[0570] In some embodiments of any of the aspects described herein, the cells in culture produce isoprene at greater than or about 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole/gwcm/hr isoprene. In some embodiments of any of the aspects described herein, the cells in culture convert greater than or about 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6%, or more of the carbon in the cell culture medium into isoprene. In some embodiments of any of the aspects described herein, the cells in culture produce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wet weight of the cells /hr (ng/gwcm/h). In some embodiments of any of the aspects described herein, the cells in culture produce a cumulative titer (total amount) of isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth (mg/Lbro , wherein the volume of broth includes the volume of the cells and the cell medium). Other exemplary rates of isoprene production and total amounts of isoprene production are disclosed herein.

[0571] In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an IDI polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding a DXS
polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding a DXS polypeptide. In some embodiments of any of the aspects described herein, the cells further comprise one or more nucleic acids encoding an IDI polypeptide and a DXS polypeptide. In some embodiments of any of the aspects described herein, one nucleic acid encodes the isoprene synthase polypeptide, IDI
polypeptide, and DXS polypeptide. In some embodiments of any of the aspects described herein, one vector encodes the isoprene synthase polypeptide, IDI polypeptide, and DXS
polypeptide. In some embodiments, the vector comprises a selective marker, such as an antibiotic resistance nucleic acid.

[0572] In some embodiments of any of the aspects described herein, the heterologous isoprene synthase nucleic acid is operably linked to a T7 promoter, such as a T7 promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase nucleic acid is operably linked to a Trc promoter, such as a Trc promoter contained in a medium or high copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase nucleic acid is operably linked to a Lac promoter, such as a Lac promoter contained in a low copy plasmid. In some embodiments of any of the aspects described herein, the heterologous isoprene synthase nucleic acid is operably linked to an endogenous promoter, such as an endogenous alkaline serine protease promoter. In some embodiments, the heterologous isoprene synthase nucleic acid integrates into a chromosome of the cells without a selective marker.

[0573] In some embodiments, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids are placed under the control of a promoter or factor that is more active in stationary phase than in the growth phase. For example, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids may be placed under control of a stationary phase sigma factor, such as RpoS. In some embodiments, one or more MVA pathway, IDI, DXP, or isoprene synthase nucleic acids are placed under control of a promoter inducible in stationary phase, such as a promoter inducible by a response regulator active in stationary phase.

[0574] In some embodiments of any of the aspects described herein, at least a portion of the cells maintain the heterologous isoprene synthase nucleic acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture (such as a continuous culture without dilution). In some embodiments of any of the aspects described herein, the nucleic acid comprising the isoprene synthase, IDI, or DXS nucleic acid also comprises a selective marker, such as an antibiotic resistance nucleic acid.

[0575] In some embodiments of any of the aspects described herein, the cells further comprise a heterologous nucleic acid encoding an MVA pathway polypeptide (such as an MVA pathway polypeptide from Saccharomyces cerevisia or Enterococcusfaecalis). In some embodiments of any of the aspects described herein, the cells further comprise an insertion of a copy of an endogenous nucleic acid encoding an MVA pathway polypeptide (such as an MVA
pathway polypeptide from Saccharomyces cerevisia or Enterococcusfaecalis). In some embodiments of any of the aspects described herein, the cells comprise an isoprene synthase, DXS, and MVA
pathway nucleic acid. In some embodiments of any of the aspects described herein, the cells comprise an isoprene synthase nucleic acid, a DXS nucleic acid, an IDI nucleic acid, and a MVA
pathway nucleic (in addition to the IDI nucleic acid).

[0576] In some embodiments of any of the aspects described herein, the isoprene synthase polypeptide is a polypeptide from a plant such as Pueraria (e.g., Pueraria montana or Pueraria lobata) or Populus (e.g., Populus tremuloides, Populus alba, Populus nigra, Populus trichocarpa, or the hybrid, Populus alba x Populus tremula).

[0577] In some embodiments of any of the aspects described herein, the cells are bacterial cells, such as gram-positive bacterial cells (e.g., Bacillus cells such as Bacillus subtilis cells or Streptomyces cells such as Streptomyces lividans, Streptomyces coelicolor, or Streptomyces griseus cells). In some embodiments of any of the aspects described herein, the cells are gram-negative bacterial cells (e.g., Escherichia cells such as Escherichia coli cells, Rhodopseudomonas sp. such as Rhodopseudomonas palustris cells, Pseudomonas sp.
such as Pseudomonasfluorescens cells or Pseudomonas putida cells, or Pantoea cells such as Pantoea citrea cells). In some embodiments of any of the aspects described herein, the cells are fungal, cells such as filamentous fungal cells (e.g., Trichoderma cells such as Trichoderma reesei cells or Aspergillus cells such as Aspergillus oryzae and Aspergillus niger) or yeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells or Sacchraomyces cells such as Saccaromyces cerevisiae).

[0578] In some embodiments of any of the aspects described herein, the microbial polypeptide carbon source includes one or more polypeptides from yeast or bacteria. In some embodiments of any of the aspects described herein, the plant polypeptide carbon source includes one or more polypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or linseed.

Production of isoprene in anaerobic microorganisms using synthesis gas as an energy source

[0579] In some embodiements, the bioisoprene composition is produced in anaerobic microorganisms using synthesis gas as an energy source as described in US
Provisional Patent Application Nos. 61/289,347 and 61/289,355, filed on December 22, 2009, the disclosures of which are incorporated herein by reference in their entireties.

[0580] Production of isoprene from syngas by anaerobic organisms may provide a number of advantages over production of isoprene from sugars by aerobic organisms.
First, the maximum theoretical mass yield of isoprene can be greater for the aerobic organisms, as discussed further below. Second, the anaerobic organisms do not have excess reducing power in the form of NAD(P)H that must be turned over via cell growth, formation of byproducts (such as glycerol, lactic acid, or ethanol) or oxidation using molecular oxygen. Without this NAD(P)H turnover requirement, anaerobic organisms can have higher energy yield, lower oxygen demand, lower heat of fermentation, and lower utility costs to run the process. Third, due to the lack of oxygen in the system, anaerobic organisms can have greater isoprene concentration in the offgas, lower probability of creating a flammable isoprene-oxygen mixture, easier recovery, and higher isoprene quality. Fourth, the anaerobic organisms can be more easily grown by using existing infrastructure, such as existing plants designed for production of bioethanol.

[0581] Anaerobic organisms useful for isoprene production can include obligate anaerobes, facultatitive anaerobes, and aerotolerant anaerobes. The obligate anaerobes can be any one or combination selected from the group consisting of Clostridium jungdahlii, Clostridium autoethanogenum, Eurobacterium limosum, Clostridium carboxydivorans, Peptostreptococcus productus, and Butyribacterium methylotrophicum. Syngas useful as energy source for production of isoprene can be derived from a feedstock by a variety of processes, including methane reforming, coal liquefaction, co-firing, fermentative reactions, enzymatic reactions, and biomass gasification.

Exemplary Purification Methods

[0582] In some embodiments, any of the methods described herein further include recovering the co-produced compounds. In some embodiments, any of the methods described herein further include recovering the isoprene. In some embodiments, any of the methods described herein further include recovering the hydrogen by cryogenic membrane, adsorption matrix-based separation methods.

[0583] The isoprene and hydrogen produced using the compositions and methods described herein can be recovered using standard techniques. such as gas stripping, membrane enhanced separation, fractionation, adsorption/desorption, pervaporation, thermal or vacuum desorption of isoprene from a solid phase, or extraction of isoprene immobilized or absorbed to a solid phase with a solvent (see, for example, U.S. Patent Nos. 4,703,007, 4,570,029, and 4,740,222 ("Recovery and Purification of Hydrogen from Refinery and Petrochemical Off-gas Streams") which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene recovery and purification methods ('007 and '029 patents) and with respect to hydrogen recovery and purification methods ('222 patent)). In particular embodiments, extractive distillation with an alcohol (such as ethanol, methanol, propanol, or a combination thereof) is used to recover the isoprene. In some embodiments, the recovery of isoprene involves the isolation of isoprene in a liquid form (such as a neat solution of isoprene or a solution of isoprene in a solvent). Gas stripping involves the removal of isoprene vapor from the fermentation off-gas stream in a continuous manner. Such removal can be achieved in several different ways including, but not limited to, adsorption to a solid phase, partition into a liquid phase, or direct condensation (such as condensation due to exposure to a condensation coil or do to an increase in pressure). In some embodiments, membrane enrichment of a dilute isoprene vapor stream above the dew point of the vapor resulting in the condensation of liquid isoprene.
In some embodiments, the isoprene is compressed and condensed.

[0584] The recovery of isoprene may involve one step or multiple steps. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed simultaneously. For example, isoprene can be directly condensed from the off-gas stream to form a liquid. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed sequentially. For example, isoprene may be adsorbed to a solid phase and then extracted from the solid phase with a solvent.

[0585] The recovery of hydrogen may involve one step or multiple steps. In some embodiments, the removal of hydrogen gas from the fermentation off-gas and the conversion of hydrogen to a liquid phase are performed simultaneously. In some embodiments, the removal of hydrogen gas from the fermentation off-gas and the conversion of hydrogen to a liquid phase are performed sequentially. For example, hydrogen may be adsorbed to a solid phase and then desorbed from the solid phase by a pressure swing. In some embodiments, recovered hydrogen gas is concentrated and compressed.

[0586] In some embodiments, any of the methods described herein further include purifying the isoprene. For example, the isoprene produced using the compositions and methods described herein can be purified using standard techniques. Purification refers to a process through which isoprene is separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is obtained as a substantially pure liquid.
Examples of purification methods include (i) distillation from a solution in a liquid extractant and (ii) chromatography. As used herein, "purified isoprene" means isoprene that has been separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is at least about 20%, by weight, free from other components that are present when the isoprene is produced. In various embodiments, the isoprene is at least or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, by weight, pure.
Purity can be assayed by any appropriate method, e.g., by column chromatography, HPLC
analysis, or GC-MS analysis.

[0587] In some embodiments, any of the methods described herein further include purifying the hydrogen. For example, the hydrogen produced using the compositions and methods described herein can be purified using standard techniques. Purification refers to a process through which hydrogen is separated from one or more components that are present when the hydrogen is produced. In some embodiments, the hydrogen is obtained as a substantially pure gas. In some embodiments, the hydrogen is obtained as a substantially pure liquid. Examples of purification methods include (i) cryogenic condensation and (ii) solid matrix adsorption. As used herein, "purified hydrogen" means hydrogen that has been separated from one or more components that are present when the hydrogen is produced. In some embodiments, the hydrogen is at least about 20%, by weight, free from other components that are present when the hydrogen is produced. In various embodiments, the hydrogen is at least or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, by weight, pure. Purity can be assayed by any appropriate method, e.g., by column chromatography or GC-MS analysis.

[0588] In some embodiments, at least a portion of the gas phase remaining after one or more recovery steps for the removal of isoprene is recycled by introducing the gas phase into a cell culture system (such as a fermentor) for the production of isoprene.

[0589] Methods and apparatus for the purification of a bioisoprene composition from fermentor off-gas is described in US Provisional Patent Application No.
61/88,142, filed December 18, 2009, which is incorporated herein by reference in its entirety.

[0590] A bioisoprene composition from a fermentor off-gas may contain bioisoprene with volatile impurities and bio-byproduct impurities. In some embodiments, a bioisoprene composition from a fermentor off-gas is purified using a method comprising:
(a) contacting the fermentor off-gas with a solvent in a first column to form: an isoprene-rich solution comprising the solvent, a major portion of the isoprene and a major portion of the bio-byproduct impurity;
and a vapor comprising a major portion of the volatile impurity; (b) transferring the isoprene-rich solution from the first column to a second column; and (c) stripping isoprene from the isoprene-rich solution in the second column to form: an isoprene-lean solution comprising a major portion of the bio-byproduct impurity; and a purified isopene composition.

[0591] Figure 169 illustrates an exemplary method of purifying isoprene and an exemplary apparatus. Fermentor off-gas comprising isoprene may be generated from renewable resources (e.g., carbon sources) by any method in the art for example, as described in U.S. provisional patent application Nos. 61/187,944, the content of which is hereby incorporated by reference, particularly with respect to the methods of generating fermentor off-gas comprising isoprene.
The fermentor off-gas generated from one or more individual fermentors 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more fermentors connected in series and/or in parallel) may be directed to a first column 14. As described below, the fermentor off-gas may be directed through an isolation unit 16 and/or compressed by a compression means, such as compression system 18.
Additionally, the temperature of the fermentor off-gas may optionally be reduced at any point, for example, to form a condensate or partial condensate prior to contact with the solvent (which may aid in solubiliztion of one or more off-gas components, such as isoprene). The fermentor off-gas may be contacted (e.g., absorbed) at column 14 with a solvent (e.g., any solvent described herein, such as a non-polar high boiling-point solvent). The volatile impurities having less propensity for absorption in the solvent (particularly with non-polar high boiling-point solvents) are separated from the remaining solvent/fermentor off-gas mixture, resulting in a vapor comprising a major portion of the volatile impurity (e.g., exiting at port 20), and an isoprene-rich solution having a major portion of the isoprene and a major portion of the bio-byproduct impurity (e.g., at port 22). The solvent may optionally be heated by any suitable means (e.g., by steam) prior to, simultaneously, and/or after contact with the fermentor off-gas, which may aid in separation of the volatile impurity from the remaining solution. Steam may be directed through the column (at any suitable location, such as near entry of the off-gas and/or the opposite end of the volatile impurity exit as shown in Figure 169) to provide a sweeping vapor phase which may aid in the removal of the volatile impurity.

[0592] The isoprene-rich solution having a major portion of the isoprene and a major portion of the bio-byproduct impurity (e.g., at port 22) may be directed to a second column 24. The second column may be isolated from the first column 14 (as shown in Figure 169) or may be part of a single column comprising both the first and second columns (e.g., a tandem column wherein the solvent enters the first column at or near one end, and exits the second column at or near an opposite end). The isoprene may be stripped from the isoprene-rich solution in the second column to generate a purified isopene composition (e.g., at port 26) and an isoprene-lean solution comprising a major portion of the bio-byproduct impurity (e.g., at port 28). The isoprene-rich solution may be heated by any suitable means (e.g., by steam), which may aid in stripping of the isoprene from the remaining solution. Steam may be directed through the column (at any suitable location, such as the opposite end of the entry point of the isoprene-rich solution and/or the near the end of the isoprene-lean solution exit as shown in Figure 169).

[0593] As described herein, the columns may be conventional and of any suitable size.
Exemplary types of columns are commercially available from manufacturers including Koch Modular Process Systems (Paramus, NJ), Fluor Corporation (Irving, TX), Kuhni USA (Mount Holly, NC). In general, columns are designed to maximize vapor/liquid contact in order to achieve the desired efficiency. This is achieved by filling the column with either a packing material, or trays spaced at regular intervals along the column. Suitable packing materials include both random and structured types based on metal, glass, polymer and ceramic materials.
Exemplary random packing types include Raschig rings, Pall rings, A-PAK rings, Saddle rings, Pro-Pak, Heli-Pak, Ceramic saddles and FLEXIRINGS . Structured packings include wire mesh and perforated metal plate type materials. Manfacturers specializing in column packings include ACS Separations & Mass-Transfer Products (Houston, TX), Johnson Bros. Metal Forming Co.

(Berkeley, IL) and Koch Glitsch, Inc. Knight Div. (East Canton, OH). The efficiency of a gas stripping column is expressed in terms of the theoretical plate height and the total number of plates in the column. In general, the greater the number of theoretical plates present, the greater the efficiency of the column. Laboratory scale columns can be purchased from Ace Glass (Vineland, NJ), Sigma-Aldrich (St. Louis, MO) and Chemglass (Vineland, NJ).
Suitable types of glass column include Vigreux, Snyder, Hemple and Perforated-plate type columns. Columns can include packing materals, or contain features designed to maximize vapor/liquid contact. A
laboratory scale gas scrubber unit (part # CG-1830-10) is available from Chemglass and consists of a packed glass column, solvent reservoir and solvent recirculation pump.

[0594] The purified isoprene composition from the second column 24 (e.g., exiting at port 26) may be further purified by any suitable means (e.g., by using a reflux condenser 34 and/or an adsorption system 36, such as a silica adsorption system). The reflux reduces the solvent composition in the isoprene product. The isoprene-lean solution may be recycled back to the first column for reuse (e.g., as shown in Figure 169 at port 30). The isoprene-lean solution may be purified by any suitable means (e.g., by liquid-liquid extraction and/or an adsorption system 32, such as a silica adsorption system) prior to recycling to the first column 14 to reduce to amount of bio-byproduct. Additionally, the temperature of the isoprene-lean solution may be reduced by any suitable means prior to recycling to the first column 14 (e.g., prior to, simultaneously, and/or after optionally purifying the isoprene solution). Figure 169 shows an example of reducing the temperature of the isoprene-lean solution at port 40 prior to purification of the isoprene-lean solution (in this case, using coolant for temperature reduction).

[0595] The vapor comprising a major portion of the volatile impurity (e.g., the vapor exiting at port 20 in Figure 169) may comprise a minor portion of isoprene (e.g., residual isoprene not remaining in the isoprene-rich solution). The residual isoprene may be recollected for use from the vapor comprising a major portion of the volatile impurity by any suitable means (e.g., an adsorption system 38, such as an activated carbon adsorption system) and in some cases, as shown in Figure 169, may be combined with the purified isoprene composition (e.g., prior to, during, or after additional purification, such as an adsorption system similar to system 36).
Figure 169 also shows an optional capture device 42 (e.g., a thermal oxidizer and/or CO2 capture system) capable of reducing the amount of undesirable components released into the atmosphere (e.g., C02) from the vapor.

Exemplary chemical transformations of isoprene

[0596] Although current industrial use of isoprene is predominantly in the production of synthetic rubber, isoprene is a reactive conjugated diene and undergoes a varieties of chemical transformations to form oxygenates and higher molecular weight hydrocarbons.
For example, Palladium(O) complexes (Pd(acac)2-Ph3P and Pd(OAc)2-Ph3P) catalyze dimerization and telomerization of isoprene in alcohol solvents to give linear isoprene dimers (e.g. 2,7-dimethyl-1,3,7-octatriene) and methoxydimethyloctadienes (Zakharkin, L. I. and Babich, S. A. Russ.
Chem. Bull. (1976), pp 1967-1968.) Adams, J. M. and Clapp, T. V. (Clay and Clay Minerals (1986), 34(3), 287-294) reported reactions of isoprene over divalent and trivalent transition metal-exchanged montmorillonites (e.g. Cr3+-montmorillonite) to give isoprene dimers and adducts with methanol. The linear dimerization of isoprene catalyzed by Ni(0)-aminophosphinite systems resulted in regioselective tail-to-tail linear dimers, accompanied by a competitive cyclodimerization reaction (Denis, Philippe; Croizy, Jean Francois; Mortreux, Andre; Petit, Francis, Journal of Molecular Catalysis (1991), 68(2), 159-75.
Denis, Philippe;
Jean, Andre; Croizy, Jean Francois; Mortreux, Andre; Petit, Francis, Journal of the American Chemical Society (1990), 112(3), 1292-4.) New chiral aminophosphinite ligands, e.g., (+)-McCH2CHMeCH(NH2)CH2OPPh2 was investigated as homogeneous catalysts in the linear dimerization of isoprene, leading to a conversion rate above 50% (Masotti, Henriette; Peiffer, Gilbert; Siv, Chhan; Courbis, Pierre; Sergent, Michelle; Phan Tan Luu, Roger, Bulletin des Societes Chimiques Belges (1991), 100(1), 63-77.)

[0597] Thermal dimerization of isoprene at 110-250 in presence of dinitrocresol as polymerization inhibitor gives high yields of dimers and little polymer (U.S.
Patent No.
4,973,787.) Ni-catalyzed dimerization of isoprene yields a dimethyl-1,5-cyclooctadiene mixture consisting of 80% 1,5-dimethyl-1,5-cyclooctadiene and 20% 1,6-dimethyl-1,5-cyclooctadiene (Doppelt, Pascal; Baum, Thomas H.; Ricard, Louis, Inorganic Chemistry (1996), 35(5), 1286-91.) Isoprene is converted to dimethylcyclooctadienes with a catalytic amt. of Cp*Ru(I14-isoprene)Cl and AgOTf (Itoh, Kenji; Masuda, Katsuyuki; Fukahori, Takahiko;
Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima, Hideo, Organometallics (1994), 13(3), 1020-9.) JP59065026A (1984) reported preparation of 1,6-dimethyl-1,5-cyclooctadiene by cyclic dimerization of isoprene in the presence of catalysts comprising Fe carboxylates or R-diketone compounds, organo-Al or Mg compounds, and 2,2'-dipyridyl derivatives having electron-donating groups. Dimethylcyclooctadiene was prepared by cyclodimerization of isoprene over 3-component catalysts containing Ni carboxylates or R-ketones, organoaluminum or organomagnesium compounds and substituted triphenylphosphite (JP58055434A, 1983.) 1,5-Dimethyl-1,5-cyclooctadiene was prepared by cyclodimerization of isoprene at 100-300 in an inert organic solvent in the presence of a homogeneous catalyst containing Fe(3) salt, organoaluminum compound and an activator (SU615056A1, 1978), in the presence of a homogeneous catalyst containing Ni acetylacetonate, a triarylphosphite and perhydroalumophenolene (SU493455A1, 1975), in the presence of a catalyst containing a mixture of a Ni carboxylate or carboxylate or chelate compounds of Ni and 1-hydroxy-3-carbonyl compounds, trialkylaluminum, dialkylmagnesium or active organo-Mg compounds obtained from conjugated dienes and Mg, triaryl phosphites and tertiary amines (JP48064049A, 1973), or in the presence of a catalyst composed of Ni naphthenate, Et3Al, and tri-o-cresyl phosphate (Suga, K.; Watanabe, S.; Fujita, T.; Shimada, T., Israel Journal of Chemistry (1972), 10(1), 15-18.) U.S. Patent No. 3,954,665 disclosed dimerization of isoprene in the presence of reaction products of [(i3-C6H5)NiBr]2 or [M(NO)2X]2 (M = Fe, Co; X = Cl, I, Br) with Fe, Co, or Ni carbonyls. European Patent No. 2411(1981) disclosed cyclodimerization of isoprene over a Fe(NO)2Cl-bis(1,5-cyclooctadiene)nickel catalyst at from -5 to +20 to give 1-methyl- and 2-methyl-4-isopropenyl-l-cyclohexene and 1,4- and 2,4-dimethyl-4-vinyl-l-cyclohexene. U.S.
Patent No. 4,189,403 disclosed preparation of 1,5-dimethyl-1,5-cyclooctadiene and 1,4-dimethyl-4-vinyl-l-cyclohexene by contacting isoprene with a mixed catalyst of a tris(substituted hydrocarbyl) phosphite, arsenite, or antimonite and a Group VIII metal(0) compound (e.g. Ni acetylacetonate). Jackstell, R.; Grotevendt, A.; Michalik, D.; El Firdoussi, L.; Beller, M. J. Organometallic Chem. (2007) 692(21), 4737-4744 cites the use of palladium/carbene catalysts for isoprene dimerization. Bowen, L.; Charemsuk, M.; Wass, D.F.
Chem. Commun. (2007) 2835-2837 describes the use of a chromium N,N-bis(diarylphosphino)amine catalyst for the production of linear and cyclic trimers of isoprene.

[0598] Isoprene was reportedly dimerized in the presence of a Ni catalyst to yield cis-2-isopropenyl-l-methylvinylcyclobutane (Billups, W. E.; Cross, J. H.; Smith, C.
V., Journal of the American Chemical Society (1973), 95(10), 3438-9.) The oligomerization of isoprene [78-79-5]
catalyzed by nickel naphthenate and isoprenemagnesium in the presence of various phosphites as electron donors gave cyclic dimers containing dimethylcyclooctadiene [39881-79-3]; in particular 1,1,1-tris(hydroxymethyl)propane phosphite [39865-19-5] gave trimethylcyclododecatriene [39881-80-6] selectively (Suga, Kyoichi; Watanabe, Shoji; Fujita, Tsutomu; Shimada, Takashi, Journal ofApplied Chemistry & Biotechnology (1973), 23(2), 131-8.) W02006/05 1011 discloses preparation of trimethylcyclododecatriene, useful in perfumes and fragrances, by the trimerization of isoprene in the presence of a catalyst system comprising Ni and/or Ti, one or more organometallic compound, and a Group VA compound, and that the reaction is conducted in a hydroxyl group-containing solvent. Ligabue, R. A.;
Dupont, J.; de Souza, R. F., Alegre, R.S. J. Mol. Cat. A: Chem. (2001), 169(1-2), 11-17, describes the selective dimerization of isoprene to six-membered dimers using an iron nitrosyl catalyst in an ionic liquid. Huchette, D.; Nicole, J.; Petit, F., Tetrahedron Letters (1979), (12), 1035-8, describes the electrochemical generation of an iron nitrosyl catalyst and subsequent use for the dimerization of isoprene to cyclohexene dimers. Zakharkin, L. I.; Zhigareva, G. G.;
Pryanishnikov, A. P.
Zhurnal Obshchei Khimii (1987), 57(11), 2551-6, describes the cyclooligomerization of isoprene on complex nickel and iron catalysts.

[0599] The highly pure isoprene starting compositions described herein are chemically transformed using each catalyst systems and reaction conditions disclosed in the references cited. Other catalysts and reaction conditions known in the art such as catalysts and reaction conditions applied to chemical transformations of 1,3-butadiene can be adapted to the isoprene starting compositions by one skilled in the art.

Dimerization and Trimerization

[0600] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes catalytic chemical transformation to give dimers and trimers.
Catalyst systems are identified using methods known in the art. Preferred catalysts include those known to convert isoprene to dimers and trimers with high efficiency. Examples include those based upon Palladium, Nickel, Cobalt, Iron and Chromium.

[0601] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes thermal or catalytic dimerization. The isoprene starting composition is converted to a mixture of dimers that includes unsaturated 6 and 8-membered rings by heating the starting composition under pressure. In some embodiments, the starting isoprene composition is heated to over or about 100, 125, 150, 175, 200, 225 or 250 C
under pressure.
In some embodiments, the starting isoprene composition is heated in the presence of an antioxidant (e.g. 2,6-di-tert-butyl-4-methylphenol) to prevent radical-mediated polymerization.
In some embodiments, the reaction is accelerated by one or more catalysts selected from catalysts known in the art for catalyzing cyclic dimerization of isoprene, e.g., iron nitrosyl halide catalysts described in US patents 4,144,278, 4,181,707, 5,545,789 and European patent EP0397266A2. The use of iron nitrosyl catalysts in ionic liquids is described by Ligabue et al.

(2001) J. Mol. Catalysis A: Chemical, 169 (2), 11-17. Examples of catalysts include but are not limited to Fe(NO)2C12 and Cr3+-montmorillonite. In another example, an isoprene starting composition is converted to C10 cyclic dimers (e.g. a mixture of dimethyl-cyclooctadienes) using a ruthenium catalyst (Itoh, Kenji; Masuda, Katsuyuki; Fukahori, Takahiko; Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima, Hideo, Organometallics (1994), 13(3), 1020-9.)

[0602] In a specific embodiment, a isoprene starting composition in a liquid state is heated to over 100 C under pressure and in the presence of an antioxidant to produce a mixture of dimers that includes 6 and 8-membered rings, for examples, limonene (1-methyl-4-(prop-l-en-2-yl)cyclohex-l-ene), 1-methyl-5-(prop-l-en-2-yl)cyclohex-l-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene, 1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta- 1,5-diene, 2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene, 3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene, 3,6-dimethyl-1,3-cyclooctadiene and other 8-membered ring isoprene dimers as described in Organometallics (1994), 13(3), 1020-9. All stereoisomers of these compounds are contemplated.
Conversion of isoprene to dimers is best performed in the absence of oxygen so as to avoid the production of undesirable reaction products.

[0603] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes photo-dimerization to give 4-membered ring dimers.
The isoprene starting composition is converted by light irradiation to a mixture comprising one or more of 1,2-di(prop-l-en-2-yl)cyclobutane, 1,3-di(prop-l-en-2-yl)cyclobutane, 1-methyl-l-vinyl-3-(prop-l-en-2-yl)cyclobutane, 1-methyl-l-vinyl-2-(prop-l-en-2-yl)cyclobutane, 1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutane and stereoisomers thereof. In some embodiments, the highly pure isoprene starting material is dimerized in the presence of a catalyst (e.g. a nickel catalyst) or a photosensitizer (e.g. benzophenone) to yield 4-membered ring dimers, e.g. cis-2-isopropenyl-l-methylvinylcyclobutane, in addition to 6- and 8-membered rings [See: Hammond, J.S. ;Turro, N.J. ; Liu, R.S.H. (1963) "Mechanisms of Photochemical Reactions in Solution. XVI. Photosensitized Dimerization of Conjugated Dienes.
J. Org. Chem., 28, 3297-3303.]

[0604] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material is converted to a cyclic trimer in the presence of a catalyst system. In some embodiments, the cyclic trimer is trimethylcyclododecatriene. In some embodiments, the catalyst system comprises a nickel catalyst. In some embodiments, the catalyst system comprises a titanium compound. In some embodiments, the catalyst system comprises a nickel catalyst and a titanium compound. In some embodiments, the catalyst system comprises one or more organometallic compound and a Group VA compound. In some embodiments, the catalytic transformation is conducted in a hydroxyl group-containing solvent.
In a particular embodiment, a starting isoprene composition is converted to trimethylcyclododecatriene in the presence of a catalyst system comprising Ni and/or Ti, one or more organometallic compound, and a Group VA compound, and that the reaction is conducted in a hydroxyl group-containing solvent.

[0605] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes thermal or catalytic conversion to hydrocarbons in the C7 to C14 range by treatment with a C2 to C5 unsaturated hydrocarbon. The C2 to C5 hydrocarbon can be an alkene, a diene or an alkyne. For example, in some embodiments, isoprene is contacted with ethylene and an appropriate catalyst to produce C7 compounds. In another embodiment, isoprene is contacted with 1,3-butadiene to form cyclic C9 hydrocarbons.
Hydrogenation of these C7 to C14 hydrocarbons results in compositions suitable for use as jet fuels and other aviation fuels. In yet another embodiment, unsaturated C7 to C14 hydrocarbons derived from isoprene and one or more unsaturated hydrocarbons undergo dehydrogenation to form aromatic derivatives suitable for use as jet fuels and other aviation fuels, or as blendstocks for such fuels.

[0606] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material is converted to a mixture of linear dimers and trimers using a catalyst system in alcohol solvents. This reaction also produces alkoxy derivatives when performed in methanol or ethanol. In some embodiments, the catalyst system is a palladium-based catalyst system, e.g., a palladium acetylacetonate - triphenylphosphine system or a palladium acetate -triphenylphosphine system. In some embodiments, the alcohol solvent is methanol, ethanol or isopropyl alcohol. The nature of the products depends on the catalyst and the solvent used. For example, when a palladium-based catalyst system, e.g., Pd(acac)2-Ph3P or Pd(OAc)2-Ph3P, is used in isopropyl alcohol solvent, linear dimers of isoprene is formed, e.g.
2,7-dimethyl-1,3,7-octatriene and 2,7-dimethyl-2,4,6-octatriene. When this reaction is performed in methanol, methoxydimethyloctadienes (e.g., 1-methoxy-2,7-dimethyl-2,7-octadiene and 3-methoxy-2,7-dimethyl-1,7-octadiene) are formed in addition to linear isoprene dimers such as dimethyloctatrienes. Some reactions produce linear trimers of isoprene, such as a-farnesene (3,7,11-trimethyl-1,3,6,10-dodecatetraene), B-farnesene (7,11-dimethyl-3-methylene-1,6,10-dodecatriene) and other positional isomers (e.g. from tail to tail and head to head addition of isoprene units). In another example, isoprene is converted to linear and cyclic C 15 trimers using a chromium N,N-bis(diarylphosphino)amine catalyst (Bowen, L.; Charernsuk, M.;
Wass, D.F.
Chem. Commun. (2007) 2835-2837.) Conversion to Oxygenates

[0607] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material is converted to fuel oxygenates by reaction with ethanol and other alcohols in the presence of an acid catalyst. In one embodiment, the acid catalyst is sulfuric acid. In another embodiment, the acid catalyst is a solid phase sulfuric acid (e.g., Dowex Marathon(g).
Other catalysts include both liquid and solid-phase fluorosulfonic acids, for example, trifluoromethanesulfonic acid and Nafion-H (DuPont). Zeolite catalysts can also been used, for example beta-zeolite, under conditions similar to those described by Hensel et al. [Hensen, K.;
Mahaim, C.; Holderich, W.F., Applied Catalysis A: General (1997) 140(2), 311-329.] for the methoxylation of limonene and related monoterpenes. In some embodiments, a highly pure isoprene starting material is converted to alcohols and esters by a hydroxylation/esterification process or other known reaction of alkenes in the art, for example peroxidation to epoxides with peracids such as peracetic acid and 3-chloroperbenzoic acid; and hydration to give alcohols and diols with i) water and acid catalysts and ii) hydroboration methods. Such reactions are described in, for example, Michael B. Smith and Jerry March, Advanced Organic Chemistry:
Reactions, Mechanisms, and Structure, Sixth Edition, John Wiley & Sons, 2007.
Figures 3 and 4 show examples of alcohols and oxygenates that can be produced from the starting isoprene compositions.

Partial Hydrogenation

[0608] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material is partially hydrogenated to a mono-olefin (e.g. 2-methylbut-l-ene, 3-methyl-but-l-ene and 2-methylbut-2-ene). In some embodiments, the mono-olefin undergoes dimerization or reaction with other olefins using traditional hydrocarbon cationic catalysis, such as that used to covert isobutylene to isooctane. See, for example, H.M.
Lybarger. Isoprene in Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed., Wiley, New York (1995),14,934-952. In some preferred embodiments, the highly pure isoprene starting material is a bioisoprene composition.

[0609] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes partial hydrogenation to form mono-olefins (e.g. 2-methylbut-l-ene, 3-methyl-but-l-ene and 2-methylbut-2-ene). In some preferred embodiments, the highly pure isoprene starting material is a bioisoprene composition. In some embodiments, partial hydrogenation of the isoprene starting material produces high yields of mono-olefins with minimal conversion to isopentane and low residual isoprene levels. In some embodiments, a mixture of mono-olefin and isopentane is produced, with low levels of residual isoprene. In some embodiments, the partial hydrogenation is selective hydrogenation where a particular mono-olefin such as 2-methylbut-2-ene, 2-methylbut-l-ene, or 3-methylbut-l-ene is preferentially produced. Preferred hydrogenation catalysts give high catalytic activity, maintain catalytic activity over time, and are highly selective for the conversion of isoprene to mono-olefins.

[0610] Suitable catalysts for partial hydrogenation of the isoprene starting composition may contain a platinum-group metal such as platinum, palladium, rhodium, or ruthenium, or a transition metal such as nickel, cobalt, copper, iron, molybdenum, or a noble metal such as silver or gold in elemental form, or a salt or complex with organic and inorganic ligands. Alloys of these metals can also be used in some circumatances. These metals and their derivatives can be pure or mixed with other active and inert materials. Catalysts may be adsorbed to a support material (such as activated carbon, alumina, or silica) in order to maximize the effective surface area. The physical morphology of hydrogenation catalysts is known to have a considerable influence on their performance. Preferred hydrogenation catalysts include heterogeneous palladium catalysts such as palladium on carbon (Pd/C), palladium on alumina (Pd/A1203), or palladium on silica (Pd/Si02) in grades ranging from 0.1 % Pd to 20% Pd (w/w) relative to the support material. An example of a suitable selective hydrogenation catalyst is LD 2773, a sulfur tolerant promoted Pd on alumina (Axens, Rueil-Malmaison Cedex, France).
Partial hydrogenation catalysts that allow the conversion of isoprene into isoamylenes with minimal conversion to isopentane include the Lindlar catalyst (Pd/BaS04 treated with quinoline), Pd/C
treated with the triphenyl derivative of a group 15 element (N, P, As), Pd/C
treated with a sulfur-containing compound, molybdenum sulfide, and Pd/Fe alloys. One preferred catalyst comprises palladium adsorbed to egg-shell alumina (d-A1203). Catalysts used in the refining industry for the removal of diolefins and alkynes from pyrolysis gasoline are particularly preferred, for example Ni/A1203 and Pd/A1203 based catalysts. Another suitable class of catalyst for the conversion of isoprene to isoamylenes are those used in industry for the hydrotreating of pyrolysis gasoline. See for example US Patent Nos. 7,014,750 and 6,9949,686, and references cited therein. In general, the catalysts used in pyrolysis gas hydrotreament allow for selective conversion of acetlyenes and diolefins (e.g. isoprene and piperylenes) into monoolefins.

[0611] The hydrogen source can be hydrogen gas or a hydrogen source including, but not limited to, hydrogen gas (H2), formic acid, hydrazine, or isopropanol. The hydrogen source can be either chemically or biologically derived. In some embodiments the hydrogen used for hydrogenation is co-produced with isoprene during fermentation. The hydrogenation can be performed at hydrogen pressures ranging from 0.5 atm to 200 atm, or higher.
The temperature can range from 0 C to 200 C.

[0612] Products from partial hydrogenation of a bioisoprene composition are expected to contain certain impurities originally present in the starting isoprene composition and/or hydrogenated derivatives of the imputities such as acetone, ethanol, amyl alcohol, ethyl acetate, isoamyl acetate, methyl ethyl ketone and other saturated polar impurities.

[0613] In some embodiments, the isoprene starting composition undergoes partial hydrogenation or selective hydrogenation in the presence of a palladium catalyst. For example, palladium catalysts, such as Pd/CaCO3, Pd/BaSO4, Pd/C, Pd black, Pd/Si03, Pd/A1203, or Pd/Si02, were shown to convert isoprene to mono-olefins with a selectivity of greater than 95%
over fully reduced C5 alkanes. Use of these palladium catalysts gave a mixture of mono-olefin products, with 5%Pd/CaCO3 having the greatest selectivity for 3-methylbut-l-ene and 5%
Pd/Si02 having the greatest selectivity for 2-methylbut-2-ene. (See G.C. Bond and A.F. Rawle.
J. Mol. Catalysis A: Chemical 109 (1996) 261-271.) Bond and Rawle have also shown that palladium-gold and palladium-silver catalysts (e.g., Pd-Au/Si02 and Pd-Ag/Si02) have high selectivity for reducing isoprene to mono-olefins over fully reduced C5 alkanes. In some embodiments, silica-supported polyamidoamine (PAMAM) dendrimer-palladium complexes have been used to selectively catalyze the reduction of cyclic and acyclic dienes to a mixture of mono-olefin isomers. Selectivity for the various mono-olefin isomers was dependent on the precise PAMAM ligand used in the catalyst. (See P.P. Zweni and H. Alper. Adv.
Synth. Catal.
348 (2006) 725-73 1.) In some embodiments, reduction of isoprene to mono-olefins can be carried out using a Group VIB metal on an inorganic support (e.g., a metal zeolite) as a catalyst.
For example, a Mo/A1203 catalyst was used to selectively reduce 1,3-butadiene to the respective mono-olefins. (See US 6,235,954 B1.) In some embodiment, isoprene can be reduced to mono-olefins using a Group VIII metal catalyst promoted by a metal from Group IB, VIB, VIIB, or zinc to reduce poisoning of the catalyst. (See US 6,949,686 B2). In some embodiments, isoprene can be reduced to mono-olefins using a monolithic catalyst bed, which may be in a honeycomb configuration. Catalyst support materials for the monolithic catalyst bed may include metals such as nickel, platinum, palladium, rhodium, ruthenium, silver, iron, copper, cobalt, chromium, iridium, tin, and alloys or mixtures thereof. (See US
7,014,750 B2). In some embodiments, isoprene can be reduced to mono-olefins using an eggshell Pd/d-A1203 catalyst, particularly if the reaction is free of water. The eggshell Pd/d-A1203 catalyst is selective for mono-olefins over fully reduced alkanes and 2-methylbut-2-ene is the thermodynamically favorable isomer. (See J.-R. Chang and C.-H. Cheng. Ind. Eng. Chem. Res. 36 (1997) 4094-4099.)

[0614] Light olefins (C3-C6) can be readily dimerized using acid catalysts to give higher olefins (C6-C12) also known as dimate. For example, the conversion of isobutylene (2-methylpropene) to isooctene is well described in the art using a variety of acid catalysts including sulfuric, phosphoric and other mineral acids, sulfonic acids, fluorosulfonic acids, zeolites and acidic clays.

[0615] In some embodiments, the isoprene starting composition undergoes partial hydrogenation or selective hydrogenation to form a mono-olefin, and the mono-olefin undergoes dimerization or reaction with other olefins using traditional hydrocarbon cationic catalysis, such as that used to covert isobutylene to isooctane, e.g. sulfuric, phosphoric and other mineral acids, sulfonic acids, fluorosulfonic acids, zeolites and acidic clays. See, for example, H.M. Lybarger.
Isoprene in Kirk-Othmer Encyclopedia of Chemical Technology, 4th ed., Wiley, New York (1995), 14, 934-952. In some preferred embodiments, the highly pure isoprene starting material is a bioisoprene composition. Acid catalyst in both liquid and solid forms can be used. Acid resins are preferred and include Amberlyst 15, 35, XE586, XN1010 (Rohm and Haas) and similar acidic ion-exchange resins. Acidic molecular sieves are also preferred catalysts including a medium-pore acid molecular sieve such as ZSM-5, ferrierite, ZSM-22 and ZSM-23.

[0616] In some embodiments, the isoprene starting composition undergoes partial hydrogenation or selective hydrogenation to form isoamylenes. In some embodiments, the isoamylenes are dimerized to give C10 dimates such as isodecenes. In some embodiments, the isoamylenes are dimerized with an olefin such as propylene, butane or isobutene to give C8-C10 dimates. In some embodiments, the mono-olefin undergoes dimerization using a resin catalyst (e.g., Amberlyst, Amberlyst 35, Amberlyst 15, Amberlyst XN1010, Amberlyst XE586) to produce diisoamylenes. Use of such resin catalysts has been shown to minimize cracking and further oligomerization reactions, and under optimal conditions the resin catalysts provide selectivity for dimers of greater than 92%, with trimer formation of less than 8%. See, for example, M. Marchionna et al. Catalysis Today 65 (2001) 397-403. In some embodiments, the mono-olefin undergoes dimerization using a catalytic material containing an acidic mesoporous molecular sieve, such as a mesoporous sieve embedded in a zeolite structure, ZSM-5, ferrierite, ZSM-22, or ZSM-23. In a particular embodiment, the catalytic material is thermally stable at high temperatures (e.g., at least 900 C). In another particular embodiment, the mono-olefin is substantially free of multi-unsaturated hydrocarbons, such as isoprene. Use of a catalytic material containing an acidic mesoporous molecular sieve for dimerization can lead to selectivity for dimers in excess of 80%. See, for example, US 2007/0191662 Al. In some embodiments, the mono-olefin undergoes dimerization using a solid acidic catalyst (e.g., a solid phosphoric acid catalyst, acidic ion exchange resins). Selectivity for dimers using a solid phosphoric acid catalyst can be at least 75%, at least 85% or at least 90%. See, for example, Al and US 6,660,898 Bl.

[0617] Efficient dimerization can sometimes require the presence of a polar component(s) such as water and oxygenated compound in the feed stream. Examples include alcohols such as methanol, ethanol and t-butanol, ethers such as methyl t-butyl ether (MTBE) and methyl t-amyl ether (TAME) or an ester such as Cl to C5 acetates. In some embodiments, isoamylenes can be converted to ethers such as TAME by treatment with alcohols in the presence of an acid catalyst.

[0618] In some embodiments, the C10 dimers of mono-olefins produced by any of the methods described herein can be reduced to fully saturated C10 alkylates (e.g., by hydrogenation). In a particular embodiment, isoprene or a mono-olefin can be reduced to an alkylate using a catalyst containing an acid component, such as sulfuric acid, a fluorosulfonic acid, a perhaloalkylsulfonic acid, an ionic liquid, or a mixture of Bronsted acids and Lewis acids, mixed with a polymer component, such as a polyacrylate. (See US 2010/0094072 Al).

[0619] In some embodiments, a commercially beneficial amount of highly pure isoprene starting material undergoes oligomerization in the presence of an acid catalysts to produce dimers, trimers, higher oligomers, aromatic products, and/or polymeric products. Kinetic control of the reaction can favor certain products, for example lower oligomers, although gum formation and coking are known issues leading to catalyst deactivation.

Exemplary unsaturated isoprene derivatives

[0620] In some embodiments, the compositions and systems for producing a fuel constituent from isoprene further comprise an unsaturated hydrocarbon or oxygenate intermediate for chemical transformation of an isoprene starting composition to a fuel constituent. In some embodiment, the unsaturated hydrocarbon intermediate comprise one or more unsaturated dimers of isoprene selected from the group consisting of 1,2-di(prop-l-en-2-yl)cyclobutane, 1,3-di(prop-l-en-2-yl)cyclobutane, 1-methyl-l-vinyl-3-(prop-l-en-2-yl)cyclobutane, 1-methyl-l-vinyl-2-(prop-l-en-2-yl)cyclobutane, 1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutane, 1-methyl-4-(prop-l-en-2-yl)cyclohex-l-ene), 1-methyl-5-(prop-l-en-2-yl)cyclohex-1-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene, 1,5-dimethylcycloocta- 1,5-diene, 1,6-dimethylcycloocta-1,5-diene, 1,4-dimethyl-4-vinyl-l-cyclohexene, 2,4-dimethyl-4-vinyl-l-cyclohexene, 2,7-dimethyl-1,3,7-octatriene, 2,7-dimethyl-2,4,6-octatriene, 2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene, 3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene and 3,6-dimethyl-1,3-cyclooctadiene. In some embodiments, the unsaturated hydrocarbon intermediate comprises one or more unsaturated trimers such as a-farnesene, B-farnesene, trimethylcyclododecatrienes (e.g.
1,5,9-trimethyl-(1E,5E,9E)-cyclododecatriene and positional and geometric isomers thereof) and trimethyldodecatetraenes and the like. In some embodiments, the unsaturated oxygenate intermediate comprise one or more unsaturated methyl ethers such as 1-methoxy-2,7-dimethyl-2,7-octadiene and 3-methoxy-2,7-dimethyl-1,7-octadiene and the like. In some embodiments, the unsaturated oxygenate intermediate comprise one or more unsaturated ethyl ethers such as ethyl 3-methyl-3-butenyl ether, ethyl 1,1-dimethyl-2-propenyl ether, ethyl 1,2-dimethyl-2-propenyl ether, ethyl 2-methyl-3-butenyl ether and the like. In some embodiments, the unsaturated oxygenate intermediate comprise one or more unsaturated alcohols such as 3-methyl-3-buten-l-ol, 2-methyl-3-buten-2-ol, 3-methyl-3-buten-2-ol, 2-methyl-3-buten-l-ol and the like. In some embodiments, the unsaturated oxygenate intermediate comprise one or more unsaturated esters such as 3-methyl-3-buten-1-yl acetate, 2-methyl-3-buten-2-yl acetate, 3-methyl-3-buten-2-yl acetate, 2-methyl-3-buten-1-yl acetate and esters of other C3-C18 aliphatic carboxylic acids. In some embodiment, the unsaturated hydrocarbon intermediate comprise one or more isoamylenes, e.g. 2-methylbut-l-ene, 3-methyl-but-l-ene and 2-methylbut-2-ene. In some embodiments, the unsaturated hydrocarbon intermediate comprise on or more diisoamylenes derived from dimerization of isoamylenes.

Scheme I. Exemplary unsaturated 6- and 8-membered ring intermediates -0-< 6- -0 1 b I

1-methyl-4-(prop-l-en- 1-methyl-5-(prop-l-en- 1,4-dimethyl-4- 2,4-dimethyl-4-2-yl)cyclohex-l-ene 2-yl)cyclohex-l-ene vinylcyclohexene vinylcyclohexene N

2,6-dimethyl-1,3- 2,6-dimethyl-1,4- 3,7-dimethyl-1,3- 3,7-dimethyl-1,5-cyclooctadiene cyclooctadiene cyclooctadiene cyclooctadiene 1,5-dimethylcycloocta-1,5- 1,6-dimethylcycloocta- 3,6-dimethyl-1,3-diene 1,5-diene cyclooctadiene Scheme II. Exemplary unsaturated 4-membered ring intermediates 1,2-di(prop-l-en-2-yl)cyclobutane 1-methyl-3-(prop- l -en-2- 1,3-dimethyl-1,3-yl)-1-vinylcyclobutane divinylcyclobutane -1-methyl-2-(prop-l-en-2- 1,2-dimethyl-l,2 divinylcyclobutane 1,3-di(prop-l-en-yl)-1-vinylcyclobutane yl)cyclobutane Scheme III. Exemplary unsaturated isoprene telomer and oxygenate intermediates 2,7-dimethylocta- 2,7-dimethylocta- 3,7,11 -trimethyldodeca-1,3,7-triene 2,4,6-triene 1,3,7,11-tetraene 3,7,11 -trimethyldodeca- 1,3,6, 1 0-tetraene 7,11 -dimethyl-3 -methylenedodeca-1,6, 1 0-triene O
O/

8-methoxy-2,7-dimethylocta-1,6-diene 3-methoxy-2,7-dimethylocta-1,7-diene Scheme IV. Exemplary unsaturated ethyl ether intermediates Ethyl 3-methyl-3-butenyl ether Ethyl 1, 1 -dimethyl-2-propenyl ether r Ethyl 1,2-dimethyl-2-propenyl ether Ethyl 2-methyl-3-butenyl ether Scheme V. Exemplary unsaturated alcohol intermediates OH
)~~OH >~\% ---y HO
OH
3-methyl-3-buten- l -ol 2-methyl-3-buten-2-ol 3-methyl-3-buten-2-ol 2-methyl-3-buten-l-ol Scheme VI. Exemplary unsaturated ester intermediates OAc OAc >[\%
3-methyl-3-buten-l-yl acetate 2-methyl-3-buten-2-yl acetate AcO
OAc 3-methyl-3-buten-2-yl acetate 2-methyl-3-buten-l-yl acetate Exemplary hydrogenation of unsaturated intermediates

[0621] The unsaturated isoprene derivatives are subject hydrogenation in the presence of a hydrogenation catalyst to produce saturated compounds. The saturated compounds are characterized and their values as fuels are assessed. In some embodiments, the hydrogen source for hydrogenation is hydrogen gas. In some embodiments, the hydrogen gas is co-produced with the bioisoprene as described in U.S. provisional patent application No.61/141,652, filed on December 30, 2008 and US 2009/0203102 Al. In some embodiments, the hydrogenation catalyst is a palladium based catalyst such as Pd/C (e.g. 5% (wt.) Pd/C). In some embodiments, the hydrogenation catalyst is a Raney Nickel catalyst. In some embodiments, the hydrogenation catalyst is a homogenous catalyst such as ruthenium or rhodium based homogenous hydrogenation catalysts. In some embodiments, unsaturated isoprene dimers and trimers are hydrogenated to produce saturated C 10 and C 15 hydrocarbons suitable for making fuels. In some embodiments, unsaturated cyclic dimers are hydrogenated to produce saturated cyclic C 10 hydrocarbons such as 1,2-bis(isopropyl)cyclobutane, 1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1-ethyl-l,4-dimethylcyclohexane, 1-ethyl-l,3-dimethylcyclohexane, 1,5-dimethylcyclooctane and 1,4-dimethylcyclooctane. In some embodiments, unsaturated cyclic trimers are hydrogenated to produce saturated cyclic C 15 hydrocarbons such as 1,5,9-trimethylcyclododecane and 1,5,10-trimethylcyclododecane (see Scheme VII). In some embodiments, unsaturated linear dimers are hydrogenated to produce saturated aliphatic C 10 hydrocarbons such as 2,6-dimethyloctane, 2,7-dimethyloctane and 3,6-dimethyloctane. In some embodiments, unsaturated linear trimers are hydrogenated to produce saturated aliphatic C15 hydrocarbons such as 2,6,10-trimethyldodecane, 2,7,10-trimethyldodecane and 3,7,10-trimethyldodecane (see Scheme VIII). In some embodiments, the product of the dimerization of isoamylenes (diamylenes or C 10 dimates) are fully hydrogenated to isoparaffins (e.g. 2,3,4,4-tetramethylhexane, 2,2,3,4-tetramethylhexane, 2,3,3,4-tetramethylhexane and 3,3,5-trimethylheptane) (see Scheme VIIIa). In some embodiments, a commercially beneficial amount of highly pure isoprene starting composition is hydrogenated to produce a product comprising 2-methylbutane. In some embodiments, the unsaturated isoprene hydroxylates are hydrogenated to produce saturated hydroxylates such as C5 alcohols and diols (e.g. 3-methyl-butan-l-ol, 2-methyl-butan-l-ol and 2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol and 2-methyl-butan-2,3-diol), C-10 alcohols and diols (e.g. 3,7-dimethyloctan-l-ol, 2,7-dimethyloctan-l-ol, 2,7-dimethyloctan-2-ol and 2,7-dimethyloctan-2,7-diol) and cyclic C-10 alcohols (e.g. 2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-l-ol, 2-(1,4-dimethylcyclohexyl)ethanol and 4-ethyl-1,4-dimethylcyclohexanol) (see Scheme IX). In some embodiments, the unsaturated isoprene oxygenates are hydrogenated to produce saturated ethers such as 1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane, 1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane (see Scheme X). It is understood that when an alkene moiety is hydrogenated, one or more stereo isomers are produced. The relative ratios between the stereo isomers depend on the reaction conditions and the catalysts used.
When applicable, each and every stereo isomer is intended for the saturated hydrocarbons and oxygenates described herein.

Scheme VII. Examples of cyclic hydrocarbons derived from isoprene bis-(isopropyl)cyclobutanes from 2+2 photodimerization Cyclohexanes from thermal or catalytic cyclodimerization ,-~a -~Cl 1,5-dimethyl- and 1,6-dimethylcyclooctanane from thermal or catalytic cyclodimerization 1,5,9- and 1,5,10-trimethylcyclododecane from cyclic trimerization of isoprene Scheme VIII. Examples of aliphatic hydrocarbons derived from isoprene 2-methylbutane 2,6, 1 0-trimethyldodecane (Farnesane) 2,6-dimethyloctane 2,7, 1 0-trimethyldodecane 2,7-dimethyloctane 3,7, 1 0-trimethyldodecane 3,6-dimethyloctane Scheme VIIIa. Examples of isodecanes derived from isoamylenes >1 2,3,4,4-tetramethylhexane 2,2,3,4-tetramethylhexane 2,3,3,4-tetramethylhexane 3,3,5-trimethylheptane Scheme IX. Examples of IsoFuelTM alcohols derived from isoprene OH HO HO OH HO
OH
HO

C5 alcohols and diols OH OH
HO

OH OH
C 10 alcohols and diols OH
OH
OH OH
Cyclic C 10 alcohols Scheme X. Examples of IsoFuelTM oxygenates derived from isoprene OAc O

3-methyltetrahydrofuran 3-methyl-2-butanone Isopentyl acetate Reaction products of isoprene and ethanol (see attached presentation from S. Reyer) O
O/

Alkoxy-derivatives produced by Pd-catalyzed telomerization of isoprene in methanol solvent.
(see Zakharkin (1976))

[0622] In some embodiment, the fuel constituent produced by chemical transformation of a commercially beneficial amount of highly pure isoprene starting composition comprises saturated isoprene derivatives. In some embodiments, the fuel constituent comprises saturated C10 and C15 hydrocarbons derived from isoprene. In some embodiments, the fuel constituent comprises one or more saturated cyclic C10 hydrocarbons selected from the group consisting of 1,2-bis(isopropyl)cyclobutane, 1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1-ethyl-l,4-dimethylcyclohexane, 1-ethyl-1,3-dimethylcyclohexane, 1,5-dimethylcyclooctane and 1,4-dimethylcyclooctane. In some embodiments, the fuel constituent comprises one or more saturated cyclic C15 hydrocarbons selected from the group consisting of 1,5,9-trimethylcyclododecane and 1,5,10-trimethylcyclododecane. In some embodiments, the fuel constituent comprises one or more saturated aliphatic C10 hydrocarbons selected from the group consisting of 2,6-dimethyloctane, 2,7-dimethyloctane and 3,6-dimethyloctane. In some embodiments, the fuel constituent comprises one or more saturated aliphatic C15 hydrocarbons selected from the group consisting of 2,6,10-trimethyldodecane, 2,7, 1 0-trimethyldodecane and 3,7,10-trimethyldodecane. In some embodiments, the fuel constituent comprises 2-methylbutane. In some embodiments, the fuel constituent comprises one or more isoparaffins selected from the group consisting of 2,3,4,4-tetramethylhexane, 2,2,3,4-tetramethylhexane, 2,3,3,4-tetramethylhexane and 3,3,5-trimethylheptane. In some embodiments, the fuel constituent comprises one or more saturated hydroxylates selected from the group consisting of 3-methyl-butan-l-ol, 2-methyl-butan-l-ol and 2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol, 2-methyl-butan-2,3-diol, 3,7-dimethyloctan-1-ol, 2,7-dimethyloctan-l-ol, 2,7-dimethyloctan-2-ol, 2,7-dimethyloctan-2,7-diol, 2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-l-ol, 2-(1,4-dimethylcyclohexyl)ethanol and 4-ethyl-1,4-dimethylcyclohexanol. In some embodiments, the fuel constituent comprises one or more saturated ethers selected from the group consisting of 1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane, 1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane. In some embodiments, the fuel constituent comprises one or more oxygenates of isoprene selected from the group consisting of 3-methyltetrahydrofuran, 3-methyl-2-butanone and isopentyl acetate.

[0623] In some embodiments, the compositions and systems for producing a fuel constituent from isoprene further comprise catalysts for catalyzing the chemical transformation of an isoprene starting composition to a fuel constituent or an intermediate for making a fuel constituent. In some embodiments, the compositions and systems for producing a fuel constituent from isoprene further comprise catalysts for catalyzing hydrogenation of an unsaturated intermediate to produce a saturated fuel constituent. In some embodiments, the catalyst is any catalyst described or a combination of one or more of the catalyst described herein.

Method and/or Processes for Producing Fuels

[0624] The invention provides methods and/or processes for producing a fuel constituent from isoprene comprising: (a) obtaining a commercially beneficial amount of highly pure isoprene;
and (b) chemically transforming at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent. In one embodiment, a highly pure isoprene composition is transformed to a fuel component in a continuous chemical process. In another embodiment, a highly pure isoprene is further purified before chemical transformation to a fuel composition. In yet another embodiment, a highly pure isoprene is chemically transformed to an intermediate composition; the intermediate composition undergoes further chemical transformations to produce a fuel or a fuel component. In a further embodiment, the fuel component produced is mixed with a petroleum distillate and other optional additives to make a fuel. In some preferred embodiments, the highly pure isoprene is a bioisoprene composition.

[0625] In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene composition useful in the invention comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene. In some embodiments, the highly pure isoprene composition comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. In some embodiments, the starting isoprene composition comprises greater than or about 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 kg of isoprene.

[0626] In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene starting composition comprises greater than or about 98.0, 98.5, 99.0, 99.5, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the highly pure isoprene composition useful in the invention comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition. In some embodiments, the highly pure isoprene composition comprises less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-l-yne) by weight compared to the total weight of all C5 hydrocarbons in the composition.
In some embodiments, the highly pure isoprene composition has less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the composition. In particular embodiments, the highly pure isoprene composition has greater than about 2 mg of isoprene and has greater than or about 98.0, 98.5, 99.0, 99.5, 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.

[0627] In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene composition useful in the invention has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a compound that inhibits the polymerization of isoprene for any compound in the composition that inhibits the polymerization of isoprene. In particular embodiments, the composition also has greater than about 2 mg of isoprene.

[0628] In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene composition useful in the invention has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the highly pure isoprene composition has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 g/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), or any two or more of the foregoing. In particular embodiments, the highly pure isoprene composition has greater than about 2 mg of isoprene and has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.

[0629] In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the highly pure isoprene composition useful in the invention includes isoprene and one or more second compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. In various embodiments, the amount of one of these second components relative to the amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110%
(w/w). In some embodiments, the amount of methanethiol relative to the amount of isoprene in units of percentage by weight is at less than 0.01 % (w/w).

[0630] In some embodiments, the method for producing a fuel constituent from isoprene comprises obtaining a commercially beneficial amount of highly pure isoprene composition. In some embodiments, the method comprises obtaining a commercially beneficial amount of highly pure isoprene composition from a biological process comprising: (a) culturing cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide under suitable culture conditions for the production of isoprene, wherein the cells (i) produce greater than about 400 nmole/gwcm/hr of isoprene, (ii) convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, or (iii) have an average volumetric productivity of isoprene greater than about 0.1 mg/Lbrofh/hr of isoprene, and (b) producing isoprene. In some embodiments, the cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide, e.g. a naturally-occurring polypeptide from a plant such as Pueraria, and (ii) is operably linked to a promoter, e.g. a T7 promoter. In some embodiments, the cells are cultured in a culture medium that includes a carbon source, such as, but not limited to, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fatty acid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide (e.g., a microbial or plant protein or peptide), yeast extract, component from a yeast extract, or any combination of two or more of the foregoing. In some embodiments, the cells are cultured under limited glucose conditions. In some embodiment, the cells further comprise a heterologous nucleic acid encoding an IDI polypeptide.
In some embodiments, the cells further comprise a heterologous nucleic acid encoding an MDV
pathway polypeptide. In some embodiment, the method comprises producing isoprene using the methods described in U.S. provisional patent application Nos. 61/134,094, filed on July 2, 2008, WO 2010/003007, and U.S. patent application No. 12/335,071, filed December 15, 2008 (US
2009/0203102 Al), which are incorporated by reference in their entireties.

[0631] In some embodiment, the method comprises obtaining a gas phase (off-gas) produced by cells in culture that produces isoprene. In some embodiments, the cells in culturing produce greater than about 400 nmole/gwcm/hr of isoprene. In some embodiments, the gas phase comprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g/L of isoprene when sparged at a rate of 1 vvm. In some embodiments, the volatile organic fraction of the gas phase comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100%
isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase comprises less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In some embodiments, the volatile organic fraction of the gas phase has less than or about 2.0, 1.5, 1.0, 0.5, 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction. In particular embodiments, the volatile organic fraction of the gas phase has greater than about 2 mg of isoprene and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction.

[0632] In some embodiment, the method comprises obtaining a gas phase (off-gas) produced by cells in culture that produces isoprene. In some embodiments, the volatile organic fraction of the gas phase has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 g/L of a compound that inhibits the polymerization of isoprene for any compound in the volatile organic fraction of the gas phase that inhibits the polymerization of isoprene. In particular embodiments, the volatile organic fraction of the gas phase also has greater than about 2 mg of isoprene. In some embodiments, the volatile organic fraction of the gas phase has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the volatile organic fraction of the gas phase has greater than or about 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 60, 80, 100, or 120 g/L of ethanol, acetone, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), or any two or more of the foregoing. In particular embodiments, the volatile organic fraction of the gas phase has greater than about 2 mg of isoprene and has one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms. In some embodiments, the volatile organic fraction of the gas phase has includes isoprene and one or more second compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine. In various embodiments, the amount of one of these second components relative to amount of isoprene in units of percentage by weight (i.e., weight of the component divided by the weight of isoprene times 100) is at greater than or about 0.01, 0.02, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 110% (w/w) in the volatile organic fraction of the gas phase. In some embodiments, the method further includes recovering the isoprene from the gas phase. For example, the isoprene in the gas phase comprising isoprene can be recovered using standard techniques, such as gas stripping, membrane enhanced separation, fractionation, adsorption/desorption, pervaporation, thermal or vacuum desorption of isoprene from a solid phase, or extraction of isoprene immobilized or absorbed to a solid phase with a solvent (see, for example, U.S. Patent Nos.
4,703,007 and 4,570,029, which are each hereby incorporated by reference in their entireties, particularly with respect to isoprene recovery and purification methods). In some particular embodiments, extractive distillation with an alcohol (such as ethanol, methanol, propanol, or a combination thereof) is used to recover the isoprene. In some embodiments, the recovery of isoprene involves the isolation of isoprene in a liquid form (such as a neat solution of isoprene or a solution of isoprene in a solvent). Gas stripping involves the removal of isoprene vapor from the fermentation off-gas stream in a continuous manner. Such removal can be achieved in several different ways including, but not limited to, adsorption to a solid phase, partition into a liquid phase, or direct condensation (such as condensation due to exposure to a condensation coil or do to an increase in pressure). In some embodiments, membrane enrichment of a dilute isoprene vapor stream above the dew point of the vapor resulting in the condensation of liquid isoprene. In some embodiments, the recovered isoprene is compressed and condensed.

[0633] The recovery of isoprene may involve one step or multiple steps. In some embodiments, the removal of isoprene vapor from a fermentation off-gas and the conversion of isoprene to a liquid phase are performed simultaneously. For example, isoprene can be directly condensed from the off-gas stream to form a liquid. In some embodiments, the removal of isoprene vapor from the fermentation off-gas and the conversion of isoprene to a liquid phase are performed sequentially. For example, isoprene may be adsorbed to a solid phase and then extracted from the solid phase with a solvent.

[0634] In some embodiments, a method is provided for producing a cyclic dimer of isoprene comprising heating neat bioisoprene. The cyclic dimers of isoprene produced may be 6-membered ring dimers (e.g. a [2+4] electrocyclization product such as limonene) or 8-membered ring dimers or a mixture thereof. Examples of 6-membered ring dimers include but not limited to 1-methyl-4-(prop-l-en-2-yl)cyclohex-l-ene, 1-methyl-5-(prop-l-en-2-yl)cyclohex-l-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene and the like.
Examples of 8-membered ring dimers include but not limited to 1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene and the like. In one embodiment, neat biologically produced isoprene is dimerized by heating to a temperature of about 100 C to about 300 C, preferably about 150 C to about 250 C. The pressure is maintained at about 2 to 3 atm.
In another embodiment, a catalyst suitable for catalyzing dimerization of conjugated dienes may be used.
The proportions of various dimers in the product mixture may be controlled by the catalyst and other reactions conditions. For example, a nickel catalyst can promote formation of 8-membered ring dimers.

[0635] In some embodiments, a method is provided for producing a cyclic dimer of isoprene comprising photo-dimerization of bioisoprene. The cyclic dimer of isoprene produced may be one or more 4-membered ring dimer or a mixture thereof. Examples of Examples of 4-membered ring dimers include but not limited to 1,2-di(prop-l-en-2-yl)cyclobutane, 1,3-di(prop-1-en-2-yl)cyclobutane, 1-methyl-l-vinyl-3-(prop-l-en-2-yl)cyclobutane, 1-methyl-l-vinyl-2-(prop-l-en-2-yl)cyclobutane, 1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutane and the like. In one embodiment, neat biologically produced isoprene is dimerized by irradiating with UV light, preferably in presence of a photosensitizer such as benzophenone.

[0636] Isoprene dimers can be hydrogenated to form saturated Cio hydrocarbons that can serve as fuels or blended into fuels. In one embodiment, the unsaturated isoprene dimers are subjected to catalytic hydrogenation to produce partially hydrogenated and/or fully hydrogenated products.
Examples of fully hydrogenated products include but not limited to 1-methyl-4-isopropylcyclohexane and 1-methyl-5-isopropylcyclohexane, 1,4-dimethyl-4-ethylcyclohexane, 2,4-dimethyl-4-ethylcyclohexane, 1,2-diisopropylcyclobutane and 1,3-diisopropylcyclobutane, 1-methyl-l-vinyl-3-(prop-l-en-2-yl)cyclobutane, 1-methyl-l-ethyl-2-(prop-2-yl)cyclobutane, 1,3-dimethyl-1,3-ethylcyclobutane, 1,2-dimethyl-1,2-diethylcyclobutane, 1,5-dimethylcyclooctane, 1,6-dimethylcyclooctane and the like.

[0637] In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) chemically transforming at least a portion of the highly pure isoprene starting composition to a fuel constituent comprising:
(i) heating the highly pure isoprene composition to over or about 100 C under pressure; (ii) converting at least a portion of the starting isoprene composition to unsaturated cyclic isoprene dimers; and (iii) hydrogenating the unsaturated cyclic isoprene dimers to produce saturated cyclic isoprene dimers. In some embodiments, the starting isoprene composition is heated to over or about 100, 125, 150, 175, 200, 225 or 250 C under pressure. In some embodiments, the starting isoprene composition is heated in the presence of an antioxidant (e.g. 2,6-di-tert-butyl-4-methylphenol) to prevent radical-mediated polymerization. In one embodiment, the thermal dimerization of isoprene is performed in presence of dinitrocresol as polymerization inhibitor. Suitable antioxidants may be used as polymerization inhibitors. In some embodiments, at least a portion of the starting isoprene composition to a mixture of unsaturated dimers that includes 6 and 8-membered rings, for examples, limonene (1-methyl-4-(prop-l-en-2-yl)cyclohex-l-ene), 1-methyl-5-(prop-l-en-2-yl)cyclohex-l-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene, 1,5-dimethylcycloocta-1,5-diene, 1,6-dimethylcycloocta-1,5-diene, 2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene, 3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene and 3,6-dimethyl-1,3-cyclooctadiene. In some embodiments, greater than or about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% of isoprene in starting isoprene composition is converted to unsaturated cyclic isoprene dimers. In some embodiments, hydrogenation of the unsaturated cyclic isoprene dimers is catalyzed by a hydrogenation catalyst such as a palladium based catalyst (e.g. Pd/C, e.g. 5%
(wt.) Pd/C.) In some embodiments, the saturated cyclic isoprene dimers comprise one or more C

hydrocarbons selected from the group consisting of 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1,5-dimethylcyclooctane and 1,4-dimethylcyclooctane.
In some preferred embodiments, the starting isoprene composition is a bioisoprene composition.

[0638] In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) chemically transforming at least a portion of the highly pure isoprene starting composition to a fuel constituent comprising:
(i) contacting the highly pure isoprene composition with a catalyst system; (ii) converting at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers;
and (iii) hydrogenating the unsaturated dimers and/or trimers to produce saturated C10 and/or C15 hydrocarbons. In some embodiments, greater than or about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% of isoprene in starting isoprene composition is converted to unsaturated dimers and/or trimers of isoprene. In some embodiments, the starting isoprene composition is contacted with appropriate catalyst systems known in the art to yield one or more of unsaturated cyclic isoprene dimers selected from the group consisting of 1,2-di(prop-l-en-2-yl)cyclobutane, 1,3-di(prop-l-en-2-yl)cyclobutane, 1-methyl-l-vinyl-3-(prop-l-en-2-yl)cyclobutane, 1-methyl-1-vinyl-2-(prop-l-en-2-yl)cyclobutane, 1,3-dimethyl-1,3-divinylcyclobutane, 1,2-dimethyl-1,2-divinylcyclobutane, 1-methyl-4-(prop-l-en-2-yl)cyclohex-l-ene), 1-methyl-5-(prop-l-en-2-yl)cyclohex-1-ene, 1,4-dimethyl-4-vinylcyclohexene, 2,4-dimethyl-4-vinylcyclohexene, 1,5-dimethylcycloocta- 1,5-diene, 1,6-dimethylcycloocta-1,5-diene, 1,4-dimethyl-4-vinyl-l-cyclohexene, 2,4-dimethyl-4-vinyl-l-cyclohexene, 2,7-dimethyl-1,3,7-octatriene, 2,7-dimethyl-2,4,6-octatriene, 2,6-dimethyl-1,3-cyclooctadiene, 2,6-dimethyl-1,4-cyclooctadiene, 3,7-dimethyl-1,5-cyclooctadiene, 3,7-dimethyl-1,3-cyclooctadiene and 3,6-dimethyl-1,3-cyclooctadiene. In some embodiments, the starting isoprene composition is contacted with appropriate catalyst systems known in the art to yield one or more of unsaturated cyclic isoprene trimers such as trimethylcyclododecatrienes and trimethyldodecatetraenes and the like. In some embodiments, the starting isoprene composition is contacted with appropriate catalyst systems known in the art to yield one or more of unsaturated linear dimers and/or trimers of isoprene such as dimethyloctatrienes (e.g. 2,7-dimethyl-1,3,7-octatriene and 2,7-dimethyl-2,4,6-octatriene) and trimethyldodecatetraenes (e.g. 2,6, 1 0-trimethyl- 1,5,9,11 -dodecatetraene, a-farnesene and B-farnesene) and the like. In some preferred embodiments, the starting isoprene composition is a bioisoprene composition.

[0639] In some embodiments, the method for producing a fuel constituent from isoprene comprises contacting the highly pure isoprene composition with a catalyst system to convert at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers. In some embodiments, the catalyst system comprise a Ni, Fe or Co catalyst and the isoprene in the starting composition is converted an 8-membered ring isoprene dimer comprising dimethylcyclooctadienes such as 1,5-dimethylcycloocta-1,5-diene and 1,6-dimethylcycloocta-1,5-diene. In some embodiments, the catalyst system comprises a nickel compound. Ni-catalyzed dimerization of isoprene yields a dimethyl-1,5-cyclooctadiene mixture consisting of about 90 to 10, 80 to 20, 70 to 30, 60 to 40, 50 to 50, 40 to 60, 30 to 70, 20 to 80 and 10 to 90 ratio of 1,5-dimethyl-1,5-cyclooctadiene to 1,6-dimethyl-1,5-cyclooctadiene.
In one embodiment, the catalyst system comprises a 3-component catalyst containing Ni carboxylates or B-ketones, organoaluminum or organomagnesium compounds and substituted triphenylphosphite. For example, a degassed solution of a highly pure isoprene starting composition in anhydrous toluene is mixed with Ni(acac)2, Et3Al, and tris(3,4-bis(dimethylamino)phenyl)phosphite under nitrogen atmosphere and the mixture is heated at 95 C to give dimethylcyclooctadiene. In another embodiment, the catalyst system comprises Fe carboxylates or B-diketone compounds, organo-Al or Mg compounds, and 2,2'-dipyridyl derivatives having electron-donating groups. For example, a starting isoprene composition is heated with a mixture of Fe acetylacetonate, 4,4'-dimethyl-2,2'-dipyridyl, and Et3Al in toluene to gave 1,6-dimethyl-1,5-cyclooctadiene in good yields. In some preferred embodiments, the starting isoprene composition is a bioisoprene composition.

[0640] In some embodiments, the method for producing a fuel constituent from isoprene comprises contacting the highly pure isoprene composition with a catalyst system to convert at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers. In some embodiments, the catalyst system comprises a Ru, Fe, Ni or Pd catalyst or a combination thereof and the isoprene in the starting composition is converted to a mixture comprising 6-membered ring isoprene dimers. In one embodiment, the catalyst system is a Fe(NO)2Cl-bis(1,5-cyclooctadiene)nickel catalyst system and the isoprene in the starting composition is cyclodimerized at low temperatures (e.g. from -5 to +20 C) to give 1-methyl-and 2-methyl-4-isopropenyl-l-cyclohexene and 1,4- and 2,4-dimethyl-4-vinyl-l-cyclohexene.
In another embodiment, contacting with a mixed catalyst of a tris(substituted hydrocarbyl) phosphite, arsenite, or antimonite and a Group VIII metal(0) compound (e.g. Ni acetylacetonate) converts the isoprene in the starting composition to 1,4-dimethyl-4-vinyl-l-cyclohexene and 1,5-dimethyl-1,5-cyclooctadiene. In another embodiment, contacting an isoprene starting composition with a ruthenium catalyst (e.g. see Itoh, Kenji;
Masuda, Katsuyuki; Fukahori, Takahiko; Nakano, Katsumasa; Aoki, Katsuyuki; Nagashima, Hideo, Organometallics (1994), 13(3), 1020-9) converts isoprene to C10 cyclic dimers (e.g. a mixture of dimethyl-cyclooctadienes). The ruthenium catalyst can be synthesized in two steps from RuC13 and pentamethylcyclopentadiene (C5Me5H). The process may be performed in batch mode and the catalyst recovered and recycled. In some preferred embodiments, the starting isoprene composition is a bioisoprene composition. One embodiment of the method is illustrated in Scheme XI.

[0641] In some embodiments, the method for producing a fuel constituent from isoprene comprises contacting the highly pure isoprene composition with a catalyst system to convert at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers. In some embodiments, the catalyst system comprises a Ni, Ti, Al, or Mg catalyst or a mixture thereof and the isoprene in the starting composition is converted to a mixture comprising cyclic isoprene trimers such as trimethylcyclododecatrienes. In one embodiment, the catalyst system for the trimerization of isoprene comprising Ni and/or Ti, one or more organometallic compound, and a Group VA compound. The reaction may be conducted in a hydroxyl group-containing solvent. In another embodiment, oligomerization of isoprene catalyzed by nickel naphthenate and isoprenemagnesium in the presence of various phosphites as electron donors give cyclic dimers containing dimethylcyclooctadiene, in particular 1,1,1-tris(hydroxymethyl)propane phosphite gives trimethylcyclododecatriene selectively.
Scheme XI

heat and/or catalyst Isoprene 6-membered 8-membered ring dimers ring dimers

[0642] In some embodiments, the method for producing a fuel constituent from isoprene comprises contacting the highly pure isoprene composition with a catalyst system to convert at least a portion of the starting isoprene composition to unsaturated isoprene dimers and/or trimers. In some embodiments, the catalyst system comprises a Ni, Pd or Cr catalyst or a mixture thereof and the isoprene in the starting composition is converted telomers or linear dimers and/or trimers. In one embodiment, a catalyst system comprising Pd(O) complexes (e.g.
Pd(acac)2-Ph3P and Pd(OAc)2-Ph3P) catalyzes dimerization and telomerization of isoprene to give linear isoprene dimers (e.g. 2,7-dimethyl-1,3,7-octatriene). In one variation, the reaction is performed in methanol as a solvent and methyl ethers such as methoxydimethyloctadienes (e.g.
1-methoxy-2,7-dimethyl-2,7-octadiene and 3-methoxy-2,7-dimethyl-1,7-octadiene) are produced. In another embodiment, the catalyst system comprises divalent and trivalent transition metal-exchanged montmorillonites (e.g. Cr3+-montmorillonite). In another embodiment, the catalyst system comprises a Ni(O)-aminophosphinite catalyst and the isoprene in the starting composition is converted to regioselective tail-to-tail linear dimers. In some variations, the linear dimer formation is accompanied by a competitive cyclodimerization reaction. In another embodiment, the catalyst system comprises a chromium N,N-bis(diarylphosphino)amine catalyst (e.g. see Bowen, L.; Charernsuk, M.; Wass, D.F. Chem.
Commun. (2007) 2835-2837) and the isoprene is converted to linear and cyclic C15 trimers. In one variation, the chromium catalyst is made in situ from CrC13(THF)3 and a PNP phosphine ligand.

[0643] In some embodiments, the method for producing a fuel constituent from a bioisoprene composition comprises chemically transforming a substantial portion of the isoprene in the bioisoprene composition by (i) contacting the bioisoprene composition with a catalyst for catalyzing trimerization of isoprene to produce an unsaturated isoprene trimer and (ii) hydrogenating the isoprene trimer to produce a fuel constituent. In some embodiments, at least about 95% of isoprene in the bioisoprene composition is converted to non-isoprene compounds during the chemical transformation.

[0644] In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) converting at least a portion of the starting isoprene composition to unsaturated isoprene derivatives; and (c) hydrogenating the unsaturated isoprene derivatives to produce saturated compounds. In some embodiments, hydrogenation of unsaturated isoprene derivatives to saturated compounds is performed in batch mode. In some embodiments, hydrogenation of unsaturated isoprene derivatives to saturated compounds is performed in continuous mode. One embodiment of the method is illustrated in Scheme XII.

Scheme XII

Metal catalyst Isoprene H2, catalyst

[0645] In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) converting at least a portion of the starting isoprene composition to oxygenated isoprene derivatives; and optionally (c) hydrogenating any unsaturated oxygenated isoprene derivatives to produce saturated oxygenates.
In some embodiments, the isoprene starting composition is contacted with a catalyst in the presence of alcohols (e.g. methanol or ethanol or mixtures thereof) to form oxygenated isoprene derivatives comprise one or more unsaturated or saturated ethers (e.g. methyl ethers or ethyl ethers or mixtures thereof). In some embodiments, the catalyst is an acid catalyst such as sulfuric acid, solid phase sulfuric acid (e.g., Dowex Marathon(g), liquid and solid-phase fluorosulfonic acids (e.g. trifluoromethanesulfonic acid and Nafion-H (DuPont)). Zeolite catalysts can also been used, for example beta-zeolite, under conditions similar to those described by Hensel et al.
[Hensen, K.; Mahaim, C.; Holderich, W.F., Applied Catalysis A: General (1997) 140(2), 311-329.] for the methoxylation of limonene and related monoterpenes. In some embodiments, the isoprene starting compositions or the unsaturated isoprene dimers or trimers undergo oxidation/hydrogenation to form the hydroxylated isoprene derivatives comprise one or more alcohols such as C5, C10 and C15 alcohols and diols. In some embodiments, the isoprene starting composition undergoes hydroxylation/esterification to form alcohols and esters. For example, bioisoprene or an unsaturated intermediate undergoes peroxidation to epoxides with peracids such as peracetic and 3-chloroperbenzoic acid; and hydration to give alcohols and diols with i) water and acid catalysts and ii) hydroboration methods. Such reactions are described in, for example, Michael B. Smith and Jerry March, Advanced Organic Chemistry:
Reactions, Mechanisms, and Structure, Sixth Edition, John Wiley & Sons, 2007. In some preferred embodiments, the starting isoprene composition is a bioisoprene composition.

[0646] In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) partially hydrogenating the starting isoprene composition to mono-olefins (e.g. 2-methylbut-l-ene, 3-methyl-but-l-ene and 2-methylbut-2-ene); and (c) producing the fuel constituent from the mono-olefins. In one embodiment, the mono-olefin undergoes dimerization. In another embodiment, the mono-olefin is reacted with other olefins using traditional hydrocarbon cationic catalysis, such as that used to covert isobutylene to isooctane. The product of dimerization or reaction with other olefins are then undergoes hydrogenation to give saturated fuel constituents. In some preferred embodiments, the highly pure isoprene is a bioisoprene composition. In some embodiment, the starting isoprene composition partially hydrogenated using a palladium catalyst, such as Pd/CaCO3, Pd/BaS04, Pd/C, Pd black, Pd/Si03, Pd/A1203, or Pd/Si02. In some embodiment, the starting isoprene composition partially hydrogenated using a silica-supported polyamidoamine (PAMAM) dendrimer-palladium complex. In some embodiment, the starting isoprene composition partially hydrogenated using a palladium-gold and palladium-silver catalysts (e.g., Pd-Au/Si02 and Pd-Ag/Si02) have high selectivity for reducing isoprene to mono-olefins over fully reduced C5 alkanes. In some embodiment, the starting isoprene composition partially hydrogenated using a Group VIB metal on an inorganic support (e.g., a metal zeolite) as a catalyst, e.g. a Mo/A1203 catalyst. In some embodiment, the starting isoprene composition partially hydrogenated using a monolithic catalyst bed, which may be in a honeycomb configuration. Catalyst support materials for the monolithic catalyst bed may include metals such as nickel, platinum, palladium, rhodium, ruthenium, silver, iron, copper, cobalt, chromium, iridium, tin, and alloys or mixtures thereof. In some embodiment, the starting isoprene composition partially hydrogenated using an eggshell Pd/d-A1203 catalyst, particularly if the reaction is free of water. In some embodiment, the starting isoprene composition partially hydrogenated using a Group VIII metal catalyst promoted by a metal from Group IB, VIB, VIIB, or zinc to reduce poisoning of the catalyst. Methods for converting isoprene into isoamylenes (mono-olefins) can be performed in continuous or batch mode using both homogeneous or heterogeneous catalysts. One embodiment of the method is illustrated in Scheme XIII.

[0647] In some embodiments, isoamylenes produce from partial hydrogenatin of isoprene undergos trimerization in liquid phase over ion exchange resins and zeolites.
See, GranGranollers, Ind. Eng. Chem. Res., 49 (8), pp 3561-3570 (2001).
Scheme XIII

Oxygenates (e.g. TAME) Fie ROH, H+

Catalyst Isoprene Acid Catalyst Diisoamylenes Isoamylenes

[0648] In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) partially hydrogenating the starting isoprene composition to a mono-olefin (e.g. 2-methylbut-l-ene, 3-methyl-but-l-ene and 2-methylbut-2-ene); (c) contacting the mono-olefin with a catalyst for dimerization of mono-olefins to form a dimate with another mono-olefin; and (d) hydrogenating the product of dimerization (dimate) to produce saturated hydrocarbon fuel constituents. In some preferred embodiments, the highly pure isoprene is a bioisoprene composition. In some embodoments, the other mono-olefin is an isoamylene derived from the highly pure isoprene starting composition. In some embodoments, the other mono-olefin is an olefin from another source such as propylene, butane or isobutene.
In some embodiements, the catalyst for dimerization is a resin catalyst (e.g., Amberlyst, Amberlyst 35, Amberlyst 15, Amberlyst XN1010, Amberlyst XE586). In some embodiements, the catalyst for dimerization is a catalytic material containing an acidic mesoporous molecular sieve, such as a mesoporous sieve embedded in a zeolite structure, ZSM-5, ferrierite, ZSM-22, or ZSM-23. In some embodiements, the catalyst for dimerization is the catalytic material is thermally stable at high temperatures (e.g., at least 900 C). In some embodiements, the catalyst for dimerization is a catalytic material containing an acidic mesoporous molecular sieve. In some embodiements, the catalyst for dimerization is a solid acidic catalyst (e.g., a solid phosphoric acid catalyst, acidic ion exchange resins). One embodiment of the method is illustrated in Scheme XIV.

Scheme XIV

H H2 2,3,4,4-tetramethylhexane 2,2,3,4-tetramethylhexane diisoamylenes 2,3,3,4-tetramethylhexane 3,3,5-trimethylheptane

[0649] In some embodiments, the method for producing a fuel constituent from isoprene comprises: (a) obtaining a commercially beneficial amount of any of the highly pure isoprene starting composition described herein; (b) partially hydrogenating the starting isoprene composition to an isoamylene (e.g. 2-methylbut-l-ene, 3-methyl-but-l-ene and 2-methylbut-2-ene); (c) contacting the isoamylene with an alcohol and a catalyst to form an oxygenate; and (c) producing producing the fuel constituent from the isoamylene oxygenate. In some preferred embodiments, the highly pure isoprene is a bioisoprene composition. In some embodiements, the isoamylene is contacted with ethanol and the fuel oxygenate formed is an ether such as TAME.

[0650] In some embodiments, any of the methods described herein further include purifying the starting isoprene composition before the chemical transformation. For example, the isoprene produced using the compositions and methods of the invention can be purified using standard techniques. Purification refers to a process through which isoprene is separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is obtained as a substantially pure liquid. Examples of purification methods include (i) distillation from a solution in a liquid extractant and (ii) chromatography.
As used herein, "purified isoprene" means isoprene that has been separated from one or more components that are present when the isoprene is produced. In some embodiments, the isoprene is at least about 20%, by weight, free from other components that are present when the isoprene is produced. In various embodiments, the isoprene is at least or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%, by weight, pure. Purity can be assayed by any appropriate method, e.g., by column chromatography, HPLC analysis, or GC-MS analysis. In some embodiments, bioisoprene is recovered from fermentation off-gas by initial adsorption to activated carbon, followed by desorption and condensation to give a liquid isoprene composition for chemical transformation to a fuel constituent.

Continuous Process for Producing Fuels

[0651] The invention also provides a continuous process for producing a fuel constituent from isoprene comprising: (a) continuously producing a commercially beneficial amount of highly pure isoprene; and (b) continuously transforming chemically at least a portion of the commercially beneficial amount of highly pure isoprene to a fuel constituent.
In a continuous process according to the invention, a stream of highly pure isoprene is produced continuously, for example, by a biological process (e.g. culturing cells producing isoprene) and is passed to a reactor for chemical transformation. In some embodiments, the isoprene from a fermentation off-gas is separated from water and other permanent gases (e.g., 02, N2 and C02) using extractive distillation, condensation, adsorption or membranes in a continuous purification unit, where the oxygen level is lowered to ppm range and other impurities of concern are removed;
the isoprene vapor is catalytically converted to dimers (C 10) and trimers (C
15) using a heterogeneous catalyst; the desired product is separated and the unreacted isoprene is returned to the reactor for further conversion. In some embodiments, the catalytic dimerization is monitored by measuring the levels of desirable C 10 hydrocarbons in the gas phase.

[0652] In some embodiments, a stream of biologically produced isoprene is passed through a chemical reactor (e.g., a reaction tube) maintained at a temperature of about 100 C to about 300 C, where the biologically produced isoprene undergoes dimerization reactions.
In one variation, the isoprene dimers are separated from the product stream and at least a portion of the unreacted isoprene is recycled back into the chemical reactor. In one embodiment, the unsaturated isoprene dimers produced is contacted with a hydrogenation catalyst and a hydrogen source in a second chemical reactor producing partially hydrogenated and/or fully hydrogenated products. The process can further comprise a step of recovering isoprene from the off-gas of a bio-reactor producing isoprene.

[0653] In some embodiments, a stream of bioisoprene containing co-produced hydrogen is first separated from the hydrogen gas, the bioisoprene stream is passed through a chemical reactor (e.g., a reaction tube) maintained at a temperature of about 100 C to about 300 C, where the biologically produced isoprene undergoes dimerization reactions. In one variation, the isoprene dimers are separated from the product stream and at least a portion of the unreacted isoprene is recycled back into the chemical reactor. In one embodiment, the unsaturated isoprene dimers produced is contacted with a hydrogenation catalyst and a hydrogen source in a second chemical reactor producing partially hydrogenated and/or fully hydrogenated products.
In one variation, the hydrogen stream isolated from the isoprene-hydrogen co-production is used in the hydrogenation step. The process can further comprise a step of recovering isoprene from the off-gas of a bio-reactor producing isoprene. The process can further comprise a step of purifying the hydrogen stream isolated from the isoprene-hydrogen co-production using purification methods known in the art such as cryogenic condensation and solid matrix adsorption.

Removal of Dienes and Polymers from Fuel Products

[0654] Fuel compositions often contain unsaturated compounds (olefins, diolefins and polyolefins) that can form gums, resins, polymers and other undesirable byproducts over time (for example, see Pereira and Pasa (2006) Fuel, 85, 1860-1865 and references therein). In general, as the degree of unsaturation increases of a given compound, the more likely that compound is to form such byproducts. Isoprene is a 1,3-diene that readily forms undesirable polymeric byproducts when present in fuel compositions. While there exist fuel additives (anti-oxidants, radical quenchers etc.) that can reduce the extent of byproduct formation, such byproducts can still form over time. Olefins can also contribute to the formation of ground-level ozone when released into the atmosphere upon evaporation from fuels, or as the result of incomplete combustion of olefin-containing fuels.

[0655] Accordingly, fuel compositions derived wholly or in part from isoprene should contain little to no free isoprene. A range of methods can be used to either remove isoprene from fuel compositions such as purification by distillation, reaction with alcohols to form ethers, or hydrogenation to convert isoprene to saturated derivatives. Alternately, isoprene can be treated with a dienophile such as malic anhydride producing inert adducts that do not contribute to the formation of undesirable byproducts.

[0656] Provided are fuel compositions comprising isoprene derivatives that are substantially free of isoprene. In some embodiements, the fuel composition contains less than 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10% or 15% isoprene. Also provided are methods for chemically transforming a bioisoprene composition to fuel compositions comprising isoprene derivatives that are substantially free of isoprene. In some embodiments, a substantial portion of the isoprene from the bioisoprene composition is converted to fuel constituents.
In some embodiments, a substantial portion of the isoprene from the bioisoprene composition is converted to intermediates that can be further converted to produce fuel constituents. In some embodiments, a substantial portion of the isoprene from the bioisoprene composition is converted to compounds other than isoprene. A "substantial portion" is 99.99%, 99.9%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%.

[0657] In some instances, isoprene and other conjugated dienes can form polymeric products with gum-like consistencies that can reduce the yields of desired products and/or deactivate catalysts. (See, e.g., R.C.C. Pereira and V.M.D. Pasa. Fuel 85 (2006) 1860-1865.) In some embodiments, methods are provided for determining the amount of conjugated diene present in a product mixture. D.F. Andrade et al. describe various methods for determining the amount of conjugated diene present (Fuel (2010), doi:10.106/j.fuel/2010.01.003), including the following:
1) UOP-326 method (maleic anhydride method), a semi-quantitative method in which the amount of maleic anhydride consumed via Diels-Alder reaction with the diene is measured; 2) polarography; 3) gas chromatography, in which the diene may be reacted with a derivatization agent, such as 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) or 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD); 4) HPLC; 5) supercritical fluid chromatography; 6) NMR; 7) UV
and near IR
spectroscopy; and 8) other spectroscopic methods, which may include first derivatizing the diene with p-nitrobenezenediazonium fluoroborate.

[0658] Method are provided herein for minimizing gum-formation and/or reducing the amount of gum that has been produced. In some embodiments, an anti-oxidant is used to minimize gum-formation. In other embodiments, polymeric by-products are recycled back to the process stream. In some embodiments, depolymerization of polymeric by-products is carried out via olefin metathesis. Olefin metathesis can be carried out using a molybdenum or tungsten catalyst, such as 2,6-diisopropylphenylimido neophylidene molybdenum bis(2-tertbutylphenoxide). (See US 5,446,102.)

[0659] In some embodiments, any of the methods described herein further include characterizing the products of these reactions and assessing the potential fuel value. For example, the products are characterized by standard methods known in the art, e.g. GC/MS, NMR, UV-Vis and IR spectroscopies, boiling temperature, density and other physical properties.
The products can be further characterized by dual carbon-isotopic fingerprinting (see, US
Patent Number 7,169,588). The potential fuel value of the products are assessed by parameters measuring fuel properties such as the energy density, heating value, water solubility, octane/cetane number, density, viscosity, surface tension, enthalpy of vaporization, vapor diffusivity, flash point, autoignition point, flammability limits, cloud point and chemical stability.

[0660] As with commercial petroleum fuels, BiolsoFuelTM products can be tested for their acidity, density, trace mineral content, benzene, total aromatics content, water content, and corrosivity. To assure that minor impurities in BiolsoFuelTM products are not adversely affecting their material properties, samples can also be tested for their corrosivity and compatibility with fuel systems by standard ASTM tests such as Karl Fischer for water and copper strip for corrosivity.

Fuel Compositions

[0661] The invention provides a fuel composition comprising a fuel constituent produced by any of the methods and processes described herein. In some embodiments, the fuel constituent comprises one or more of the hydrocarbons selected from the group consisting of saturated C10 and C15 hydrocarbons derived from isoprene and oxygenated derivatives of isoprene. The hydrocarbon BiolsoFuelTM products are expected to be completely compatible with petroleum gasoline and diesel. Blends of BiolsoFuelTM with commercial petroleum fuels are expected to have more desirable properties, as they do not contain acids, sulfur, aromatics, or other undesirable impurities.

[0662] In some preferred embodiments, the fuel constituent comprises derivatives of bioisoprene. It is anticipated that there will be a significant demand for fuels derived from bioisoprene which is made from renewable, non-petrochemical based resources.
It is believed that industrial customers and consumers would prefer to purchase fuel components derived from such environmentally friendly sources to those that are made with isoprene derived from a petrochemical process. It is further believed that customers would be willing to pay premium prices for such environmentally friendly products that are made with renewable resources. Fuel components derived from bioisoprene compositions described herein offer the benefit of being verifiable as to be derived from non-petrochemical based resources.

[0663] In some embodiments, the fuel constituent comprises one or more of the saturated cyclic C10 hydrocarbons such as 1,2-bis(isopropyl)cyclobutane, 1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1-ethyl-1,4-dimethylcyclohexane, 1-ethyl-l,3-dimethylcyclohexane, 1,5-dimethylcyclooctane and 1,4-dimethylcyclooctane. In some embodiments, the fuel constituent comprises one or more of the saturated cyclic C15 hydrocarbons such as 1,5,9-trimethylcyclododecane and 1,5,10-trimethylcyclododecane. In some embodiments, the fuel constituent comprises one or more of the saturated aliphatic C10 hydrocarbons such as 2,6-dimethyloctane, 2,7-dimethyloctane and 3,6-dimethyloctane. In some embodiments, the fuel constituent comprises one or more of the saturated aliphatic C15 hydrocarbons such as 2,6,10-trimethyldodecane, 2,7,10-trimethyldodecane and 3,7,10-trimethyldodecane. In some embodiments, the fuel constituent comprises 2-methylbutane. In some embodiments, the fuel constituent comprises one or more of the hydrocarbons selected from the group consisting of 1,2-bis(isopropyl)cyclobutane, 1,2-bis(isopropyl)cyclobutane, 1-methyl-4-isopropylcyclohexane, 1-methyl-3-isopropylcyclohexane, 1-ethyl-l,4-dimethylcyclohexane, 1-ethyl-l,3-dimethylcyclohexane, 1,5-dimethylcyclooctane, 1,4-dimethylcyclooctane, 1,5,9-trimethylcyclododecane, 1,5,10-trimethylcyclododecane, 2,6-dimethyloctane, 2,7-dimethyloctane, 3,6-dimethyloctane, 2,6, 1 0-trimethyldodecane, 2,7,10-trimethyldodecane, 3,7,10-trimethyldodecane and 2-methylbutane.

[0664] In some embodiments, the fuel constituent comprises one or more of the saturated hydroxylates such as C5 alcohols and diols (e.g. 3-methyl-butan-l-ol, 2-methyl-butan-l-ol and 2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol and 2-methyl-butan-2,3-diol), C-10 alcohols and diols (e.g. 3,7-dimethyloctan-l-ol, 2,7-dimethyloctan-l-ol, 2,7-dimethyloctan-2-ol and 2,7-dimethyloctan-2,7-diol) and cyclic C-10 alcohols (e.g. 2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-l-ol, 2-(1,4-dimethylcyclohexyl)ethanol and 4-ethyl-1,4-dimethylcyclohexanol). In some embodiments, the fuel constituent comprises one or more of the saturated ethers such as 1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane, 1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane. In some embodiments, the fuel constituent comprises one or more of the isoprene derived oxygenates selected from the group consisting of 3-methyl-butan-l-ol, 2-methyl-butan-l-ol, 2-methyl-butan-2-ol, 3-methyl-butan-1,3-diol, 2-methyl-butan-2,3-diol, 3,7-dimethyloctan-l-ol, 2,7-dimethyloctan-l-ol, 2,7-dimethyloctan-2-ol, 2,7-dimethyloctan-2,7-diol, 2-(4-methylcyclohexyl)propan-2-ol, 2-(4-methylcyclohexyl)propan-l-ol, 2-(1,4-dimethylcyclohexyl)ethanol, 4-ethyl-1,4-dimethylcyclohexanol, 1,3-diethoxy-3-methylbutane, 1-ethoxy-3-methylbutane, 1-methoxy-2,7-dimethyloctane and 3-methoxy-2,7-dimethyloctane.

[0665] In some embodiments, the fuel constituent comprises less than or about 0.5 g/L a product from a C5 hydrocarbon other than isoprene after undergoing the steps according to any of the methods and processes described herein. In one embodiment, he fuel constituent is substantially free of a product from a C5 hydrocarbon other than isoprene after undergoing the steps according to any of the methods and processes described herein. In some embodiments, the fuel constituent comprises less than or about 0.2, 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-l,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-l-yne) by weight compared to the total weight of all C5 hydrocarbons in the starting composition. In some embodiments, the fuel constituent comprises C5 hydrocarbons other than isoprene (such 1,3-cyclopentadiene, trans-l,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-l-butene, 3-methyl-l-butyne, pent-4-ene-l-yne, trans-pent-3-ene-l-yne, or cis-pent-3-ene-l-yne) at a concentration of less than 100, 10, 1, 0.1 or 0.01 ppm.

[0666] It is understood that components other than isoprene in the bioisoprene compositions described herein will be converted to different products after undergoing various processes described herein. The sensitivities of metal-based catalysts used in the methods/processes to the components other than isoprene are different depending on the nature and levels of these components. For the thermal dimerization process, a far wider range of compounds will be tolerated. In some cases, a component other than isoprene will react with isoprene to produce adducts, for example the Diels-Alder reactions of methacrolein and methyl vinyl ketone with isoprene to give 6-membered products. The unsaturated adducts can be further hydrogenated to saturated derivatives which are present in the fuel constituents and compositions, e.g. 1,4-dimethyl-l-(hydroxymethyl)cyclohexane and 1-(1-hydroxyethyl)-4-methylcyclohexane (see Scheme XV).

Scheme XV. Examples of trace impurities derived from BioisopreneTM

i) Hydrogenated Diels-Alder adducts OH
OH

1,4-dimethyl-l-(hydroxymethyl)cyclohexane 1-(1-hydroxyethyl)-4-methylcyclohexane ii) Alkanes 2-methylbutane 3-methylpentane iii) Alcohols OH OH
OH
OH
2-propanol 2-methyl- l -propanol 2-butanol 3-methyl- l -butanol iv) Esters O O
~O O
ethyl acetate 3-methyl-l-butyl acetate

[0667] In some embodiments, the fuel constituent comprises a product from a compound selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms after undergoing the steps according to any of the methods and processes described herein. In some embodiments, the fuel constituent comprises one or more products from one or more compounds selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), 2-heptanone, 6-methyl-5 -hepten-2 -one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine after undergoing the steps according to any of the methods and processes described herein. In some embodiments, the fuel constituent comprises one or more compounds selected from the group consisting of 1,4-dimethyl-1-(hydroxymethyl)cyclohexane, 1-(1-hydroxyethyl)-4-methylcyclohexane, methylbutane, 3-methylpentane, 2-propanol, 2-methyl-l-propanol, 2-butanol, 3-methyl-l-butanol, ethyl acetate and 3-methyl-l-butyl acetate. In some embodiments, the fuel constituent comprises one or more compounds selected from the group consisting of 1,4-dimethyl-l-(hydroxymethyl)cyclohexane, 1-(1-hydroxyethyl)-4-methylcyclohexane, 2-methylbutane, 3-methylpentane, 2-propanol, 2-methyl-l-propanol, 2-butanol, 3-methyl-l-butanol, ethyl acetate and 3-methyl-l-butyl acetate at levels greater than or about 10 ppm, 1 ppm, 100 ppb, 10 ppb or 1 ppb.

[0668] In some embodiments, the fuel constituent comprises one or more compounds selected from the group consisting of ethanol, acetone, methanol, acetaldehyde, methacrolein, methyl vinyl ketone, 2-methyl-2-vinyloxirane, cis- and trans-3-methyl-1,3-pentadiene, a C5 prenyl alcohol (such as 3-methyl-3-buten-l-ol or 3-methyl-2-buten-l-ol), 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-l-propanol, 3-methyl-l-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-l-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-buten-1-yl acetate, 3-methyl-2-buten-1-yl acetate, 3-hexen-l-ol, 3-hexen-1-yl acetate, limonene, geraniol (trans-3,7-dimethyl-2,6-octadien-l-ol), citronellol (3,7-dimethyl-6-octen-l-ol), (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine at levels greater than or about 10 ppm, 1 ppm, 100 ppb, 10 ppb or 1 ppb.

Carbon fingerprinting

[0669] BiolsoFuels derived from bioisoprene can be distinguished from fuels derived form petrochemical carbon on the basis of dual carbon-isotopic fingerprinting.
Additionally, the specific source of biosourced carbon (e.g. glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see, US Patent Number 7,169,588, which is herein incorporated by reference).

[0670] This method usefully distinguishes chemically-identical materials, and apportions carbon in products by source (and possibly year) of growth of the biospheric (plant) component.
The isotopes, 14C and 13C, bring complementary information to this problem.
The radiocarbon dating isotope (14C), with its nuclear half life of 5730 years, clearly allows one to apportion specimen carbon between fossil ("dead") and biospheric ("alive") feedstocks [Currie, L. A.
"Source Apportionment of Atmospheric Particles," Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc) (1992) 3 74]. The basic assumption in radiocarbon dating is that the constancy of 14C concentration in the atmosphere leads to the constancy of 14C
in living organisms. When dealing with an isolated sample, the age of a sample can be deduced approximately by the relationship t = (-5730/0.693)ln(A/Ao) (Equation 14) where t = age, 5730 years is the half-life of radiocarbon, and A and AO are the specific 14C
activity of the sample and of the modem standard, respectively [Hsieh, Y., Soil Sci. Soc. Am J., 56, 460, (1992)].
However, because of atmospheric nuclear testing since 1950 and the burning of fossil fuel since 1850, 14C has acquired a second, geochemical time characteristic. Its concentration in atmospheric CO2 - and hence in the living biosphere - approximately doubled at the peak of nuclear testing, in the mid-1960s. It has since been gradually returning to the steady-state cosmogenic (atmospheric) baseline isotope rate (14C /12C) of ca. 1.2x10-12, with an approximate relaxation "half-life" of 7-10 years. (This latter half-life must not be taken literally; rather, one must use the detailed atmospheric nuclear input/decay function to trace the variation of atmospheric and biospheric 14C since the onset of the nuclear age.) It is this latter biospheric 14C
time characteristic that holds out the promise of annual dating of recent biospheric carbon. 14C
can be measured by accelerator mass spectrometry (AMS), with results given in units of "fraction of modem carbon" (fM). fm is defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the 14Ci 12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fm - 1.1.

[0671] The stable carbon isotope ratio (13C /12C) provides a complementary route to source discrimination and apportionment. The 13C /12C ratio in a given biosourced material is a consequence of the 13C /12C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed and also reflects the precise metabolic pathway. Regional variations also occur.
Petroleum, C3 plants (the broadleaf), C4 plants (the grasses), and marine carbonates all show significant differences in 13C /12C and the corresponding d13C values.
Furthermore, lipid matter of C3 and C4 plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway. Within the precision of measurement, 13C shows large variations due to isotopic fractionation effects, the most significant of which for the instant invention is the photosynthetic mechanism. The major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation, i. e., the initial fixation of atmospheric CO2. Two large classes of vegetation are those that incorporate the "C3" (or Calvin-Benson) photosynthetic cycle and those that incorporate the "C4" (or Hatch-Slack) photosynthetic cycle. C3 plants, such as hardwoods and conifers, are dominant in the temperate climate zones. In C3 plants, the primary CO2 fixation or carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase and the first stable product is a 3-carbon compound. C4 plants, on the other hand, include such plants as tropical grasses, corn and sugar cane. In C4 plants, an additional carboxylation reaction involving another enzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction. The first stable carbon compound is a 4-carbon acid which is subsequently decarboxylated. The CO2 thus released is refixed by the C3 cycle.

[0672] Both C4 and C3 plants exhibit a range of 13C/12C isotopic ratios, but typical values are ca. -10 to -14 per mil (C4) and -21 to -26 per mil (C3) [Weber et al., J.
Agric. Food Chem., 45, 2942 (1997)]. Coal and petroleum fall generally in this latter range. The 13C
measurement scale was originally defined by a zero set by pee dee belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material. The "d 13C,,, values are in parts per thousand (per mil), abbreviated %o, and are calculated as follows (Equation 15):

(13,1!2 C)Swta . - (13 Ci12. C)sVT
6t3 M X 1()0% (Equation 15) Since the PDB reference material (RM) has been exhausted, a series of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the per mil deviations from PDB is d13C.
Measurements are made on CO2 by high precision stable ratio mass spectrometry (IRMS) on molecular ions of masses 44, 45 and 46.

[0673] For isoprene derived from extractive distillation of C5 streams from petroleum refineries, 813C is about -22%o to about -24%0. This range is typical for light, unsaturated hydrocarbons derived from petroleum, and products derived from petroleum-based isoprene typically contain isoprenic units with the same 813C. Bioisoprene produced by fermentation of corn-derived glucose (813C -10.73%o) with minimal amounts of other carbon-containing nutrients (e.g., yeast extract) produces isoprene which can be polymerized into polyisoprene with 813C -14.66%o to -14.85%0. Products produced from such bioisoprene are expected to have 813C values that are less negative than those derived from petroleum-based isoprene. For isoprene derived from the reaction of isobutylene with formaldehyde, 813C
values can be about -34.4%o because formaldehyde is often derived from feedstocks with much more negative 813C
values.

[0674] The fuels and fuel constituents of this invention which are made with isoprene from the cell cultures that utilize bio-renewable carbon sources can be identified as such by virtue of their d13C value and other fuel characteristics. In some embodiments, the fuel constituent derived from bioisoprene has 813C values of greater (less negative) than -22%0. In some embodiments, the fuel constituent derived from bioisoprene has 813C values of greater than -20, -18, -16, -14, -12, or -10 %o. In some embodiments, the fuel constituent derived from bioisoprene has a 813C
value which is within the range of -22 to -10, -21 to -12, or -20 to -14 %o.
In some embodiments, the fuel constituent derived from bioisoprene has a 813C value which is within the range of -34 to -24, -34 to -25, -33 to -25, -32 to -24, -32 to -25, -31 to -25, -30 to -29, -30.0 to -29.5, -29.5 to -28.5, or -29.0 to -28.5 %o.

[0675] In some embodiments, the fuel constituent derived from bioisoprene comprises radioactive carbon-14. In some embodiments, the 14C/12C ratio is greater than or about 1.0 x 10-12, 1.05 x 10-12, 1.1 x 10-12, 1.15 x 10-12 or 1.2 x 10-12. In some embodiments, the fuel constituent derived from bioisoprene has an fm value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1. In some embodiments, the fuel constituent derived from bioisoprene has an fm value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1 and 813C values of greater (less negative) than -22%o. In some embodiments, the fuel constituent derived from bioisoprene has an fm value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1 and a 813C value which is within the range of -22 to -10, -21 to -12, or -20 to -14 %o. In some embodiments, the fuel constituent derived from bioisoprene has an fm value of greater than or about 0.9, 0.95, 1.0, 1.05 or 1.1 and a 813C value which is within the range of -34 to -24, -34 to -25, -33 to -25, -32 to -24, -32 to -25, -31 to -25, -30 to -29, -30.0 to -29.5, -29.5 to -28.5, or -29.0 to -28.5 %o.

[0676] The bioisoprene derivatives and the associated BiolsoFuels, intermediates, and mixtures may be completely distinguished from their petrochemical derived counterparts on the basis of 14C (fm) and dual carbon-isotopic fingerprinting, indicating new compositions of matter.

[0677] In some embodiments, the fuel constituent of the invention has an energy density higher than that of ethanol. In some embodiments, the fuel constituent boosts the cetane number of a fuel, e.g., a petroleum-based fuel. In some embodiments, the fuel constituent reduces emission of petroleum based fuels. In some embodiments, the fuel composition has an octane number in the range between about 80 to about 120. In some embodiments, the fuel composition has a cetane number in the range between about 30 to about 130.

[0678] In some embodiments, a fuel composition of the invention comprises one or more dimethylcyclooctane compounds. In some embodiments, a fuel composition of the invention comprises one or more C 10 hydrocarbons such as substitued cyclohexanes. In some embodiments, a fuel composition of the invention comprises one or more isoprene derived oxygenates described herein. In some embodiments, any of the fuel compositions described herein further comprises a petroleum based fuel in the amount of from about I%
to about 95%
by weight or volume, based on the total weight or volume of the total fuel composition.

[0679] The invention further provides methods for making a fuel composition comprising obtaining a petroleum distillate and adding a fuel constituent of the invention.

[0680] The invention can be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting.

EXAMPLES
Example 1: Production of isoprene in E. coli expressing recombinant kudzu isoprene synthase 1. Construction of vectors for expression of the kudzu isoprene synthase in E.
coli

[0681] The protein sequence for the kudzu (Pueraria montana) isoprene synthase gene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprene synthase gene, optimized for E.
coli codon usage, was purchased from DNA2.0 (SEQ ID NO: 1). The isoprene synthase gene was removed from the supplied plasmid by restriction endonuclease digestion with BspLUI II /PstI, gel-purified, and ligated into pTrcHis2B (Invitrogen) that had been digested with NcoI/Pstl. The construct was designed such that the stop codon in the isoprene synthase gene 5' to the Pstl site.
As a result, when the construct was expressed the His-Tag is not attached to the isoprene synthase protein. The resulting plasmid, pTrcKudzu, was verified by sequencing (Figures 2 and 3; SEQ ID NO:2).

[0682] The isoprene synthase gene was also cloned into pET16b (Novagen). In this case, the isoprene synthase gene was inserted into pETl6b such that the recombinant isoprene synthase protein contained the N-terminal His tag. The isoprene synthase gene was amplified from pTrcKudzu by PCR using the primer set pET-His-Kudzu-2F: 5'-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:49) and pET-His-Kudzu-R: 5'-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:50).
These primers added an Ndel site at the 5'-end and a BamHl site at the 3' end of the gene respectively. The plasmid pTrcKudzu, described above, was used as template DNA, Herculase polymerase (Stratagene) was used according to manufacture's directions, and primers were added at a concentration of 10 pMols. The PCR was carried out in a total volume of 25 l. The PCR product was digested with NdellBamHI and cloned into pETl6b digested with the same enzymes. The ligation mix was transformed into E. coli Top 10 (Invitrogen) and the correct clone selected by sequencing. The resulting plasmid, in which the kudzu isoprene synthase gene was expressed from the T7 promoter, was designated pETNHisKudzu (Figures 4 and 5;
SEQ ID
NO:51).

[0683] The kudzu isoprene synthase gene was also cloned into the low copy number plasmid pCL 1920. Primers were used to amplify the kudzu isoprene synthase gene from pTrcKudzu described above. The forward primer added a HindIll site and an E. coli consensus RBS to the 5' end. The Pstl cloning site was already present in pTrcKudzu just 3' of the stop codon so the reverse primer was constructed such that the final PCR product includes the Pstl site. The sequences of the primers were: Hindlll-rbs-Kudzu F: 5'-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQ ID NO:51) and BamH 1-Kudzu R:

[0684] 5'- CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:50).
The PCR product was amplified using Herculase polymerase with primers at a concentration of pmol and with 1 ng of template DNA (pTrcKudzu). The amplification protocol included 30 cycles of (95 C for 1 minute, 60 C for 1 minute, 72 C for 2 minutes). The product was digested with HindIll and Pstl and ligated into pCL 1920 which had also been digested with HindIII and Pstl. The ligation mix was transformed into E. coli Top 10.
Several transformants were checked by sequencing. The resulting plasmid was designated pCL-lac-Kudzu (Figures 6 and 7; SEQ ID NO:4).

It Determination of isoprene production

[0685] For the shake flask cultures, one ml of a culture was transferred from shake flasks to 20 ml CTC headspace vials (Agilent vial cat# 5188 2753; cap cat# 5188 2759). The cap was screwed on tightly and the vials incubated at the equivalent temperature with shaking at 250 rpm. After 30 minutes the vials were removed from the incubator and analyzed as described below (see Table 1 for some experimental values from this assay).

[0686] In cases where isoprene production in fermentors was determined, samples were taken from the off-gas of the fermentor and analyzed directly as described below (see Table 2 for some experimental values from this assay).

[0687] The analysis was performed using an Agilent 6890 GC/MS system interfaced with a CTC Analytics (Switzerland) CombiPAL autosampler operating in headspace mode.
An Agilent HP-5MS GC/MS column (30 in x 0.25 mm; 0.25 m film thickness) was used for separation of analytes. The sampler was set up to inject 500 L of headspace gas. The GC/MS
method utilized helium as the carrier gas at a flow of 1 ml/min. The injection port was held at 250 C with a split ratio of 50:1. The oven temperature was held at 37 C for the 2 minute duration of the analysis.
The Agilent 5793N mass selective detector was run in single ion monitoring (SIM) mode on mlz 67. The detector was switched off from 1.4 to 1.7 minutes to allow the elution of permanent gases. Under these conditions isoprene (2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A calibration table was used to quantify the absolute amount of isoprene and was found to be linear from 1 g/L to 70,000 g/L. The limit of detection was estimated to be 50 to 100 ng/L using this method.

III. Production of isoprene in shake flasks containing E. coli cells expressing recombinant isoprene synthase

[0688] The vectors described above were introduced to E. coli strain BL21 (Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu and BL21/pETHisKudzu. The strains were spread for isolation onto LA (Luria agar) + carbenicillin (50 g/ml) and incubated overnight at 37 C. Single colonies were inoculated into 250 ml baffled shake flasks containing 20 ml Luria Bertani broth (LB) and carbenicillin (100 g/ml). Cultures were grown overnight at 20 C with shaking at 200 rpm. The OD600 of the overnight cultures were measured and the cultures were diluted into a 250 ml baffled shake flask containing 30 ml MagicMedia (Invitrogen) + carbenicillin (100 g/ml) to an OD600 - 0.05. The culture was incubated at 30 C
with shaking at 200 rpm. When the OD600 - 0.5 - 0.8, 400 M IPTG was added and the cells were incubated for a further 6 hours at 30 C with shaking at 200 rpm. At 0, 2, 4 and 6 hours after induction with IPTG, 1 ml aliquots of the cultures were collected, the OD600 was determined and the amount of isoprene produced was measured as described above. Results are shown in Figure 8.

IV. Production of Isoprene from BL21/ptrcKudzu in 14 liter fermentation

[0689] Large scale production of isoprene from E. coli containing the recombinant kudzu isoprene synthase gene was determined from a fed-batch culture. The recipe for the fermentation media (TM2) per liter of fermentation medium was as follows: K2HPO4 13.6 g, KH2PO4 13.6 g, MgS04 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, (NH4)2504 3.2 g, yeast extract 5 g, 1000X Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH2O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) and q.s. to volume. The final product was filter sterilized with 0.22 filter (only, do not autoclave). The recipe for 1000X Modified Trace Metal Solution was as follows:
Citric Acids *
H2O 40 g, MnS04 * H2O 30 g, NaCl 10 g, FeS04 * 7H20 1 g, CoC12 * 6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3BO3 100 mg, NaMoO4 * 2H20 100 mg. Each component was dissolved one at a time in diH2O, pH to 3.0 with HC1/NaOH, then q.s. to volume and filter sterilized with a 0.22 filter.

[0690] This experiment was carried out in 14 L bioreactor to monitor isoprene formation from glucose at the desired fermentation, pH 6.7 and temperature 34 C. An inoculum of E. coli strain BL21/ptrcKudzu taken from a frozen vial was prepared in soytone-yeast extract-glucose medium. After the inoculum grew to OD550 = 0.6, two 600 ml flasks were centrifuged and the contents resuspended in 70 ml supernatant to transfer the cell pellet (70 ml of OD 3.1 material) to the bioreactor. At various times after inoculation, samples were removed and the amount of isoprene produced was determined as described above. Results are shown in Figure 9.
Example 2: Production of isoprene in E. coli expressing recombinant poplar isoprene synthase

[0691] The protein sequence for the poplar (Populus alba x Populus tremula) isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786) was obtained from GenBank (CAC35696). A gene, codon optimized for E. coli, was purchased from DNA2.0 (p9796-poplar, Figures 30 and 31; SEQ ID NO: 14). The isoprene synthase gene was removed from the supplied plasmid by restriction endonuclease digestion with BspLUI II /Pstl, gel-purified, and ligated into pTrcHis2B that had been digested with NcoI/Pstl. The construct is cloned such that the stop codon in the insert is before the Pstl site, which results in a construct in which the His-Tag is not attached to the isoprene synthase protein. The resulting plasmid pTrcPoplar (Figures 32 and 33;
SEQ ID NO:15), was verified by sequencing.

Example 2B: Demonstration of isoprene synthase activity from several Populus isoprene synthases

[0692] The following isoprene synthases were examined; Populus alba (Accession number BAD98243; Figures 137A and B; SEQ ID NO:30), Populus nigra (Accession number CAL69918; Figures 137C and D; SEQ ID NO:31), Populus tremuloides (Accession number AAQ16588; Figure 137 E, F, and G; SEQ ID NOs:32-33), Populus trichocarpa (Accession number ACD70404; Figures 137H and I; SEQ ID NO:34), Populus alba x Populus tremula (Accession number CAJ29303; Figures 137J and K; SEQ ID NO:35), and MCM112-Kudzu.

[0693] pET24Kudzu (also referred to as MCM112) was constructed as follows: the kudzu isoprene synthase gene was subcloned into the pET24d vector (Novagen) from the pCR2.1 vector (Invitrogen). The kudzu IspS gene was amplified from pTrcKudzu template DNA using primers MCM50 5'-GATCATGCAT TCGCCCTTAG
GAGGTAAAAAAACATGTGTGCGACCTCTTC TCAATTTACT (SEQ ID NO:52); and MCM53 5'-CGGTCGACGGATCCCTGCAG TTAGACATAC ATCAGCTG (SEQ ID NO:50).
PCR reactions were carried out using Taq DNA Polymerase (Invitrogen), and the resulting PCR
product was cloned into pCR2.1-TOPO TA cloning vector (Invitrogen), and transformed into E.
coli Top 10 chemically competent cells (Invitrogen). Transformants were plated on L-agar containing carbenicillin (50 g/ml) and incubated overnight at 37 C. Five ml Luria Broth cultures containing carbenicillin 50 g/ml were inoculated with single transformants and grown overnight at 37 C. Five colonies were screened for the correct insert by sequencing of plasmid DNA isolated from 1 ml of liquid culture (Luria Broth) and purified using the QlAprep Spin Mini-prep Kit (Qiagen). The resulting plasmid, designated MCM93, contains the kudzu IspS
coding sequence in a pCR2.1 backbone (Figure 137L). The sequence of MCM93 (SEQ
ID
NO:36) is shown in Figures 137M and N.

[0694] The kudzu coding sequence was removed by restriction endonuclease digestion with Pcil and BamHl (Roche) and gel purified using the QlAquick Gel Extraction kit (Qiagen). The pET24d vector DNA was digested with Ncol and BamHI (Roche), treated with shrimp alkaline phosphatase (Roche), and purified using the QlAprep Spin Mini-prep Kit (Qiagen). The kudzu IspS fragment was ligated to the NcoI/BamHl digested pET24d using the Rapid DNA Ligation Kit (Roche) at a 5:1 fragment to vector ratio in a total volume of 20 pd. A
portion of the ligation mixture (5 l) was transformed into E. coli Top 10 chemically competent cells and plated on L
agar containing kanamycin (50 g/ml). The correct transformant was confirmed by sequencing and transformed into chemically competent BL21(),DE3)pLysS cells (Novagen). A
single colony was selected after overnight growth at 37 C on L agar containing kanamycin (50 g/ml).
A map of the resulting plasmid designated as pET24D-Kudzu is shown in Figure 1370. The sequence of pET24D-Kudzu (SEQ ID NO:37) is shown in Figures 137P and Q.

[0695] Escherichia coli optimized isoprene synthase genes cloned into the pET24a expression vector (Novagen) were purchased from DNA2.0 (Menlo Park, CA) for Populus tremuloides, Populus alba, Populus nigra and Populus trichocarpa. Genes were synthesized with the chloroplast transit peptide sequence removed, resulting in expression of mature proteins.

[0696] The construct for the Kudzu isoprene synthase was used as control in this example. The plasmids were transformed into the E. coli expression host BL21(DE3)plysS and transformants were grown in 0.6 ml TM3 medium. The recipe for TM3 medium is as follows:
K2HPO4 (13.6 g/l) KH2PO4 (13.6 g/1), MgS04 * 7H20 (2 g/L) Citric Acid Monohydrate (2 g/L) Ferric Ammonium Citrate (0.3 g/L) (NH4)2504 (3.2 g/L) yeast extract (0.2 g/L) 1 ml of 1000x Trace Elements solution, pH adjusted to 6.8 with ammonium hydroxide qs to volume with sterile DI
H2O and filter sterilized with a 0.22 micron filter. The recipe for 1000X
Trace Elements solution is as follows: Citric Acids * H2O (40 g/L), MnS04 * H2O (30 g/L), NaCl (10 g/L), FeS04 * 7 H2O (1 g/L), CoC12 * 6 H2O (1 g/L), ZnS04 * 7 H2O (1 g/L), CuS04 * 5 H2O (100 mg/L), H3BO3 (100 mg/L), NaMoO4 * 2 H2O (100 mg/L). Each component was dissolved one at a time in DI H20, pH adjusted to 3.0 with HCl/NaOH, qs to volume and filter sterilized with a 0.22 micron filter.

[0697] The cultures were induced with 400 M IPTG and growth was continued to OD600 of about 5. Aliquots of culture were transferred to a deep well glass plate and wells were sealed with aluminum plate sealer. The plate was incubated at 25 C for 30 minutes with shaking at 450 rpm. The reactions were heat inactivated by raising the temperature to 70 C
for 5 minutes.
Whole cell head space was measured by the GCMS method as described in Example 1, Part II.

[0698] Km values were obtained from cultures grown in similar manner but cells were harvested and lysed by a freeze/thaw lysozyme protocol. A volume of 400 L of culture was transferred into a new 96-well plate (Perkin Elmer, Catalog No. 6008290) and cells were harvested by centrifugation in a Beckman Coulter Allegra 6R centrifuge at 2500 x g. The pellet was resuspended in 200 mL of hypotonic buffer (5 mM MgCL2, 5 mM Tris HCI, 5 mM
DTT pH
8.0) and the plate was frozen at -80 C for a minimum time of 60 minutes. Cell lysate was prepared by thawing the plate and adding 32 mL of isoprene synthase DMAPP
assay buffer (57 mM Tris HCI, 19 mM MgC12, 74 mg/mL DNase I (Sigma Catalog No. DN-25), 2.63x105 U/mL
of ReadyLyse lysozyme solution (Epicentre Catalog No. R1802M), and 5 mg/mL of molecular biology grade BSA. The plate was incubated with shaking at 25 C for 30 minutes and then placed on ice. DMAPP and lysate were added at desired concentration in a sealed deep well glass block for the whole cell head space assay described above. The reactions were allowed to proceed for 1 hour and then terminated by the heat step described above and head space activity was measured also as described.

[0699] In an alternate approach, the activity of the enzymes was measured from cells cultured in 25 mL volume and induced similarly as described above. Cells were harvested by centrifugation and the pellets were lysed by French pressing in buffer consisting of 50% glycerol mixed 1:lwith 20 mM Tris/HCI pH 7.4, 20 mM MgC12, 200 mM KCI, 1 mM DTT. A
lysate volume of 25 L was assayed for isoprene synthase activity in 2 mL screw cap vials containing 75 L of assay buffer (66.6 mM Tris/HCI pH 8, 6.66 mM DMAPP, 43 mM, MgC12).
The reaction was incubated for 15 minutes at 30 C and was quenched by the addition of 100 L of 250 mM EDTA through the septum of the vial. Isoprene was measured by GC/MS as described in Example 1, Part II.

[0700] All methods for the determination of activity showed that the poplar enzyme derived from the pure bred poplars were several-fold higher than the Populus [alba x tremula]. Figures 138 and 139 showed these results for the whole cell head space assay and the DMAPP assay, respectively, and surprisingly indicate that enzymes from P. nigra, P.
tremuloides, P.
trichocarpa, and P. alba all had significantly higher activity than hybrid [P.
alba x P. tremula].

[0701] The DMAPP assay was performed as follows: a volume of 400 L of culture was transferred into a new 96-well plate (Perkin Elmer, Catalog No. 6008290) and cells were harvested by centrifugation in a Beckman Coulter Allegra 6R centrifuge at 2500 x g. The pellet was resuspended in 200 mL of hypotonic buffer (5 mM MgCL2, 5 mM Tris HCI, 5 mM
DTT pH
8.0) and the plate was frozen at -80 C for a minimum time of 60 minutes. Cell lysate was prepared by thawing the plate and adding 32 mL of isoprene synthase DMAPP
assay buffer (57 mM Tris HCI, 19 mM MgCl2, 74 mg/mL DNase I (Sigma Catalog No. DN-25), 2.63x105 U/mL
of ReadyLyse lysozyme solution (Epicentre Catalog No. R1802M), and 5 mg/mL of molecular biology grade BSA. The plate was incubated with shaking at 25 C for 30 minutes and then placed on ice. For isoprene production an 80 mL aliquot of lysate was transferred to a 96-deep well glass plate (Zinsser Catalog No. 3600600) and 20 mL of a 10 MM DMAPP
solution in 100 mM KHPO4, pH 8.2 (Cayman Chemical Catalog No. 63180) was added. The plate was sealed with an aluminum plate seal (Beckman Coultor Catalog No. 538619) and incubated with shaking at 30 C for 60 minutes. The enzymatic reactions were terminated by heating the glass block (70 C for 5 minutes). The cell head space of each well was quantitatively analyzed as described in Example 1, Part II.

[0702] Notably, P. alba, P. tremulo ides, P. trichocarpa had higher activity than the isoprene synthase from Kudzu. The enzyme from P. alba was expressed with the greatest activity of all enzymes tested. The higher activities observed with the cell lysate compared to the whole cell head space assay was likely due to limitations in DMAPP, the substrate for these enzymes, delivered by the endogenous deoxyxylulose 5-phosphate (DXP) pathway of the cell.

[0703] Km kinetic parameter was measured to be about 2 to 3 mM for all enzymes for which the value was determined.

Example 3: Production of isoprene in Panteoa citrea expressing recombinant kudzu isoprene synthase

[0704] The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 1 were electroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformants were selected on LA
containing carbenicillin (200 g/ml) or spectinomycin (50 g/ml) respectively.
Production of isoprene from shake flasks and determination of the amount of isoprene produced was performed as described in Example 1 for E. coli strains expressing recombinant kudzu isoprene synthase. Results are shown in Figure 10.

Example 4: Production of isoprene in Bacillus subtilis expressing recombinant kudzu isoprene synthase 1. Construction of a B. subtilis replicating plasmid for the expression of kudzu isoprene synthase

[0705] The kudzu isoprene synthase gene was expressed in Bacillus subtilis aprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid (pBS19 with a chloramphenicol resistance cassette) under control of the aprE promoter. The isoprene synthase gene, the aprE
promoter and the transcription terminator were amplified separately and fused using PCR. The construct was then cloned into pBS19 and transformed into B. subtilis.
a) Amplification of the aprE promoter

[0706] The aprE promoter was amplified from chromosomal DNA from Bacillus subtilis using the following primers:

CF 797 (+) Start aprE promoter Mfel 5'- GACATCAATTGCTCCATTTTCTTCTGCTATC (SEQ ID NO:53) CF 07-43 (-) Fuse aprE promoter to Kudzu ispS
5'- ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTA (SEQ ID NO:54) b) Amplification of the isoprene synthase gene

[0707] The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu (SEQ ID
NO:2). The gene had been codon optimized for E. coli and synthesized by DNA
2Ø The following primers were used:
CF 07-42 (+) Fuse the aprE promoter to kudzu isoprene synthase gene (GTG start codon) 5'- TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT (SEQ ID NO:55) CF 07-45 (-) Fuse the 3' end of kudzu isoprene synthase gene to the terminator 5'- CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC (SEQ ID NO:56) c) Amplification of the transcription terminator

[0708] The terminator from the alkaline serine protease of Bacillus amyliquefaciens was amplified from a previously sequenced plasmid pJHPms382 using the following primers:
CF 07-44 (+) Fuse the 3' end of kudzu isoprene synthase to the terminator 5'- GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGG (SEQ ID NO:57) CF 07-46 (-) End of B. amyliquefaciens terminator (BamHI) 5'- GACATGACGGATCCGATTACGAATGCCGTCTC (SEQ ID NO:58)

[0709] The kudzu fragment was fused to the terminator fragment using PCR with the following primers:
CF 07-42 (+) Fuse the aprE promoter to kudzu isoprene synthase gene (GTG start codon) 5'- TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT (SEQ ID NO:55) CF 07-46 (-) End of B. amyliquefaciens terminator (BamHI) 5'- GACATGACGGATCCGATTACGAATGCCGTCTC (SEQ ID NO:58)

[0710] The kudzu-terminator fragment was fused to the promoter fragment using PCR with the following primers:

CF 797 (+) Start aprE promoter Mfel 5'- GACATCAATTGCTCCATTTTCTTCTGCTATC (SEQ ID NO:53) CF 07-46 (-) End of B. amyliquefaciens terminator (BamHI) 5'- GACATGACGGATCCGATTACGAATGCCGTCTC (SEQ ID NO:58)

[0711] The fusion PCR fragment was purified using a Qiagen kit and digested with the restriction enzymes Mfel and BamHI. This digested DNA fragment was gel purified using a Qiagen kit and ligated to a vector known as pBS 19, which had been digested with EcoRl and BamHI and gel purified.

[0712] The ligation mix was transformed into E. coli Top 10 cells and colonies were selected on LA+50 carbenicillin plates. A total of six colonies were chosen and grown overnight in LB+50 carbenicillin and then plasmids were isolated using a Qiagen kit. The plasmids were digested with EcoRI and BamHI to check for inserts and three of the correct plasmids were sent in for sequencing with the following primers:
CF 149 (+) EcoRl start of aprE promoter 5'- GACATGAATTCCTCCATTTTCTTCTGC (SEQ ID NO:59) CF 847 (+) Sequence in pXX 049 (end of aprE promoter) 5'- AGGAGAGGGTAAAGAGTGAG (SEQ ID NO:60) CF 07-45 (-) Fuse the 3' end of kudzu isoprene synthase to the terminator 5'- CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC (SEQ ID NO:56) CF 07-48 (+) Sequencing primer for kudzu isoprene synthase 5'- CTTTTCCATCACCCACCTGAAG (SEQ ID NO:61) CF 07-49 (+) Sequencing in kudzu isoprene synthase 5'- GGCGAAATGGTCCAACAACAAAATTATC (SEQ ID NO:62)

[0713] The plasmid designated pBS Kudzu #2 (Figures 52 and 12; SEQ ID NO:5) was correct by sequencing and was transformed into BG 3594 comK, a Bacillus subtilis host strain.
Selection was done on LA + 5 chloramphenicol plates. A transformant was chosen and struck to single colonies on LA + 5 chloramphenicol, then grown in LB+5 chloramphenicol until it reached an OD600 of 1.5. It was stored frozen in a vial at -80 C in the presence of glycerol. The resulting strain was designated CF 443.

II. Production of isoprene in shake flasks containing B. subtilis cells expressing recombinant isoprene synthase

[0714] Overnight cultures were inoculated with a single colony of CF 443 from a LA +
Chloramphenicol (Cm, 25 g/ml). Cultures were grown in LB + Cm at 37 C with shaking at 200 rpm. These overnight cultures (1 ml) were used to inoculate 250 ml baffled shake flasks containing 25 ml Grants II media and chloramphenicol at a final concentration of 25 g/ml.
Grants II Media recipe was 10 g soytone, 3 ml 1M K2HPO4, 75 g glucose, 3.6 g urea, 100 ml l OX MOPS, q.s. to 1 L with H20, pH 7.2; lOX MOPS recipe was 83.72 g MOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K2SO4 solution, 10 ml 0.528M MgC12 solution, 29.22 g NaCl, 100 ml 100X micronutrients, q.s. to 1 L with H20; and 100X micronutrients recipe was 1.47 g CaC12*2H2O, 0.4 g FeS04*7H20, 0.1 g MnS04*H2O, 0.1 g ZnSO4*H20, 0.05 g CuC12*2H20, 0.1 g COC12*6H20, 0.1 g Na2MoO4*2H2O, q.s. to 1 L with H20. Shake flasks were incubated at 37 C and samples were taken at 18, 24, and 44 hours. At 18 hours the headspaces of CF443 and the control strain were sampled. This represented 18 hours of accumulation of isoprene. The amount of isoprene was determined by gas chromatography as described in Example 1.
Production of isoprene was enhanced significantly by expressing recombinant isoprene synthase (Figure 11).

III. Production of isoprene by CF443 in 14 L fermentation

[0715] Large scale production of isoprene from B. subtilis containing the recombinant kudzu isoprene synthase gene on a replication plasmid was determined from a fed-batch culture.
Bacillus strain CF 443, expressing a kudzu isoprene synthase gene, or control stain which does not express a kudzu isoprene synthase gene were cultivated by conventional fed-batch fermentation in a nutrient medium containing soy meal (Cargill), sodium and potassium phosphate, magnesium sulfate and a solution of citric acid, ferric chloride and manganese chloride. Prior to fermentation the media is macerated for 90 minutes using a mixture of enzymes including cellulases, hemicellulases and pectinases (see, W095/04134).
14-L batch fermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADM
Versadex greens or Danisco invert sugar) and 99% wt/wt oil (Western Family soy oil, where the 99%
wt/wt is the concentration of oil before it was added to the cell culture medium). Feed was started when glucose in the batch was non-detectable. The feed rate was ramped over several hours and was adjusted to add oil on an equal carbon basis. The pH was controlled at 6.8 -7.4 using 28% w/v ammonium hydroxide. In case of foaming, antifoam agent was added to the media.
The fermentation temperature was controlled at 37 C and the fermentation culture was agitated at 750 rpm. Various other parameters such as pH, DO%, airflow, and pressure were monitored throughout the entire process. The DO% is maintained above 20. Samples were taken over the time course of 36 hours and analyzed for cell growth (OD55o) and isoprene production. Results of these experiments are presented in Figures 53A and 53B.
IV. Integration of the kudzu isoprene synthase (ispS) in B. subtilis.

[0716] The kudzu isoprene synthase gene was cloned in an integrating plasmid (pJH101-cmpR) under the control of the aprE promoter. Under the conditions tested, no isoprene was detected.

Example 5: Production of isoprene in Trichoderma 1. Construction of vectors for expression of the kudzu isoprene synthase in Trichoderma reesei

[0717] The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized by DNA 2.0 (SEQ ID NO:6) (Figure 13). This plasmid served as the template for the following PCR
amplification reaction: 1 l plasmid template (20 ng/ul), 1 l Primer EL-945 (10 M) 5'-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:63), 1 l Primer EL-965 (10 M) 5'-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID NO:64), 1 gl dNTP (10mM), 5 gl l Ox PfuUltra II Fusion HS DNA Polymerase Buffer, 1 gl PfuUltra II
Fusion HS DNA Polymerase, 40 l water in a total reaction volume of 50 l. The forward primer contained an additional 4 nucleotides at the 5'-end that did not correspond to the Y. lipolytica codon-optimized kudzu isoprene synthase gene, but was required for cloning into the pENTR/D-TOPO vector. The reverse primer contained an additional 21 nucleotides at the 5'-end that did not correspond to the Y. lipolytica codon-optimized kudzu isoprene synthase gene, but were inserted for cloning into other vector backbones. Using the MJ Research PTC-Thermocycler, the PCR reaction was performed as follows: 95 C for 2 minutes (first cycle only), 95 C for 30 seconds, 55 C for 30 seconds, 72 C for 30 seconds (repeat for 27 cycles), 72 C for 1 minute after the last cycle. The PCR product was analyzed on a 1.2% E-gel to confirm successful amplification of the Y. lipolytica codon-optimized kudzu isoprene synthase gene.

[0718] The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kit following manufacturer's protocol: 1 gl PCR reaction, 1 gl Salt solution, 1 gl TOPO pENTR/D-TOPO vector and 3 l water in a total reaction volume of 6 l. The reaction was incubated at room temperature for 5 minutes. One microliter of TOPO reaction was transformed into TOP 10 chemically competent E. coli cells. The transformants were selected on LA + 50 g/ml kanamycin plates. Several colonies were picked and each was inoculated into a 5 ml tube containing LB + 50 g/ml kanamycin and the cultures grown overnight at 37 C
with shaking at 200 rpm. Plasmids were isolated from the overnight culture tubes using QlAprep Spin Miniprep Kit, following manufacturer's protocol. Several plasmids were sequenced to verify that the DNA
sequence was correct.

[0719] A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimized kudzu isoprene synthase gene, was used for Gateway Cloning into a custom-made pTrex3g vector.
Construction of pTrex3g is described in WO 2005/001036 A2. The reaction was performed following manufacturer's protocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen):
1 pl Y. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPO
donor vector, 1 l pTrex3g destination vector, 6 l TE buffer, pH 8.0 in a total reaction volume of 8 l. The reaction was incubated at room temperature for 1 hour and then 1 l proteinase K solution was added and the incubation continued at 37 C for 10 minutes. Then 1 l of reaction was transformed into TOP 10 chemically competent E. coli cells. The transformants were selected on LA + 50 g/ml carbenicillin plates. Several colonies were picked and each was inoculated into a ml tube containing LB + 50 1g/ml carbenicillin and the cultures were grown overnight at 37 C with shaking at 200 rpm. Plasmids were isolated from the overnight culture tubes using QlAprep Spin Miniprep Kit (Qiagen, Inc.), following manufacturer's protocol.
Several plasmids were sequenced to verify that the DNA sequence was correct.

[0720] Biolistic transformation of Y. lipolytica codon-optimized kudzu isoprene synthase pTrex3g plasmid (Figure 14) into a quad delete Trichoderma reesei strain was performed using the Biolistic PDS-1000/HE Particle Delivery System (see WO 2005/001036 A2).
Isolation of stable transformants and shake flask evaluation was performed using protocol listed in Example 11 of patent publication WO 2005/001036 A2.

II. Production of isoprene in recombinant strains of T. reesei

[0721] One ml of 15 and 36 hour old cultures of isoprene synthase transformants described above were transferred to head space vials. The vials were sealed and incubated for 5 hours at 30 C. Head space gas was measured and isoprene was identified by the method described in Example 1. Two of the transformants showed traces of isoprene. The amount of isoprene could be increased by a 14 hour incubation. The two positive samples showed isoprene at levels of about 0.5 g/L for the 14 hour incubation. The untransformed control showed no detectable levels of isoprene. This experiment shows that T. reesei is capable of producing isoprene from endogenous precursor when supplied with an exogenous isoprene synthase.

Example 6: Production of isoprene in Yarrowia 1. Construction of vectors for expression of the kudzu isoprene synthase in Yarrowia lipolytica.

[0722] The starting point for the construction of vectors for the expression of the kudzu isoprene synthase gene in Yarrowia lipolytica was the vector pSPZ I
(MAP29Spb). The complete sequence of this vector (SEQ ID NO:7) is shown in Figure 15.

[0723] The following fragments were amplified by PCR using chromosomal DNA of a Y
lipolytica strain GICC 120285 as the template: a promotorless form of the URA3 gene, a fragment of 18S ribosomal RNA gene, a transcription terminator of the Y.
lipolytica XPR2 gene and two DNA fragments containing the promoters of XPR2 and ICL1 genes. The following PCR
primers were used:

5'- GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACTGCAGGTGAC (SEQ
ID NO:65) 5'- GCAGGTGGGAAACTATGCACTCC (SEQ ID NO:66) 5'- CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG (SEQ ID NO:67) 5'- GGTGTCGACGTACGGTCGAGCTTATTGACC (SEQ ID NO:68) 5'- GGTGGGCCCGCATTTTGCCACCTACAAGCCAG (SEQ ID NO:69) 5'- GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG (SEQ ID NO:70) 5'- GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG (SEQ ID NO:71) 5'- GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG (SEQ ID NO:72) 5'- GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG (SEQ ID NO:73) 5'- GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG (SEQ ID NO:74) 5'- GCGGCCGCAGACTAAATTTATTTCAGTCTCC (SEQ ID NO:75)

[0724] For PCR amplification the PfuUltrall polymerase (Stratagene), supplier-provided buffer and dNTPs, 2.5 M primers and the indicated template DNA were used as per the manufacturer's instructions. The amplification was done using the following cycle: 95 C for 1 min; 34x (95 C for 30 sec; 55 C for 30 sec; 72 C for 3 min) and 10 min at 72 C followed by a 4 C incubation.

[0725] Synthetic DNA molecules encoding the kudzu isoprene synthase gene, codon-optimized for expression in Yarrowia, was obtained from DNA 2.0 (Figure 16;
SEQ ID NO:8).
Full detail of the construction scheme of the plasmids pYLA(KZ1) and pYLI(KZ1) carrying the synthetic kudzu isoprene synthase gene under control of XPR2 and ICL1 promoters respectively is presented in Figure 18. Control plasmids in which a mating factor gene (MAP29) is inserted in place of an isoprene synthase gene were also constructed (Figure 18E and 18F).

[0726] A similar cloning procedure can be used to express a poplar (Populus alba x Populus tremula) isoprene synthase gene. The sequence of the poplar isoprene is described in Miller B. et al. (2001) Planta 213, 483-487 and shown in Figure 17 (SEQ ID NO:9). A
construction scheme for the generation the plasmids pYLA(POP1) and pYLI(POP1) carrying synthetic poplar isoprene synthase gene under control of XPR2 and ICL1 promoters respectively is presented in Figure 18A and B.

II. Production of isoprene by recombinant strains of Y. lipolytica.

[0727] Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digested with SacII and used to transform the strain Y. lipolytica CLIB 122 by a standard lithium acetate/polyethylene glycol procedure to uridine prototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract, 2% peptone, 2% glucose) overnight, were collected by centrifugation (4000 rpm, 10 min), washed once with sterile water and suspended in 0.1 M
lithium acetate, pH
6Ø Two hundred l aliquots of the cell suspension were mixed with linearized plasmid DNA
solution (10-20 g), incubated for 10 minutes at room temperature and mixed with 1 ml of 50%
PEG 4000 in the same buffer. The suspensions were further incubated for 1 hour at room temperature followed by a 2 minutes heat shock at 42 C. Cells were then plated on SC his leu plates (0.67% yeast nitrogen base, 2% glucose, 100 mg/L each of leucine and histidine).
Transformants appeared after 3-4 days of incubation at 30 C.

[0728] Three isolates from the pYLA(KZ1) transformation, three isolates from the pYLI(KZ1) transformation, two isolates from the pYLA(MAP29) transformation and two isolates from the pYLI(MAP29) transformation were grown for 24 hours in YEP7 medium (I% yeast extract, 2%
peptone, pH 7.0) at 30 C with shaking. Cells from 10 ml of culture were collected by centrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 ml screw cap vials. The vials were incubated overnight at room temperature with gentle (60 rpm) shaking. Isoprene content in the headspace of these vials was analyzed by gas chromatography using mass-spectrometric detector as described in Example 1. All transformants obtained with pYLA(KZ 1) and pYLI(KZ1) produced readily detectable amounts of isoprene (0.5 g/L to 1 g/L, Figure 20). No isoprene was detected in the headspace of the control strains carrying phytase gene instead of an isoprene synthase gene.

Example 7: Production of isoprene in E. coli expressing kudzu isoprene synthase and idi, or dxs, or idi and dxs 1. Construction of vectors encoding kudzu isoprene synthase and idi, or dxs, or idi and dxs for the production of isoprene in E. coli i) Construction of pTrcKudzuKan

[0729] The bla gene of pTrcKudzu (described in Example 1) was replaced with the gene conferring kanamycin resistance. To remove the bla gene, pTrcKudzu was digested with BspHI, treated with Shrimp Alkaline Phosphatase (SAP), heat killed at 65 C, then end-filled with Klenow fragment and dNTPs. The 5 kbp large fragment was purified from an agarose gel and ligated to the kanr gene which had been PCR amplified from pCR-Blunt-II-TOPO
using primers MCM22 5'- GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:76) and MCM23 5'-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:77), digested with HindIII and Pvul, and end-filled. A transformant carrying a plasmid conferring kanamycin resistance (pTrcKudzuKan) was selected on LA containing kanamycin 50 g/ml.

ii) Construction of pTrcKudzu yIDI Kan

[0730] pTrcKudzuKan was digested with Pstl, treated with SAP, heat killed and gel purified.
It was ligated to a PCR product encoding idi from S. cerevisiae with a synthetic RBS. The primers for PCR were Nsil-YIDI 1 F 5'-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:78) and Pstl-YIDI 1 R 5'- CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:79); and the template was S. cerevisiae genomic DNA. The PCR product was digested with Nsil and Pstl and gel purified prior to ligation. The ligation mixture was transformed into chemically competent TOP 10 cells and selected on LA containing 50 g/ml kanamycin. Several transformants were isolated and sequenced and the resulting plasmid was called pTrcKudzu-yIDI(kan) (Figures 34 and 35; SEQ ID NO:16).

iii) Construction of pTrcKudzu DXS Kan

[0731] Plasmid pTrcKudzuKan was digested with Pstl, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding dxs from E. coli with a synthetic RBS. The primers for PCR were MCM13 5'-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATAC
CCG (SEQ ID NO:80) and MCM14 5'- CATGCTGCAGTTATGCCAGCCAGGCCTTGAT
(SEQ ID NO:81); and the template was E. coli genomic DNA. The PCR product was digested with Nsil and Pstl and gel purified prior to ligation. The resulting transformation reaction was transformed into TOP 10 cells and selected on LA with kanamycin 50 g/ml.
Several transformants were isolated and sequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (Figures 36 and 37; SEQ ID NO:17).

iv) Construction of pTrcKudzu-ylDl-dxs (kan)

[0732] pTrcKudzu-yIDI(kan) was digested with Pstl, treated with SAP, heat killed and gel purified. It was ligated to a PCR product encoding E. coli dxs with a synthetic RBS (primers MCM13 5'-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATAC
CCG (SEQ ID NO:80) and MCM14 5'- CATGCTGCAGTTATGCCAGCCAGGCCTTGAT
(SEQ ID NO:81); template TOP 10 cells) which had been digested with Nsil and Pstl and gel purified. The final plasmid was called pTrcKudzu-ylDl-dxs (kan) (Figures 21 and 22; SEQ ID
NO:10).

v) Construction of pCL PtrcKudzu

[0733] A fragment of DNA containing the promoter, structural gene and terminator from Example 1 above was digested from pTrcKudzu using Sspl and gel purified. It was ligated to pCL 1920 which had been digested with PvuII, treated with SAP and heat killed.
The resulting ligation mixture was transformed into TOP 10 cells and selected in LA
containing spectinomycin 50 g/ml. Several clones were isolated and sequenced and two were selected.
pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insert in opposite orientations (Figures 38-41; SEQ ID
NOs:18-19).

vi) Construction of pCL PtrcKudzu yIDI

[0734] The Nsil-Pstl digested, gel purified, IDI PCR amplicon from (ii) above was ligated into pCL PtrcKudzu which had been digested with Pstl, treated with SAP, and heat killed. The ligation mixture was transformed into TOP 10 cells and selected in LA
containing spectinomycin 50 g/ml. Several clones were isolated and sequenced and the resulting plasmid is called pCL
PtrcKudzu yIDI (Figures 42 and 43; SEQ ID NO:20).

vii) Construction of pCL PtrcKudzu DXS

[0735] The Nsil-Pstl digested, gel purified, DXS PCR amplicon from (iii) above was ligated into pCL PtrcKudzu (A3) which had been digested with Pstl, treated with SAP, and heat killed.
The ligation mixture was transformed into TOP 10 cells and selected in LA
containing spectinomycin 50 g/ml. Several clones were isolated and sequenced and the resulting plasmid is called pCL PtrcKudzu DXS (Figures 44 and 45; SEQ ID NO:21).

II. Measurement of isoprene in headspace from cultures expressing kudzu isoprene synthase, idi, and/or dxs at different copy numbers.

[0736] Cultures of E. coli BL21(XDE3) previously transformed with plasmids pTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B), pTrcKudzu-DXS kan (C), pTrcKudzu-ylDl-DXS kan (D) were grown in LB kanamycin 50 g/mL. Cultures of pCL PtrcKudzu (E), pCL
PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCL PtrcKudzu-DXS (G) were grown in LB
spectinomycin 50 g/mL. Cultures were induced with 400 M IPTG at time 0 (OD600 approximately 0.5) and samples taken for isoprene headspace measurement (see Example 1).
Results are shown in Figure 23A-23G.

[0737] Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21 by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS was grown overnight in LB
containing kanamycin (50 g/ml) at 20 C and used to inoculate shake flasks of TM3 (13.6 g K2P04, 13.6 g KH2PO4, 2.0 g MgS04*7H2O), 2.0 g citric acid monohydrate, 0.3 g ferric ammonium citrate, 3.2 g (NH4)2504, 0.2 g yeast extract, 1.0 ml 1000x Modified Trace Metal Solution, adjusted to pH 6.8 and q.s. to H20, and filter sterilized) containing 1% glucose. Flasks were incubated at 30 C until an OD600 of 0.8 was reached, and then induced with 400 M IPTG.

Samples were taken at various times after induction and the amount of isoprene in the head space was measured as described in Example 1. Results are shown in Figure 23H.

III. Production of isoprene from biomass in E. coli/pTrcKudzu yIDI DXS

[0738] The strain BL21 pTrcKudzuIDIDXS was tested for the ability to generate isoprene from three types of biomass; bagasse, corn stover and soft wood pulp with glucose as a control.
Hydrolysates of the biomass were prepared by enzymatic hydrolysis (Brown, L
and Torget, R., 1996, NREL standard assay method Lap-009 "Enzymatic Saccharification of Lignocellulosic Biomass") and used at a dilution based upon glucose equivalents. In this example, glucose equivalents were equal to I% glucose. A single colony from a plate freshly transformed cells of BL21 (DE3) pTrcKudzu yIDI DXS (kan) was used to inoculate 5 ml of LB plus kanamycin (50 g/ml). The culture was incubated overnight at 25 C with shaking. The following day the overnight culture was diluted to an OD600 of 0.05 in 25 ml of TM3 + 0.2% YE +
I% feedstock.
The feedstock was corn stover, bagasse, or softwood pulp. Glucose was used as a positive control and no glucose was used as a negative control. Cultures were incubated at 30 C with shaking at 180 rpm. The culture was monitored for OD600 and when it reached an OD600 of -0.8, cultures were analyzed at 1 and 3 hours for isoprene production as described in Example 1.
Cultures are not induced. All cultures containing added feedstock produce isoprene equivalent to those of the glucose positive control. Experiments were done in duplicate and are shown in Figure 46.

IV. Production of isoprene from invert sugar in E. coli/pTrcKudzuIDIDXS

[0739] A single colony from a plate freshly transformed cells of BL2l (XDE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5 mL of LB + kanamycin (50 g/ml). The culture was incubated overnight at 25 C with shaking. The following day the overnight culture was diluted to an OD600 of 0.05 in 25 ml of TM3 + 0.2% YE + I% feedstock. Feedstock was glucose, inverted glucose or corn stover. The invert sugar feedstock (Danisco Invert Sugar) was prepared by enzymatically treating sucrose syrup. AFEX corn stover was prepared as described below (Part V). The cells were grown at 30 C and the first sample was measured when the cultures reached an OD600 -0.8-1.0 (0 hour). The cultures were analyzed for growth as measured by OD600 and for isoprene production as in Example 1 at 0, 1 and 3 hours. Results are shown in Figure 47.

V. Preparation of hydrolysate from AFEX pretreated corn stover

[0740] AFEX pretreated corn stover was obtained from Michigan Biotechnology Institute. The pretreatment conditions were 60% moisture, 1:1 ammonia loading, and 90 C for 30 minutes, then air dried. The moisture content in the AFEX pretreated corn stover was 21.27%. The contents of glucan and xylan in the AFEX pretreated corn stover were 31.7% and 19.1 % (dry basis), respectively. The saccharification process was as follows; 20 g of AFEX pretreated corn stover was added into a 500 ml flask with 5 ml of 1 M sodium citrate buffer pH
4.8, 2.25 ml of Accellerase 1000, 0.1 ml of Grindamyl H121 (Danisco xylanase product from Aspergillus niger for bread-making industry), and 72.65 ml of DI water. The flask was put in an orbital shaker and incubated at 50 C for 96 hours. One sample was taken from the shaker and analyzed using HPLC. The hydrolysate contained 38.5 g/l of glucose, 21.8 g/l of xylose, and 10.3 g/l of oligomers of glucose and/or xylose.

VI. The effect of yeast extract on isoprene production in E. coli grown in fed-batch culture

[0741] Fermentation was performed at the 14-L scale as previously described with E. coli cells containing the pTrcKudzu yIDI DXS plasmid described above. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed at an exponential rate. The total amount of yeast extract delivered to the fermentor was varied between 70-830 g during the 40 hour fermentation. Optical density of the fermentation broth was measured at a wavelength of 550 nm. The final optical density within the fermentors was proportional to the amount of yeast extract added (Figure 48A). The isoprene level in the off-gas from the fermentor was determined as previously described. The isoprene titer increased over the course of the fermentation (Figure 48B). The amount of isoprene produced was linearly proportional to the amount of fed yeast extract (Figure 48C).

VII. Production of isoprene in 500 L fermentation of pTrcKudzu DXS yIDI

[0742] A 500 liter fermentation of E. coli cells with a kudzu isoprene synthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21 (XDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels of isoprene varied from 50 to 300 g/L over a time period of 15 hours. On the basis of the average isoprene concentrations, the average flow through the device and the extent of isoprene breakthrough, the amount of isoprene collected was calculated to be approximately 17 g.

VIII. Production of isoprene in 500 L fermentation of E. coli grown in fed-batch culture Medium Recipe (per liter fermentation medium):

[0743] K2HPO4 7.5 g, MgSO4 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000X Modified Trace Metal Solution 1 ml.
All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH
was adjusted to 7.0 with ammonium gas (NH3) and q.s. to volume. Glucose 10 g, thiamine * HCI
0.1 g, and antibiotic were added after sterilization and pH adjustment.
1000X Modified Trace Metal Solution:

[0744] Citric Acids * H2O 40 g, MnSO4 * H2O 30 g, NaCI 10 g, FeSO4 * 7H20 1 g, CoCl2 *
6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3BO3 100 mg, NaMoO4 * 2H20 100 mg.
Each component is dissolved one at a time in DI H20, pH to 3.0 with HCI/NaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.

[0745] Fermentation was performed in a 500-L bioreactor with E. coli cells containing the pTrcKudzu yIDI DXS plasmid. This experiment was carried out to monitor isoprene formation from glucose and yeast extract at the desired fermentation pH 7.0 and temperature 30 C. An inoculum of E. coli strain taken from a frozen vial was prepared in soytone-yeast extract-glucose medium. After the inoculum grew to OD 0.15, measured at 550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-L soytone-yeast extract-glucose medium. The 2.5-L
bioreactor was grown at 30 C to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.

[0746] Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose were fed at exponential rates. The total amount of glucose and yeast extract delivered to the bioreactor during the 50 hour fermentation was 181.2 kg and 17.6 kg, respectively. The optical density within the bioreactor over time is shown in Figure 49A. The isoprene level in the off-gas from the bioreactor was determined as previously described. The isoprene titer increased over the course of the fermentation (Figure 49B). The total amount of isoprene produced during the 50 hour fermentation was 55.1 g and the time course of production is shown in Figure 49C.
Example 8: Production of isoprene in E. coli expressing kudzu isoprene synthase and recombinant mevalonic acid pathway genes 1. Cloning the lower MVA pathway

[0747] The strategy for cloning the lower mevalonic pathway was as follows.
Four genes of the mevalonic acid biosynthesis pathway; mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase (MVD) and isopentenyl diphosphate isomerase genes were amplified by PCR from S. cerevisiae chromosomal DNA and cloned individually into the pCR BluntlI TOPO plasmid (Invitrogen). In some cases, the idi gene was amplified from E. coli chromosomal DNA. The primers were designed such that an E. coli consensus RBS
(AGGAGGT (SEQ ID NO:82) or AAGGAGG (SEQ ID NO:83)) was inserted at the 5' end, 8 bp upstream of the start codon and a Pstl site was added at the 3' end. The genes were then cloned one by one into the pTrcHis2B vector until the entire pathway was assembled.

[0748] Chromosomal DNA from S. cerevisiae S288C was obtained from ATCC (ATCC
204508D). The MVK gene was amplified from the chromosome of S. cerevisiae using primers MVKF (5'-AGGAGGTAAAAAAACATGTCATTACCGTTCTTAACTTCTGC, SEQ ID
NO:84) and MVK-Pstl-R (5'-ATGGCTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTG,SEQ
ID NO:85) using PfuTurbo as per manufacturer's instructions. The correct sized PCR product (1370 bp) was identified by electrophoresis through a 1.2% E-gel (Invitrogen) and cloned into pZeroBLUNT TOPO. The resulting plasmid was designated pMVK1. The plasmid pMVK1 was digested with SacI and Taq 1 restriction endonucleases and the fragment was gel purified and ligated into pTrcHis2B digested with SacI and BstBI. The resulting plasmid was named pTrcMVKl.

[0749] The second gene in the mevalonic acid biosynthesis pathway, PMK, was amplified by PCR using primers: Pstl-PMK1 R (5'-GAATTCGCCCTTCTGCAGCTACC, SEQ ID NO:86) and BsiHKA I-PMK1 F (5'-CGACTGGTGCACCCTTAAGGAGGAAAAAAACATGTCAG, SEQ ID NO:87). The PCR reaction was performed using Pfu Turbo polymerase (Stratagene) as per manufacturer's instructions. The correct sized product (1387 bp) was digested with Pstl and BsiHKI and ligated into pTrcMVKl digested with Pstl. The resulting plasmid was named pTrcKK. The MVD and the idi genes were cloned in the same manner. PCR was carried out using the primer pairs PstI-MVD 1 R (5'-GTGCTGGAATTCGCCCTTCTGCAGC, SEQ ID
NO:88) and Nsil-MVD 1 F (5'-GTAGATGCATGCAGAATTCGCCCTTAAGGAGG, SEQ ID
NO:89) to amplify the MVD gene and Pstl-YIDI 1 R (5'-CCTTCTGCAGGACGCGTTGTTATAGC, SEQ ID NO:79) and Nsil-YIDI 1 F (5'-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC, SEQ ID NO:78) to amplify the yIDI gene. In some cases the IPP isomerase gene, idi from E. coli was used. To amplify idi from E. coli chromosomal DNA, the following primer set was used: Pstl-CIDI 1 R
(5'-GTGTGATGGATATCTGCAGAATTCG, SEQ ID NO:90) and Nsil-CIDI 1 F (5'-CATCAATGCATCGCCCTTAGGAGGTAAAAAAACATG, SEQ ID NO:91). Template DNA
was chromosomal DNA isolated by standard methods from E. coli FM5 (WO 96/35796 and WO
2004/033646, which are each hereby incorporated by reference in their entireties, particularly with respect to isolation of nucleic acids). The final plasmids were named pKKDIy for the construct encoding the yeast idi gene or pKKDIc for the construct encoding the E. coli idi gene.
The plasmids were transformed into E. coli hosts BL21 for subsequent analysis.
In some cases the isoprene synthase from kudzu was cloned into pKKDIy yielding plasmid pKKDIyIS.

[0750] The lower MVA pathway was also cloned into pTrc containing a kanamycin antibiotic resistance marker. The plasmid pTrcKKDIy was digested with restriction endonucleases Apal and Pstl, the 5930 bp fragment was separated on a 1.2% agarose E-gel and purified using the Qiagen Gel Purification kit according to the manufacturer's instructions. The plasmid pTrcKudzuKan, described in Example 7, was digested with restriction endonucleases Apal and Pstl, and the 3338 bp fragment containing the vector was purified from a 1.2%
E-gel using the Qiagen Gel Purification kit. The 3338 bp vector fragment and the 5930 bp lower MVA pathway fragment were ligated using the Roche Quick Ligation kit. The ligation mix was transformed into E. coli TOP 10 cells and tranformants were grown at 37 C overnight with selection on LA
containing kanamycin (50 g/ml). The transformants were verified by restriction enzyme digestion and one was frozen as a stock. The plasmid was designated pTrcKanKKDIy.

II. Cloning a kudzu isoprene synthase gene into pTrcKanKKDIy

[0751] The kudzu isoprene synthase gene was amplified by PCR from pTrcKudzu, described in Example 1, using primers MCM50 5'-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGTGTGCGACCTCTTCTCAATTT
ACT (SEQ ID NO:52) and MCM53 5'-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:50). The resulting PCR fragment was cloned into pCR2.1 and transformed into E. coli TOP 10. This fragment contains the coding sequence for kudzu isoprene synthase and an upstream region containing a RBS from E. coli. Transformants were incubated overnight at 37 C with selection on LA
containing carbenicillin (50 g/ml). The correct insertion of the fragment was verified by sequencing and this strain was designated MCM93.

[0752] The plasmid from strain MCM93 was digested with restriction endonucleases Nsil and Pstl to liberate a 1724 bp insert containing the RBS and kudzu isoprene synthase. The 1724 bp fragment was separated on a 1.2% agarose E-gel and purified using the Qiagen Gel Purification kit according to the manufacturer's instructions. Plasmid pTrcKanKKDIy was digested with the restriction endonuclease Pstl, treated with SAP for 30 minutes at 37 C and purified using the Qiagen PCR cleanup kit. The plasmid and kudzu isoprene synthase encoding DNA
fragment were ligated using the Roche Quick Ligation kit. The ligation mix was transformed into E. coli TOP 10 cells and transformants were grown overnight at 37 C with selection on LA containing Kanamycin at 50 g/ml. The correct transformant was verified by restriction digestion and the plasmid was designated pTrcKKDyIkISKan (Figures 24 and 25; SEQ ID NO: 11).
This plasmid was transformed into BL21(XDE3) cells (Invitrogen).

III. Isoprene production from mevalonate in E. coli expressing the recombinant lower mevalonate pathway and isoprene synthase from kudzu.

[0753] Strain BL21/pTrcKKDyIkISKan was cultured in MOPS medium (Neidhardt et al., (1974) J. Bacteriology 119:736-747) adjusted to pH 7.1 and supplemented with 0.5% glucose and 0.5% mevalonic acid. A control culture was also set up using identical conditions but without the addition of 0.5% mevalonic acid. The culture was started from an overnight seed culture with a I% inoculum and induced with 500 M IPTG when the culture had reached an OD600 of 0.3 to 0.5. The cultures were grown at 30 C with shaking at 250 rpm.
The production of isoprene was analyzed 3 hours after induction by using the head space assay described in Example 1. Maximum production of isoprene was 6.67 x 10-4 mol/Lbroth/OD600/hr where Lbroth is the volume of broth and includes both the volume of the cell medium and the volume of the cells. The control culture not supplemented with mevalonic acid did not produce measurable isoprene.

IV. Cloning the upper MVA pathway

[0754] The upper mevalonate biosynthetic pathway, comprising two genes encoding three enzymatic activities, was cloned from Enterococcus faecalis. The mvaE gene encodes a protein with the enzymatic activities of both acetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the first and third proteins in the pathway, and the mvaS gene encodes second enzyme in the pathway, HMG-CoA synthase. The mvaE
gene was amplified from E. faecalis genomic DNA (ATCC 700802D-5) with an E. coli ribosome binding site and a spacer in front using the following primers:
CF 07-60 (+) Start of mvaE w/ RBS + ATG start codon SacI

5'- GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATTATTG (SEQ
ID NO:93) CF 07-62 (-) Fuse mvaE to mvaS with RBS in between 5'- TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTCTTAAATC
(SEQ ID NO:94)

[0755] The mvaS gene was amplified from E. faecalis genomic DNA (ATCC 700802D-5) with a RBS and spacer from E. coli in front using the following primers:
CF 07-61 (+) Fuse mvaE to mvaS with RBS in between 5' -GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGATTGATAAA
(SEQ ID NO:95) CF 07-102 (-) End of mvaS gene BgIII
5' -GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT (SEQ ID NO:96)

[0756] The PCR fragments were fused together with PCR using the following primers:
CF 07-60 (+) Start of mvaE w/ RBS + ATG start codon SacI
5' -GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATTATTG (SEQ
ID NO:93) CF 07-102 (-) End of mvaS gene BgIII
5'-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT (SEQ ID NO:96)

[0757] The fusion PCR fragment was purified using a Qiagen kit and digested with the restriction enzymes SacI and BgIII. This digested DNA fragment was gel purified using a Qiagen kit and ligated into the commercially available vector pTrcHis2A, which had been digested with SacI and BgIII and gel purified.

[0758] The ligation mix was transformed into E. coli Top 10 cells and colonies were selected on LA+50 g/ml carbenicillin plates. A total of six colonies were chosen and grown overnight in LB+50 g/ml carbenicillin and plasmids were isolated using a Qiagen kit. The plasmids were digested with SacI and BgIII to check for inserts and one correct plasmid was sequenced with the following primers:
CF 07-58 (+) Start of mvaE gene 5' - ATGAAAACAGTAGTTATTATTGATGC (SEQ ID NO:97) CF 07-59 (-) End of mvaE gene 5' - ATGTTATTGTTTTCTTAAATCATTTAAAATAGC (SEQ ID NO:98) CF 07-82 (+) Start of mvaS gene 5' - ATGACAATTGGGATTGATAAAATTAG (SEQ ID NO:99) CF 07-83 (-) End of mvaS gene 5' - TTAGTTTCGATAAGAACGAACGGT (SEQ ID NO:100) CF 07-86 (+) Sequence in mvaE
5' - GAAATAGCCCCATTAGAAGTATC (SEQ ID NO:101) CF 07-87 (+) Sequence in mvaE
5' - TTGCCAATCATATGATTGAAAATC (SEQ ID NO:102) CF 07-88 (+) Sequence in mvaE
5' - GCTATGCTTCATTAGATCCTTATCG (SEQ ID NO:103) CF 07-89 (+) Sequence mvaS
5' - GAAACCTACATCCAATCTTTTGCCC (SEQ ID NO:104)

[0759] The plasmid called pTrcHis2AUpperPathway#l was correct by sequencing and was transformed into the commercially available E. coli strain BL21. Selection was done on LA+ 50 g/ml carbenicillin. Two transformants were chosen and grown in LB+ 50 g/ml carbenicillin until they reached an OD600 of 1.5. Both strains were frozen in a vial at -80 C in the presence of glycerol. Strains were designated CF 449 for pTrcHis2AUpperPathway#l in BL21, isolate #1 and CF 450 for pTrcHis2AUpperPathway#l in BL21, isolate #2. Both clones were found to behave identically when analyzed.

V. Cloning of UpperMVA Pathway into pCL1920

[0760] The plasmid pTrcHis2AUpperPathway was digested with the restriction endonuclease Sspl to release a fragment containing pTrc-mvaE-mvaS-(His tag)-terminator. In this fragment, the his-tag was not translated. This blunt ended 4.5 kbp fragment was purified from a 1.2% E-gel using the Qiagen Gel Purification kit. A dephosphorylated, blunt ended 4.2 kbp fragment from pCL 1920 was prepared by digesting the vector with the restriction endonuclease PvuII, treating with SAP and gel purifying from a 1.2% E-gel using the Qiagen Gel Purification kit. The two fragments were ligated using the Roche Quick Ligation Kit and transformed into chemically competent cells. Transformants were selected on LA containing spectinomycin (50 g/ml). A correct colony was identified by screening for the presence of the insert by PCR. The plasmid was designated pCL PtrcUpperPathway (Figures 26 and 27A-27D; SEQ ID
NO: 12).

VI. Strains expressing the combined Upper and Lower Mevalonic Acid Pathways

[0761] To obtain a strain with a complete mevalonic acid pathway plus kudzu isoprene synthase, plasmids pTrcKKDylklSkan and pCLpTrcUpperPathway were both transformed into BL21(? DE3) competent cells (Invitrogen) and transformants were selected on LA
containing kanamycin (50 g/ml) and Spectinomycin (50 g/ml). The transformants were checked by plasmid prep to ensure that both plasmids were retained in the host. The strain was designated MCM127.

VII. Production of mevalonic acid from glucose in E. coli/pUpperpathway

[0762] Single colonies of the BL21/pTrcHis2A-mvaE/mvaS or FM5/p pTrcHis2A-mvaE/mvaS
are inoculated into LB + carbenicillin (100 g/ml) and are grown overnight at 37 C with shaking at 200 rpm. These cultures were diluted into 50 ml medium in 250 ml baffled flasks to an OD600 of 0.1. The medium was TM3 + 1 or 2% glucose + carbenicillin (100 g/ml) or TM3 +
I% glucose + hydrolyzed soy oil + carbenicillin (100 g/ml) or TM3 + biomass (prepared bagasse, corn stover or switchgrass). Cultures were grown at 30 C with shaking at 200 rpm for approximately 2-3 hours until an OD600 of 0.4 was reached. At this point the expression from the mvaE mvaS construct was induced by the addition of IPTG (400 M). Cultures were incubated for a further 20 or 40 hours with samples taken at 2 hour intervals to 6 hour post induction and then at 24, 36 and 48 hours as needed. Sampling was done by removing 1 ml of culture, measuring the OD600, pelleting the cells in a microfuge, removing the supernatant and analyzing it for mevalonic acid.

[0763] A 14 liter fermentation of E. coli cells with nucleic acids encoding Enterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase polypeptides produced 22 grams of mevalonic acid with TM3 medium and 2% glucose as the cell medium. A
shake flask of these cells produced 2-4 grams of mevalonic acid per liter with LB medium and I% glucose as the cell culture medium. The production of mevalonic acid in these strains indicated that the MVA pathway was functional in E. coli.

VIII. Production of isoprene from E. coli BL21 containing the upper and lower MVA pathway plus kudzu isoprene synthase.

[0764] The following strains were created by transforming in various combinations of plasmids containing the upper and lower MVA pathway and the kudzu isoprene synthase gene as described above and the plasmids containing the idi, dxs, and dxr and isoprene synthase genes described in Example 7. The host cells used were chemically competent BL21(?
DE3) and the transformations were done by standard methods. Transformants were selected on L agar containing kanamycin (50 g/ml) or kanamycin plus spectinomycin (both at a concentration of 50 g/ml). Plates were grown at 37 C. The resulting strains were designated as follows:

Grown on Kanamycin plus Spectinomycin (50 g/ml each) MCM 127 - pCL Upper MVA + pTrcKKDyIkIS (kan) in BL21(XDE3) MCM131 - pCL1920 + pTrcKKDyIkIS (kan) in BL21(ADE3) MCM 125 - pCL Upper MVA + pTrcHis2B (kan) in BL21(XDE3) Grown on Kanamycin (50 g/ml) MCM64 - pTrcKudzu yIDI DXS (kan) in BL21(A.DE3) MCM50 - pTrcKudzu (kan) in BL21(XDE3) MCM 123 - pTrcKudzu yIDI DXS DXR (kan) in BL21(? DE3)

[0765] The above strains were streaked from freezer stocks to LA + appropriate antibiotic and grown overnight at 37 C. A single colony from each plate was used to inoculate shake flasks (25 ml LB + the appropriate antibiotic). The flasks were incubated at 22 C
overnight with shaking at 200 rpm. The next morning the flasks were transferred to a 37 C
incubator and grown for a further 4.5 hours with shaking at 200 rpm. The 25 ml cultures were centrifuged to pellet the cells and the cells were resuspended in 5 ml LB + the appropriate antibiotic. The cultures were then diluted into 25 ml LB+1 % glucose + the appropriate antibiotic to an OD600 of 0.1. Two flasks for each strain were set up, one set for induction with IPTG
(800 M) the second set was not induced. The cultures were incubated at 37 C with shaking at 250 rpm. One set of the cultures were induced after 1.50 hours (immediately following sampling time point 1). At each sampling time point, the OD600 was measured and the amount of isoprene determined as described in Example 1. Results are presented in Table 3. The amount of isoprene made is presented as the amount at the peak production for the particular strain.

Table 3. Production of isoprene in E. coli strains Strain Isoprene ( g/liter/OD/hr) MCM50 23.8 MCM131 Trace ND: not detected Trace: peak present but not integratable.
IX. Analysis of mevalonic acid

[0766] Mevalonolactone (1.0 g, 7.7 mmol) (CAS# 503-48-0) was supplied from Sigma-Aldrich (WI, USA) as a syrup that was dissolved in water (7.7 mL) and was treated with potassium hydroxide (7.7 mmol) in order to generate the potassium salt of mevalonic acid. The conversion to mevalonic acid was confirmed by 1H NMR analysis. Samples for HPLC analysis were prepared by centrifugation at 14,000 rpm for 5 minutes to remove cells, followed by the addition of a 300 l aliquot of supernatant to 900 l of H20. Perchloric acid (36 l of a 70%
solution) was then added followed by mixing and cooling on ice for 5 minutes.
The samples were then centrifuged again (14,000 rpm for 5 min) and the supernatant transferred to HPLC.
Mevalonic acid standards (20, 10, 5, 1 and 0.5 g/L) were prepared in the same fashion. Analysis of mevalonic acid (20 L injection volume) was performed by HPLC using a BioRad Aminex 87-H+ column (300 mm by 7.0 mm) eluted with 5 mM sulfuric acid at 0.6 mL/min with refractive index (RI) detection. Under these conditions mevalonic acid eluted as the lactone form at 18.5 minutes.

X. Production of isoprene from E. coli BL21 containing the upper MVA pathway plus kudzu isoprene synthase

[0767] A 15-L scale fermentation of E. coli expressing mevalonic acid pathway polypeptides and Kudzu isoprene synthase was used to produce isoprene from cells in fed-batch culture. This experiment demonstrates that growing cells under glucose limiting conditions resulted in the production of 2.2 g/L of isoprene.

Medium Recipe (per liter fermentation medium):

[0768] The medium was generated using the following components per liter fermentation medium: K2HPO4 7.5 g, MgSO4 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000X modified trace metal solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH
was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine * HCl 0.1 g, and antibiotics were added after sterilization and pH
adjustment.

1000X Modified Trace Metal Solution:

[0769] The 1000X modified trace metal solution was generated using the following components: citric acids * H2O 40 g, MnS04 * H2O 30 g, NaCl 10 g, FeS04 * 7H20 1 g, CoC12 * 6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3B03 100 mg, and NaMoO4 *

100 mg. Each component was dissolved one at a time in diH2O, pH to 3.0 with HC1/NaOH, then q.s. to volume, and filter sterilized with a 0.22 micron filter.

[0770] Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pCL PtrcUpperPathway (Figure 26) and pTrcKKDyIkIS plasmids.
This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30 C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37 C. A single colony was inoculated into soytone-yeast extract-glucose medium. After the inoculum grew to OD 1.0 when measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

[0771] Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 54 hour fermentation was 3.7 kg. Induction was achieved by adding isopropyl-beta-D-l-thiogalactopyranoside (IPTG). The IPTG
concentration was brought to 25 M when the optical density at 550 nm (OD550) reached a value of 10. The IPTG concentration was raised to 50 M when OD550 reached 190. IPTG
concentration was raised to 100 M at 38 hours of fermentation. The OD550 profile within the bioreactor over time is shown in Figure 54. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 2.2 g/L (Figure 55). The total amount of isoprene produced during the 54 hour fermentation was 15.9 g, and the time course of production is shown in Figure 56.

XI. Isoprene fermentation from E. coli expressing genes from the mevalonic acid pathway and grown in fed-batch culture at the 15-L scale

[0772] A 15-L scale fermentation of E. coli expressing mevalonic acid pathway polypeptides and Kudzu isoprene synthase was used to produce isoprene from cells in fed-batch culture. This experiment demonstrates that growing cells under glucose limiting conditions resulted in the production of 3.0 g/L of isoprene.

Medium Recipe (per liter fermentation medium):

[0773] The medium was generated using the following components per liter fermentation medium: K2HP04 7.5 g, MgSO4 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000X Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH
was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine * HCl 0.1 g, and antibiotics were added after sterilization and pH
adjustment.

1000X Modified Trace Metal Solution:

[0774] The 1000X modified trace metal solution was generated using the following components: citric acids * H2O 40 g, MnS04 * H2O 30 g, NaCl 10 g, FeS04 * 7H20 1 g, CoC12 * 6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3BO3 100 mg, and NaMoO4 *

100 mg. Each component was dissolved one at a time in diH2O, pH to 3.0 with HC1/NaOH, then q.s. to volume, and filter sterilized with a 0.22 micron filter.

[0775] Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH
7.0 and temperature 30 C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37 C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

[0776] Glucose was fed at an exponential rate until cells reached the stationary phase. After this time, the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 59 hour fermentation was 2.2 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 25 M when the optical density at 550 nm (OD550) reached a value of 10. The IPTG concentration was raised to 50 M
when OD550 reached 190. The OD550 profile within the bioreactor over time is shown in Figure 93. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 3.0 g/L (Figure 94). The total amount of isoprene produced during the 59 hour fermentation was 22.8 g, and the time course of production is shown in Figure 95. The molar yield of utilized carbon that went into producing isoprene during fermentation was 2.2%. The weight percent yield of isoprene from glucose was 1.0%.

XII. Isoprene fermentation from E. coli expressing genes from the mevalonic acid pathway and grown in fed-batch culture at the 15-L scale

[0777] A 15-L scale fermentation of E. coli expressing mevalonic acid pathway polypeptides, Pueraria lobata isoprene synthase, and Kudzu isoprene synthase was used to produce isoprene from cells in fed-batch culture. This experiment demonstrates that growing cells under glucose limiting conditions resulted in the production of 3.3 g/L of isoprene.

i) Construction of pCLPtrcUpperPathwayHGS2

[0778] The gene encoding isoprene synthase from Pueraria lobata was PCR-amplified using primers Nsil-RBS-HGS F (CTTGATGCATCCTGCATTCGCCCTTAGGAGG, SEQ ID
NO:105) and pTrcR (CCAGGCAAATTCTGTTTTATCAG, SEQ ID NO:106), and pTrcKKDyIkIS as a template. The PCR product thus obtained was restriction-digested with Nsil and Pstl and gel-purified. The plasmid pCL PtrcUpperPathway was restriction-digested with Pstl and dephosphorylated using rAPid alkaline phosphatase (Roche) according to manufacturer's instructions.

[0779] These DNA fragments were ligated together and the ligation reaction was transformed into E. coli Top 10 chemcially competent cells (Invitrogen), plated on L agar containing spectinomycin (50 g/ml) and incubated overnight at 37 C. Plasmid DNA was prepared from 6 clones using the Qiaquick Spin Mini-prep kit. The plasmid DNA was digested with restriction enzymes EcoRV and Mlul to identify a clone in which the insert had the right orientation (i.e., the gene oriented in the same way as the pTrc promoter).

[0780] The resulting correct plasmid was designated pCLPtrcUpperPathwayHGS2.
This plasmid was assayed using the headspace assay described herein and found to produce isoprene in E. coli Top 10, thus validating the functionality of the gene. The plasmid was transformed into BL21(LDE3) containing pTrcKKDyIkIS to yield the strain BL21/pCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS. This strain has an extra copy of the isoprene synthase compared to the BL21/pCL PtrcUpperMVA and pTrc KKDyIkIS strain (Example 8, part XI). This strain also had increased expression and activity of HMGS compared to the BL21/pCL
PtrcUpperMVA and pTrc KKDyIkIS strain used in Example 8, part XI.

ii) Isoprene fermentation from E. coli expressing pCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS
and grown in fed-batch culture at the 15-L scale Medium Recipe (per liter fermentation medium):

[0781] The medium was generated using the following components per liter fermentation medium: K2HPO4 7.5 g, MgSO4 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000X modified trace metal solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH
was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine * HCl 0.1 g, and antibiotics were added after sterilization and pH
adjustment.

1000X Modified Trace Metal Solution:

[0782] The 1000X modified trace metal solution was generated using the following components: citric acids * H2O 40 g, MnS04 * H2O 30 g, NaCl 10 g, FeS04 * 7H20 1 g, CoC12 * 6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3BO3 100 mg, and NaMoO4 *

100 mg. Each component is dissolved one at a time in Di H20, pH to 3.0 with HC1/NaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.

[0783] Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pCLPtrcUpperPathwayHGS2 and pTrc KKDyIkIS plasmids. This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30 C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37 C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0 measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

[0784] Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 58 hour fermentation was 2.1 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 25 M when the optical density at 550 nm (OD550) reached a value of 9. The IPTG concentration was raised to 50 M
when OD550 reached 170. The OD550 profile within the bioreactor over time is shown in Figure 104. The isoprene level in the off gas from the bioreactor was determined as described herein.
The isoprene titer increased over the course of the fermentation to a final value of 3.3 g/L
(Figure 105). The total amount of isoprene produced during the 58 hour fermentation was 24.5 g and the time course of production is shown in Figure 106. The molar yield of utilized carbon that went into producing isoprene during fermentation was 2.5%. The weight percent yield of isoprene from glucose was 1.2%. Analysis showed that the activity of the isoprene synthase was increased by approximately 3-4 times that compared to BL21 expressing CL
PtrcUpperMVA
and pTrc KKDyIkIS plasmids (data not shown).

XIII. Chromosomal Integration of the Lower Mevalonate Pathway in E. coli.

[0785] A synthetic operon containing mevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, and the IPP isomerase was integrated into the chromosome of E. coli. If desired, expression may be altered by integrating different promoters 5' of the operon.

[0786] Table 4 lists primers used for this experiment.
Table 4. Primers MCM78 attTn7 up rev for gcatgctcgagcggccgcTTTTAATCAAACATCCTGCCAA
rote ration construct CTC (SEQ ID NO:107) MCM79 attTn7 down rev for gatcgaagggcgatcgTGTCACAGTCTGGCGAAACCG
rote ration construct (SEQ ID NO:108) MCM88 attTn7 up forw for ctgaattctgcagatatcTGTTTTTCCACTCTTCGTTCACTT
integration construct T (SEQ ID NO:109) MCM89 attTn7 down forw for tctagagggcccAAGAAAAATGCCCCGCTTACG (SEQ
integration construct ID NO:110) MCM 104 GI1.2 promoter - Gatcgcggccgcgcccttgacgatgccacatcctgagcaaataattcaaccac MVK taattgtgagcggataacacaaggaggaaacagctatgtcattaccgttcttaact tc SEQ ID NO:111) MCM 105 aspA terminator -Gatcgggccccaagaaaaaaggcacgtcatctgacgtgccttttttatttgtaga IDI c c ttttata cattcta (SEQ ID NO: 112) MCM120 Forward of attTn7:
aaagtagccgaagatgacggtttgtcacatggagttggcaggatgtttgattaaa attTn7 homology, GB agcAATTAACCCTCACTAAAGGGCGG (SEQ ID

marker homology NO:113) MCM127 Rev complement of AGAGTGTTCACCAAAAATAATAACCTTTCCCGG
1.2 GI: GB marker TGCAgaagttaagaacggtaatgacatagctgtttcctccttgtgttatccgct homology(extra long), cacaattagtggttgaattatttgctcaggatgtggcatcgtcaagggcTAAT
promoter, RBS, ATG ACGACTCACTATAGGGCTCG (SEQ ID NO: 114) i) Target vector construction

[0787] The attTn7 site was selected for integration. Regions of homology upstream (attTn7 up) (primers MCM78 and MCM79) and downstream (attTn7 down) (primers MCM88 and MCM89) were amplified by PCR from MG1655 cells. A 50 L reaction with 1 L 10 M
primers, 3 L ddH2O, 45 L Invitrogen Platinum PCR Supermix High Fidelity, and a scraped colony of MG1655 was denatured for 2:00 at 94 C, cycled 25 times (2:00 at 94 C, 0:30 at 50 C, and 1:00 at 68 C), extended for 7:00 at 72 C, and cooled to 4 C. This resulting DNA was cloned into pCR2.1 (Invitrogen) according to the manufacturer's instructions, resulting in plasmids MCM278 (attTn7 up) and MCM252 (attTn7 down). The 832bp Apal-Pvul fragment digested and gel purified from MCM252 was cloned into Apal-Pvul digested and gel purified plasmid pR6K, creating plasmid MCM276. The 825bp Pstl-Notl fragment digested and gel purified from MCM278 was cloned into Pstl-Notl digested and gel purified MCM276, creating plasmid MCM281.

ii) Cloning of lower pathway and promoter

[0788] MVK-PMK-MVD-IDI genes were amplified from pTrcKKDyIkIS with primers MCM 104 and MCM 105 using Roche Expand Long PCR System according to the manufacturer's instructions. This product was digested with Notl and Apal and cloned into MCM281 which had been digested with Notl and Apal and gel purified. Primers MCM 120 and MCM127 were used to amplify CMR cassette from the GeneBridges FRT-gb2-Cm-FRT
template DNA using Stratagene Pfu Ultra II. A PCR program of denaturing at 95 C for 4:00, 5 cycles of 95 C for 0:20, 55 C for 0:20, 72 C for 2:00, 25 cycles of 95 C
for 0:20, 58 C for 0:20, 72 C for 2:00, 72 C for 10:00, and then cooling to 4 C was used with four 50 L PCR
reactions containing 1 L - 10ng/ L template, 1 L each primer, 1.25 L l 0mM
dNTPs, 5 L l Ox buffer, 1 L enzyme, and 39.75 L ddH2O. Reactions were pooled, purified on a Qiagen PCR
cleanup column, and used to electroporate water-washed Pirl cells containing plasmid MCM296. Electroporation was carried out in 2mM cuvettes at 2.5V and 200 ohms.

Electroporation reactions were recovered in LB for 3hr at 30 C. Transformant MCM330 was selected on LA with CMP5, Kan50 (Figures 107 and 108A-108C; SEQ ID NO:25).

iii) Integration into E. coli chromosome

[0789] Miniprepped DNA (Qiaquick Spin kit) from MCM330 was digested with SnaBI
and used to electroporate BL21(DE3) (Novagen) or MG 1655 containing GeneBridges plasmid pRedET Carb. Cells were grown at 30 C to -OD1 then induced with 0.4% L-arabinose at 37 C
for 1.5 hours. These cells were washed three times in 4 C ddH2O before electroporation with 2 L of DNA. Integrants were selected on L agar with containing chloramphenicol (5 g/ml) and subsequently confirmed to not grow on L agar + Kanamycin (50 g/ml). BL21 integrant MCM331 and MG1655 integrant MCM333 were frozen.

iv) Construction of pET24D-Kudzu encoding Kudzu isoprene synthase

[0790] The kudzu isoprene synthase gene was subcloned into the pET24d vector (Novagen) from the pCR2.1 vector (Invitrogen). In particular, the kudzu isoprene synthase gene was amplified from the pTrcKudzu template DNA using primers MCM50 5'-GATCATGCAT
TCGCCCTTAG GAGGTAAAAA AACATGTGTG CGACCTCTTC TCAATTTACT (SEQ ID
NO:52) and MCM53 5'-CGGTCGACGG ATCCCTGCAG TTAGACATAC ATCAGCTG
(SEQ ID NO:50). PCR reactions were carried out using Taq DNA Polymerase (Invitrogen), and the resulting PCR product was cloned into pCR2.1-TOPO TA cloning vector (Invitrogen), and transformed into E. coli Top 10 chemically competent cells (Invitrogen).
Transformants were plated on L agar containing carbenicillin (50 g/ml) and incubated overnight at 37 C. Five ml Luria Broth cultures containing carbenicillin 50 g/ml were inoculated with single transformants and grown overnight at 37 C. Five colonies were screened for the correct insert by sequencing of plasmid DNA isolated from 1 ml of liquid culture (Luria Broth) and purified using the QlAprep Spin Mini-prep Kit (Qiagen). The resulting plasmid, designated MCM93, contains the kudzu isoprene synthase coding sequence in a pCR2.1 backbone.

[0791] The kudzu coding sequence was removed by restriction endonuclease digestion with Pcil and BamHl (Roche) and gel purified using the QlAquick Gel Extraction kit (Qiagen). The pET24d vector DNA was digested with Ncol and BamHI (Roche), treated with shrimp alkaline phosphatase (Roche), and purified using the QlAprep Spin Mini-prep Kit (Qiagen). The kudzu isoprene synthase fragment was ligated to the NcoI/BamHl digested pET24d using the Rapid DNA Ligation Kit (Roche) at a 5:1 fragment to vector ratio in a total volume of 20 l. A portion of the ligation mixture (5 l) was transformed into E. coli Top 10 chemically competent cells and plated on L agar containing kanamycin (50 g/ml). The correct transformant was confirmed by sequencing and transformed into chemically competent BL21(? DE3)pLysS cells (Novagen).
A single colony was selected after overnight growth at 37 C on L agar containing kanamycin (50 g/ml). A map of the resulting plasmid designated as pET24D-Kudzu is shown in Figure 109.
The sequence of pET24D-Kudzu (SEQ ID NO:26) is shown in Figures 11 OA and 1 l OB.
Isoprene synthase activity was confirmed using a headspace assay.
v) Production strains

[0792] Strains MCM331 and MCM333 were cotransformed with plasmids pCLPtrcupperpathway and either pTrcKudzu or pETKudzu, resulting in the strains shown in Table 5.

Table 5. Production Strains Background Integrated Upper MVA Isoprene Production Lower plasmid synthase Stain plasmid BL21(DE3) MCM331 pCLPtrcUpper pTrcKudzu MCM343 Pathway BL21(DE3) MCM331 pCLPtrcUpper pET24D- MCM335 Pathway Kudzu MG1655 MCM333 pCLPtrcUpper pTrcKudzu MCM345 Pathway vi) Isoprene fermentation from E. coli expressing genes from the mevalonic acid pathway and grown in fed-batch culture at the 15-L scale.

Medium Recipe (per liter fermentation medium):

[0793] The medium was generated using the following components per liter fermentation medium: K2HPO4 7.5 g, Mg504 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000X modified trace metal solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH
was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine * HCl 0.1 g, and antibiotics were added after sterilization and pH
adjustment.

1000X Modified Trace Metal Solution:

[0794] The 1000X modified trace metal solution was generated using the following components: citric acids * H2O 40 g, MnS04 * H2O 30 g, NaCl 10 g, FeS04 * 7H20 1 g, CoC12 * 6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3B03 100 mg, and NaMoO4 *

100 mg. Each component is dissolved one at a time in Di H20, pH to 3.0 with HC1/NaOH, then q.s. to volume and filter sterilized with a 0.22 micron filter.

[0795] Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the gil.2 integrated lower MVA pathway described above and the pCL
PtrcUpperMVA and pTrcKudzu plasmids. This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30 C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37 C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

[0796] Glucose was fed at an exponential rate until cells reached the stationary phase. After this time, the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 57 hour fermentation was 3.9 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 100 M when the carbon dioxide evolution rate reached 100 mmol/L/hr. The OD550 profile within the bioreactor over time is shown in Figure 11 IA. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 1.6 g/L (Figure 111 B). The specific productivity of isoprene over the course of the fermentation is shown in Figure 111 C and peaked at 1.2 mg/OD/hr. The total amount of isoprene produced during the 57 hour fermentation was 16.2 g. The molar yield of utilized carbon that went into producing isoprene during fermentation was 0.9%. The weight percent yield of isoprene from glucose was 0.4%.

XIV. Production of isoprene from E. coli BL21 containing the kudzu isoprene synthase using glycerol as a carbon source

[0797] A 15-L scale fermentation of E. coli expressing Kudzu isoprene synthase was used to produce isoprene from cells fed glycerol in fed-batch culture. This experiment demonstrates that growing cells in the presence of glycerol (without glucose) resulted in the production of 2.2 mg/L of isoprene.

Medium Recipe (per liter fermentation medium):

[0798] The medium was generated using the following components per liter fermentation medium: K2HPO4 7.5 g, MgSO4 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, and 1000X modified trace metal solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glycerol 5.1 g, thiamine * HCl 0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000X Modified Trace Metal Solution:

[0799] The medium was generated using the following components per liter fermentation medium: citric acids * H2O 40 g, MnS04 * H2O 30 g, NaCl 10 g, FeS04 * 7H20 1 g, CoC12 6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3B03 100 mg, and NaMoO4 *

mg. Each component was dissolved one at a time in diH2O, pH to 3.0 with HC1/NaOH, then q.s.
to volume and filter sterilized with a 0.22 micron filter.

[0800] Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pTrcKudzu plasmid. This experiment was carried out to monitor isoprene formation from glycerol at the desired fermentation pH 7.0 and temperature 35 C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LA broth agar plate (with antibiotics) and incubated at 37 C. A single colony was inoculated into soytone-yeast extract-glucose medium and grown at 35 C. After the inoculum grew to OD 1.0, measured at 550 nm, 600 mL was used to inoculate a 7.5-L bioreactor.

[0801] Glycerol was fed at an exponential rate until cells reached an optical density at 550 nm (OD550) of 153. The total amount of glycerol delivered to the bioreactor during the 36 hour fermentation was 1.7 kg. Other than the glucose in the inoculum, no glucose was added to the bioreactor. Induction was achieved by adding IPTG. The IPTG concentration was brought to 20 M when the OD550 reached a value of 50. The OD550 profile within the bioreactor over time is shown in Figure 57. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 2.2 mg/L (Figure 58). The total amount of isoprene produced during the 54 hour fermentation was 20.9 mg, and the time course of production is shown in Figure 59.

XV. Isoprene fermentation from E. coli expressing genes from the mevalonic acid pathway and grown in fed-batch culture at the 15-L scale using invert sugar as a carbon source

[0802] A 15-L scale fermentation of E. coli expressing mevalonic acid pathway polypeptides and Kudzu isoprene synthase was used to produce isoprene from cells fed invert sugar in fed-batch culture. This experiment demonstrates that growing cells in the presence of invert sugar resulted in the production of 2.4 g/L of isoprene.

Medium Recipe (per liter fermentation medium):

[0803] The medium was generated using the following components per liter fermentation medium: K2HPO4 7.5 g, MgSO4 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000X Modified Trace Metal Solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH
was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Invert sugar 10 g, thiamine * HCl 0.1 g, and antibiotics were added after sterilization and pH
adjustment.

1000X Modified Trace Metal Solution:

[0804] The 1000X modified trace metal solution was generated using the following components: citric acids * H2O 40 g, MnS04 * H2O 30 g, NaCl 10 g, FeS04 * 7H20 1 g, CoC12 * 6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3B03 100 mg, and NaMoO4 *

100 mg. Each component is dissolved one at a time in Di H2O, pH to 3.0 with HCUNaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.

[0805] Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. coli cells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. This experiment was carried out to monitor isoprene formation from invert sugar at the desired fermentation pH 7.0 and temperature 30 C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37 C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

[0806] Invert sugar was fed at an exponential rate until cells reached the stationary phase.
After this time the invert sugar feed was decreased to meet metabolic demands.
The total amount of invert sugar delivered to the bioreactor during the 44 hour fermentation was 2.4 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 25 M when the optical density at 550 nm (OD550) reached a value of 9. The IPTG concentration was raised to 50 M
when OD550 reached 200. The OD550 profile within the bioreactor over time is shown in Figure 96. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 2.4 g/L (Figure 97). The total amount of isoprene produced during the 44 hour fermentation was 18.4 g and the time course of production is shown in Figure 98. The molar yield of utilized carbon that went into producing isoprene during fermentation was 1.7%. The weight percent yield of isoprene from glucose was 0.8%.

Example 9. Construction of the upper and lower MVA pathway for integration into Bacillus subtilis 1. Construction of the Upper MVA pathway in Bacillus subtilis

[0807] The upper pathway from Enterococcusfaecalis is integrated into B.
subtilis under control of the aprE promoter. The upper pathway consists of two genes; mvaE, which encodes for AACT and HMGR, and mvaS, which encodes for HMGS. The two genes are fused together with a stop codon in between, an RBS site in front of mvaS, and are under the control of the aprE promoter. A terminator is situated after the mvaE gene. The chloramphenicol resistance marker is cloned after the mvaE gene and the construct is integrated at the aprE locus by double cross over using flanking regions of homology.

[0808] Four DNA fragments are amplified by PCR such that they contain overhangs that will allowed them to be fused together by a PCR reaction. PCR amplifications are carried out using Herculase polymerase according to manufacturer's instructions.

1. PaprE
CF 07-134 (+) Start of aprE promoter Pstl 5'- GACATCTGCAGCTCCATTTTCTTCTGC (SEQ ID NO: 115) CF 07-94 (-) Fuse PaprE to mvaE
5'- CAATAATAACTACTGTTTTCACTCTTTACCCTCTCCTTTTAA (SEQ ID NO:116) Template: Bacillus subtilis chromosomal DNA

2. mvaE
CF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon) 5'- TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTG (SEQ ID NO:117) CF 07-62 (-) Fuse mvaE to mvaS with RBS in between 5'- TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTCTTAAATC
(SEQ ID NO:94) Template: Enterococcusfaecalis chromosomal DNA (from ATCC) 3. mvaS
CF 07-61 (+) Fuse mvaE to mvaS with RBS in between 5'-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGATTGATAAA
(SEQ ID NO:95) CF 07-124 (-) Fuse the end of mvaS to the terminator 5'- CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT (SEQ ID
NO:118) Template: Enterococcusfaecalis chromosomal DNA

4. B. amyliquefaciens alkaline serine protease terminator CF 07-123 (+) Fuse the end of mvaS to the terminator 5'- ACCGTTCGTTCTTATCGAAACTAAAAAAAACCGGCCTTGGCCCCG (SEQ ID
NO:119) CF 07-46 (-) End of B. amyliquefaciens terminator BamHI
5'- GACATGACGGATCCGATTACGAATGCCGTCTC (SEQ ID NO:58) Template: Bacillus amyliquefaciens chromosomal DNA

PCR Fusion Reactions 5. Fuse mvaE to mvaS
CF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon) 5'- TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTG (SEQ ID NO:117) CF 07-124 (-) Fuse the end of mvaS to the terminator 5'- CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT (SEQ ID
NO:118) Template: #2 and 3 from above 6. Fuse mvaE-mvaS to aprE promoter CF 07-134 (+) Start of aprE promoter Pstt 5'- GACATCTGCAGCTCCATTTTCTTCTGC (SEQ ID NO:115) CF 07-124 (-) Fuse the end of mvaS to the terminator 5'- CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT (SEQ ID
NO:118) Template #1 and #4 from above 7. Fuse PaprE-mvaE-mvaS to terminator CF 07-134 (+) Start of aprE promoter Pstl 5'- GACATCTGCAGCTCCATTTTCTTCTGC (SEQ ID NO:115) CF 07-46 (-) End of B. amyliquefaciens terminator BamHI
5'- GACATGACGGATCCGATTACGAATGCCGTCTC (SEQ ID NO:58) Template: #4 and #6

[0809] The product is digested with restriction endonucleases PstI/BamHI and ligated to pJM 102 (Perego, M. 1993. Integrational vectors for genetic manipulation in Bacillus subtilis, p.
615-624. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington,D.C.) which is digested with PstJBamHl. The ligation is transformed into E.coli TOP 10 chemically competent cells and transformants are selected on LA containing carbenicillin (50 g/ml). The correct plasmid is identified by sequencing and is designated pJMUpperpathway2 (Figures 50 and 51). Purified plasmid DNA is transformed into Bacillus subtilis aprEnprE Pxyl-comK and transformants are selected on L agar containing chloramphenicol (5 g/ml). A correct colony is selected and is plated sequentially on L agar containing chloramphenicol 10, 15 and 25 g/ml to amplify the number of copies of the cassette containing the upper pathway.

[0810] The resulting strain is tested for mevalonic acid production by growing in LB
containing I% glucose and I%. Cultures are analyzed by GC for the production of mevalonic acid.

[0811] This strain is used subsequently as a host for the integration of the lower mevalonic acid pathway.

[0812] The following primers are used to sequence the various constructs above.
Sequencing primers:
CF 07-134 (+) Start of aprE promoter Pstl 5'- GACATCTGCAGCTCCATTTTCTTCTGC (SEQ ID NO:115) CF 07-58 (+) Start of mvaE gene 5'- ATGAAAACAGTAGTTATTATTGATGC (SEQ ID NO:97) CF 07-59 (-) End of mvaE gene 5'- ATGTTATTGTTTTCTTAAATCATTTAAAATAGC (SEQ ID NO:98) CF 07-82 (+) Start of mvaS gene 5'- ATGACAATTGGGATTGATAAAATTAG (SEQ ID NO:99) CF 07-83 (-) End of mvaS gene 5'- TTAGTTTCGATAAGAACGAACGGT (SEQ ID NO:100) CF 07-86 (+) Sequence in mvaE
5'- GAAATAGCCCCATTAGAAGTATC (SEQ ID NO:101) CF 07-87 (+) Sequence in mvaE
5'- TTGCCAATCATATGATTGAAAATC (SEQ ID NO:102) CF 07-88 (+) Sequence in mvaE
5'- GCTATGCTTCATTAGATCCTTATCG (SEQ ID NO:103) CF 07-89 (+) Sequence mvaS
5'- GAAACCTACATCCAATCTTTTGCCC (SEQ ID NO:104)

[0813] Transformants are selected on LA containing chloramphenicol at a concentration of 5 g/ml. One colony is confirmed to have the correct integration by sequencing and is plated on LA containing increasing concentrations of chloramphenicol over several days, to a final level of 25 g/ml. This results in amplification of the cassette containing the genes of interest. The resulting strain is designated CF 455: pJMupperpathway#l X Bacillus subtilis aprEnprE Pxyl comK (amplified to grow on LA containing chloramphenicol 25 g/ml).

II. Construction of the Lower MVA pathway in Bacillus subtilis

[0814] The lower MVA pathway, consisting of the genes mvkl, pmk, mpd and idi are combined in a cassette consisting of flanking DNA regions from the nprE region of the B.
subtilis chromosome (site of integration), the aprE promoter, and the spectinomycin resistance marker (see Figures 28 and 29; SEQ ID NO:13). This cassette is synthesized by DNA2.0 and is integrated into the chromosome of B. subtilis containing the upper MVA pathway integrated at the aprE locus. The kudzu isoprene synthase gene is expressed from the replicating plasmid described in Example 4 and is transformed into the strain with both upper and lower pathways integrated.

Example 10: Exemplary isoprene compositions and methods of making them 1. Compositional analysis of fermentation off-gas containing isoprene

[0815] A 14 L scale fermentation was performed with a recombinant E. coli BL21 (DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkIS encoding the full mevalonate pathway for isoprenoid precursor biosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and an isoprene synthase from Kudzu. Fermentation off-gas from the 14 L
tank was collected into 20 mL headspace vials at around the time of peak isoprene productivity (27.9 hours elapsed fermentation time, "EFT") and analyzed by headspace GC/MS for volatile components.

[0816] Headspace analysis was performed with an Agilent 6890 GC/MS system fitted with an Agilent HP-5MS GC/MS column (30 in x 250 m; 0.25 m film thickness). A
combiPAL
autoinjector was used for sampling 500 L aliquots from 20 mL headspace vials.
The GC/MS
method utilized helium as the carrier gas at a flow of 1 mL/min. The injection port was held at 250 C with a split ratio of 50:1. The oven temperature was held at 37 C for an initial 2 minute period, followed an increase to 237 C at a rate of 25 C/min for a total method time of 10 minutes. The Agilent 5793N mass selective detector scanned from mlz 29 to mlz 300. The limit of detection of this system is approximately 0.1 g/Lgas or approximately 0.1 ppm. If desired, more sensitive equipment with a lower limit of detection may be used.

[0817] The off-gas consisted of 99.925 % (v/v) permanent gases (N2, CO2 and 02), approximately 0.075% isoprene (2-methyl-1,3-butadiene) (-750 ppmv, 2100 g/L) and minor amounts (<50 ppmv) of ethanol, acetone, and two C5 prenyl alcohols. The amount of water vapor was not determined but was estimated to be equal to the equilibrium vapor pressure at 0 T. The composition of the volatile organic fraction was determined by integration of the area under the peaks in the GC/MS chromatogram (Figs. 86A and 86B) and is listed in Table 6.
Calibration curves for ethanol and acetone standards enabled the conversion of GC area to gas phase concentration in units of g/L using standard methods.

Table 6. Composition of volatile organic components in fermentation off-gas.
The off-gas was analyzed at the 27.9 hour time point of a fermentation using an E. coli BL21 (DE3) strain expressing a heterologous mevalonate pathway, an isoprenyl pyrophosphate isomerase from yeast, and an isoprene synthase from Kudzu.

Compound RT (min) GC area Area % Cone. L) Ethanol 1.669 239005 0.84 62 +/- 6 Acetone 1.703 288352 1.02 42 +/- 4 Isoprene (2-methyl-1,3- 1.829 27764544 97.81 2000 +/- 200 butadiene) 3-methyl-3-buten-l-ol 3.493 35060 0.12 <10 3-meth l-2-buten-l-ol 4.116 58153 0.20 <10 II. Measurement of trace volatile organic compounds (VOCs) co-produced with isoprene during fermentation of a recombinant E. coli strain

[0818] A 14 L scale fermentation was performed with a recombinant E. coli BL21 (DE3) strain containing two plasmids (pCL upperMev; pTrcKKDyIkIS) encoding the full mevalonate pathway for isoprenoid precursor biosynthesis, an isoprenyl pyrophosphate isomerase from yeast, and an isoprene synthase from Kudzu.

[0819] Fermentation off-gas was passed through cooled headspace vials in order to concentrate and identify trace volatile organic components. The off-gas from this fermentation was sampled at a rate of 1 L/min for 10 minutes through a 20 mL headspace vial packed with quartz wool (2g) and cooled to -78 C with dry ice. The vial was recapped with a fresh vial cap and analyzed by headspace GC/MS for trapped VOCs using the conditions described in Example 10, part I. The ratios of compounds observed in Figs. 87A-87D are a combination of overall level in the fermentation off-gas, the relative vapor pressure at -78 C, and the detector response of the mass spectrometer. For example, the low level of isoprene relative to oxygenated volatiles (e.g., acetone and ethanol) is a function of the high volatility of this material such that it does not accumulate in the headspace vial at -78 T.

[0820] The presence of many of these compounds is unique to isoprene compositions derived from biological sources. The results are depicted in Figs. 87A-87D and summarized in Tables 7Aand7B.

Table 7A: Trace volatiles present in off-gas produced by E. coli BL21 (DE3) (pCL
upperMev; pTrcKKDyIkIS) following cryo-trapping at -78oC.

Compound (miRT GC n) Areal Area%2 Ratio%3 Acetaldehyde 1.542 4019861 4.841 40.14 Ethanol 1.634 10553620 12.708 105.39 Acetone 1.727 7236323 8.714 72.26 2-methyl-1,3-butadiene 1.777 10013714 12.058 100.00 1- ro anol 1.987 163574 0.197 1.63 Diacetyl 2.156 221078 0.266 2.21 2-methyl-3 -buten-2-ol 2.316 902735 1.087 9.01 2-methyl-l-propanol 2.451 446387 0.538 4.46 3-meth l-l-butanal 2.7 165162 0.199 1.65 1-butanol 2.791 231738 0.279 2.31 3-meth l-3-buten-1-ol 3.514 14851860 17.884 148.32 3-meth l-l-butanol 3.557 8458483 10.185 84.47 3-methyl-2-buten- l -ol 4.042 18201341 21.917 181.76 3-methyl-2-butenal 4.153 1837273 2.212 18.35 3-meth lbut l acetate 5.197 196136 0.236 1.96 3-methyl-3-buten-l-yl 5.284 652132 0.785 6.51 acetate 2-heptanone 5.348 67224 0.081 0.67 2,5-dimeth 1 razine 5.591 58029 0.070 0.58 3-methyl-2-buten-1-yl 5.676 1686507 2.031 16.84 acetate 6-methyl-5-hepten-2-one 6.307 101797 0.123 1.02 2,4,5-trimeth 1 ridine 6.39 68477 0.082 0.68 2,3,5-trimeth 1 razine 6.485 30420 0.037 0.30 (E)-3,7-dimethyl-1,3,6- 6.766 848928 1.022 8.48 octatriene (Z)-3,7-dimethyl-1,3,6- 6.864 448810 0.540 4.48 octatriene 3-methyl-2-buten-l-yl 7.294 105356 0.127 1.05 butyrate Citronellal 7.756 208092 0.251 2.08 2,3-c clohe tenol ridine 8.98 1119947 1.349 11.18 1 GC area is the uncorrected area under the peak corresponding to the listed compound.
2 Area % is the peak area expressed as a % relative to the total peak area of all compounds.
3 Ratio % is the peak area expressed as a % relative to the peak area of 2-methyl-1,3-butadiene.
Table 7B. Trace volatiles present in off-gas produced by E. coli BL21 (DE3) (pCL
upperMev; pTrcKKDyIkIS) following cryo-trapping at -196oC.

Compound (min) GC Areal Area%2 Ratio%3 Compound min GC Areal Area%2 Ratio%3 Acetaldehyde 1.54 1655710 0.276 0.33 Methanethiol 1.584 173620 0.029 0.03 Ethanol 1.631 10259680 1.707 2.03 Acetone 1.722 73089100 12.164 14.43 2-methyl-1,3-butadiene 1.771 506349429 84.269 100.00 methyl acetate 1.852 320112 0.053 0.06 1- ro anol 1.983 156752 0.026 0.03 Diacetyl 2.148 67635 0.011 0.01 2-butanone 2.216 254364 0.042 0.05 2-methyl-3-buten-2-ol 2.312 684708 0.114 0.14 ethyl acetate 2.345 2226391 0.371 0.44 2-meth l-l- ro anol 2.451 187719 0.031 0.04 3-meth l-l-butanal 2.696 115723 0.019 0.02 3-methyl-2-butanone 2.751 116861 0.019 0.02 1-butanol 2.792 54555 0.009 0.01 2-pentanone 3.034 66520 0.011 0.01 3-meth l-3-buten-l-ol 3.516 1123520 0.187 0.22 3-meth l-l-butanol 3.561 572836 0.095 0.11 ethyl isobutyrate 3.861 142056 0.024 0.03 3-meth l-2-buten-l-ol 4.048 302558 0.050 0.06 3-methyl-2-butenal 4.152 585690 0.097 0.12 butyl acetate 4.502 29665 0.005 0.01 3-meth lbut l acetate 5.194 271797 0.045 0.05 3-meth l-3-buten-l- l acetate 5.281 705366 0.117 0.14 3-meth l-2-buten-l- l acetate 5.675 815186 0.136 0.16 (E)-3,7-dimethyl-1,3,6- 6.766 207061 0.034 0.04 octatriene (Z)-3,7-dimethyl-1,3,6- 6.863 94294 0.016 0.02 octatriene 2,3-c clohe tenol ridine 8.983 135104 0.022 0.03 1 GC area is the uncorrected area under the peak corresponding to the listed compound.
2 Area % is the peak area expressed as a % relative to the total peak area of all compounds.
3 Ratio % is the peak area expressed as a % relative to the peak area of 2-methyl-1,3-butadiene.
III. Absence of C5 hydrocarbon isomers in isoprene derived from fermentation.

[0821] Cryo-trapping of isoprene present in fermentation off-gas was performed using a 2 mL
headspace vial cooled in liquid nitrogen. The off-gas (1 L/min) was first passed through a 20 mL
vial containing sodium hydroxide pellets in order to minimize the accumulation of ice and solid CO2 in the 2 mL vial (-196 C). Approximately I OL of off-gas was passed through the vial, after which it was allowed to warm to -78 C with venting, followed by resealing with a fresh vial cap and analysis by GC/MS.

[0822] GC/MS headspace analysis was performed with an Agilent 6890 GC/MS
system using a 100 L gas tight syringe in headspace mode. A Zebron ZB-624 GC/MS column (30 m x 250 m; 1.40 m film thickness) was used for separation of analytes. The GC
autoinjector was fitted with a gas-tight 100 L syringe, and the needle height was adjusted to allow the injection of a 50 L headspace sample from a 2 mL GC vial. The GC/MS method utilized helium as the carrier gas at a flow of 1 mL/min. The injection port was held at 200 C with a split ratio of 20:1. The oven temperature was held at 37 C for the 5 minute duration of the analysis.
The Agilent 5793N
mass selective detector was run in single ion monitoring (SIM) mode on mlz 55, 66, 67 and 70.
Under these conditions, isoprene was observed to elute at 2.966 minutes (Figure 88B). A
standard of petroleum derived isoprene (Sigma-Aldrich) was also analyzed using this method and was found to contain additional C5 hydrocarbon isomers, which eluted shortly before or after the main peak and were quantified based on corrected GC area (Figure 88A).

Table 8A: GC/MS analysis of petroleum-derived isoprene Area % of total Compound (min) GC area C5 hydrocarbons 2-methyl-l-butene 2.689 18.2 x 103 0.017%
(Z)-2-pentene 2.835 10.6x 104 0.101%
Isoprene 2.966 10.4x 107 99.869%
1,3-cyclopentadiene 3.297 12.8 x 103 0.012%
(CPD) Table 8B: GC/MS analysis of fermentation-derived isoprene (% total C5 hydrocarbons) Compound RT (min) Corrected GC % of total C5 Area hydrocarbons Isoprene 2.966 8.1 x 107 100%

[0823] In a separate experiment, a standard mixture of C5 hydrocarbons was analyzed to determine if the detector response was the same for each of the compounds. The compounds were 2-methyl-l-butene, 2-methyl-1,3-butadiene, (E)-2-pentene, (Z)-2-pentene and (E)-1,3-pentadiene. In this case, the analysis was performed on an Agilent DB-Petro column (100 m x 0.25 mm, 0.50 m film thickness) held at 50 C for 15 minutes. The GC/MS
method utilized helium as the carrier gas at a flow of 1 mL/min. The injection port was held at 200 C with a split ratio of 50:1. The Agilent 5793N mass selective detector was run in full scan mode from m/z 19 to m/z 250. Under these conditions, a 100 g/L concentration of each standard produced the same detector response within experimental error.

IV. Compositions comprising isoprene adsorbed to a solid phase.

[0824] Biologically-produced isoprene was adsorbed to activated carbon resulting in a solid phase containing 50 to 99.9% carbon, 0.1% to 50% isoprene, 0.01% to 5% water, and minor amounts (<0.1 %) of other volatile organic components.

[0825] Fermentation off-gas was run through a copper condensation coil held at 0 C, followed by a granulated silica desiccant filter in order to remove water vapor. The dehumidified off-gas was then run through carbon containing filters (Koby Jr, Koby Filters, MA) to the point at which breakthrough of isoprene was detected in the filter exhaust by GC/MS.
The amount of isoprene adsorbed to the cartridge can be determined indirectly by calculating the concentration in the off-gas, the overall flow rate and the percent breakthrough over the collection period.
Alternately the adsorbed isoprene can be recovered from the filters by thermal, vacuum, or solvent-mediated desorption.

V. Collection and analysis of condensed isoprene.

[0826] Fermentation off-gas is dehumidified, and the CO2 removed by filtration through a suitable adsorbent (e.g., ascarite). The resulting off-gas stream is then run through a liquid nitrogen-cooled condenser in order to condense the VOCs in the stream. The collection vessel contains t-butyl catechol to inhibit the resulting isoprene condensate. The condensate is analyzed by GC/MS and NMR in order to determine purity using standard methods, such as those described herein.

VI. Production of prenyl alcohols by fermentation

[0827] Analysis of off-gas from an E. coli BL21 (DE3) strain expressing a Kudzu isoprene synthase revealed the presence of both isoprene and 3-methyl-3-buten-l-ol (isoprenol). The levels of the two compounds in the fermentation off-gas over the fermentation are shown in Figure 89 as determined by headspace GC/MS. Levels of isoprenol (3-methyl-3-buten-l-ol, 3-MBA) attained was nearly 10 g/L ggas in this experiment. Additional experiments produced levels of approximately 20 g/L ffgas in the fermentation off-gas.

Example 11: The de-coupling of growth and production of isoprene in E. coli expressing genes from the mevalonic acid pathway and fermented in a fed-batch culture

[0828] Example 11 illustrates the de-coupling of cell growth from mevalonic acid and isoprene production.

1. Fermentation Conditions Medium Recipe (per liter fermentation medium):

[0829] The medium was generated using the following components per liter fermentation medium: K2HPO4 7.5 g, MgSO4 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000X modified trace metal solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH
was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine * HCl 0.1 g, and antibiotics were added after sterilization and pH
adjustment.

1000X Modified Trace Metal Solution:

[0830] The 1000X modified trace metal solution was generated using the following components: citric acids * H2O 40 g, MnS04 * H2O 30 g, NaCl 10 g, FeS04 * 7H20 1 g, CoC12 * 6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3B03 100 mg, and NaMoO4 *

100 mg. Each component was dissolved one at a time in Di H20, pH to 3.0 with HC1/NaOH, then q.s. to volume, and filter sterilized with a 0.22 micron filter.

[0831] Fermentation was performed with E. coli cells containing the pTrcHis2AUpperPathway (also called pTrcUpperMVA, Figures 91 and 92A-92C; SEQ
ID
NO:23) (50 g/ml carbenicillin) or the pCL PtrcUpperMVA (also called pCL
PtrcUpperPathway (Figure 26)) (50 g/ml spectinomycin) plasmids. For experiments in which isoprene was produced, the E. coli cells also contained the pTrc KKDyIkIS (50 g/ml kanamycin) plasmid.
These experiments were carried out to monitor mevalonic acid or isoprene formation from glucose at the desired fermentation pH 7.0 and temperature 30 C. An inoculum of an E. coli strain taken from a frozen vial was streaked onto an LA broth agar plate (with antibiotics) and incubated at 37 C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to optical density 1.0 when measured at 550 nm, it was used to inoculate the bioreactor.

[0832] Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. Induction was achieved by adding IPTG. The mevalonic acid concentration in fermentation broth was determined by applying perchloric acid (Sigma-Aldrich # 244252) treated samples (0.3 M
incubated at 4 C for minutes) to an organic acids HPLC column (BioRad # 125-0140). The concentration was determined by comparing the broth mevalonic acid peak size to a calibration curve generated from mevalonolacetone (Sigma-Aldrich # M4667) treated with perchloric acid to form D,L-mevalonate. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer is defined as the amount of isoprene produced per liter of fermentation broth.

II. Mevalonic acid production from E. coli BL21 (DE3) cells expressing the pTrcUpperMVA plasmid at a 150-L scale

[0833] BL21 (DE3) cells that were grown on a plate as explained above in Example 11, part I
were inoculated into a flask containing 45 mL of tryptone-yeast extract medium and incubated at 30 C with shaking at 170 rpm for 5 hours. This solution was transferred to a 5-L bioreactor of tryptone-yeast extract medium, and the cells were grown at 30 C and 27.5 rpm until the culture reached an OD550 of 1Ø The 5 L of inoculum was seeded into a 150-L
bioreactor containing 45-kg of medium. The IPTG concentration was brought to 1.1 mM when the OD550 reached a value of 10. The OD550 profile within the bioreactor over time is shown in Figure 60A. The mevalonic acid titer increased over the course of the fermentation to a final value of 61.3 g/L (Figure 60B).
The specific productivity profile throughout the fermentation is shown in Figure 60C and a comparison to Figure 60A illustrates the de-coupling of growth and mevalonic acid production.
The total amount of mevalonic acid produced during the 52.5 hour fermentation was 4.0 kg from 14.1 kg of utilized glucose. The molar yield of utilized carbon that went into producing mevalonic acid during fermentation was 34.2%.

III. Mevalonic acid production from E. coli BL21 (DE3) cells expressing the pTrcUpperMVA plasmid at a 15-L scale

[0834] BL21 (DE3) cells that were grown on a plate as explained above in Example 11, part I
were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30 C at 160 rpm to OD550 1Ø This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 1.0 mM when the OD550 reached a value of 10. The OD550 profile within the bioreactor over time is shown in Figure 61A. The mevalonic acid titer increased over the course of the fermentation to a final value of 53.9 g/L (Figure 61B).
The specific productivity profile throughout the fermentation is shown in Figure 61 C and a comparison to Figure 61A illustrates the de-coupling of growth and mevalonic acid production.
The total amount of mevalonic acid produced during the 46.6 hour fermentation was 491 g from 2.1 kg of utilized glucose. The molar yield of utilized carbon that went into producing mevalonic acid during fermentation was 28.8%.

IV. Mevalonic acid production from E. coli FM5 cells expressing the pTrcUpperMVA
plasmid at a 15-L scale

[0835] FM5 cells that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30 C
at 160 rpm to OD550 1Ø This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 1.0 mM when the OD550 reached a value of 30.
The OD550 profile within the bioreactor over time is shown in Figure 62A. The mevalonic acid titer increased over the course of the fermentation to a final value of 23.7 g/L (Figure 62B). The specific productivity profile throughout the fermentation is shown in Figure 62C and a comparison to Figure 62A illustrates the de-coupling of growth and mevalonic acid production.
The total amount of mevalonic acid produced during the 51.2 hour fermentation was 140 g from 1.1 kg of utilized glucose. The molar yield of utilized carbon that went into producing mevalonic acid during fermentation was 15.2%.

V. Isoprene production from E. coli BL21 (DE3) cells expressing the pCL
PtrcUpperMVA
and pTrc KKDyIkIS plasmids at a 15-L scale

[0836] BL21 (DE3) cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS
plasmids that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30 C at 160 rpm to OD550 1Ø This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG
concentration was brought to 25 pM when the OD550 reached a value of 10. The IPTG
concentration was raised to 50 M when OD550 reached 190. The IPTG
concentration was raised to 100 M at 38 hours of fermentation. The OD550 profile within the bioreactor over time is shown in Figure 63A. The isoprene titer increased over the course of the fermentation to a final value of 2.2 g/L broth (Figure 63B). The specific productivity profile throughout the fermentation is shown in Figure 63C and a comparison to Figure 63A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 54.4 hour fermentation was 15.9 g from 2.3 kg of utilized glucose. The molar yield of utilized carbon that went into producing isoprene during fermentation was 1.53%.

VI. Isoprene production from E. coli BL21 (DE3) tuner cells expressing the pCL
PtrcUpperMVA and pTrc KKDyIkIS plasmids at a 15-L scale

[0837] BL21 (DE3) tuner cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS
plasmids that were grown on a plate as explained above in Example 11, part I
were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30 C at 160 rpm to OD550 1Ø This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 26 M when the OD550 reached a value of 10.
The IPTG
concentration was raised to 50 M when OD550 reached 175. The OD550 profile within the bioreactor over time is shown in Figure 64A. The isoprene titer increased over the course of the fermentation to a final value of 1.3 g/L broth (Figure 64B). The specific productivity profile throughout the fermentation is shown in Figure 64C and a comparison to Figure 64A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 48.6 hour fermentation was 9.9 g from 1.6 kg of utilized glucose.
The molar yield of utilized carbon that went into producing isoprene during fermentation was 1.34%.

VII. Isoprene production from E. coli MG 1655 cells expressing the pCL
PtrcUpperMVA and pTrc KKDyIkIS plasmids at a 15-L scale

[0838] MG1655 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30 C at 160 rpm to OD550 1Ø This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG
concentration was brought to 24 M when the OD550 reached a value of 45. The OD550 profile within the bioreactor over time is shown in Figure 65A. The isoprene titer increased over the course of the fermentation to a final value of 393 mg/L broth (Figure 65B).
The specific productivity profile throughout the fermentation is shown in Figure 65C and a comparison to Figure 65A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 67.4 hour fermentation was 2.2 g from 520 g of utilized glucose.
The molar yield of utilized carbon that went into producing isoprene during fermentation was 0.92%.

VIII. Isoprene production from E. coli MG1655ack-pta cells expressing the pCL
PtrcUpperMVA and pTrc KKDyIkIS plasmids at a 15-L scale

[0839] MG1655ack-pta cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS
plasmids that were grown on a plate as explained above in Example 11, part I
were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30 C at 160 rpm to OD550 1Ø This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG concentration was brought to 30 M when the OD550 reached a value of 10.
The OD550 profile within the bioreactor over time is shown in Figure 66A. The isoprene titer increased over the course of the fermentation to a final value of 368 mg/L broth (Figure 66B). The specific productivity profile throughout the fermentation is shown in Figure 66C and a comparison to Figure 66A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 56.7 hour fermentation was 1.8 g from 531 g of utilized glucose.
The molar yield of utilized carbon that went into producing isoprene during fermentation was 0.73%.

IX. Isoprene production from E. coli FM5 cells expressing the pCL PtrcUpperMVA
and pTrc KKDyIkIS plasmids at a 15-L scale

[0840] FM5 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids that were grown on a plate as explained above in Example 11, part I were inoculated into a flask containing 500 mL of tryptone-yeast extract medium and grown at 30 C at 160 rpm to OD550 1Ø This material was seeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTG
concentration was brought to 27 M when the OD550 reached a value of 15. The OD550 profile within the bioreactor over time is shown in Figure 67A. The isoprene titer increased over the course of the fermentation to a final value of 235 mg/L broth (Figure 67B).
The specific productivity profile throughout the fermentation is shown in Figure 67C and a comparison to Figure 67A illustrates the de-coupling of growth and isoprene production. The total amount of isoprene produced during the 52.3 hour fermentation was 1.4 g from 948 g of utilized glucose.
The molar yield of utilized carbon that went into producing isoprene during fermentation was 0.32%.

Example 12: Production of isoprene during the exponential growth phase of E.
coli expressing genes from the mevalonic acid pathway and fermented in a fed-batch culture

[0841] Example 12 illustrates the production of isoprene during the exponential growth phase of cells.

Medium Recipe (per liter fermentation medium):

[0842] The medium was generated using the following components per liter fermentation medium: K2HPO4 7.5 g, MgSO4 * 7H20 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and 1000X modified trace metal solution 1 ml. All of the components were added together and dissolved in diH2O. This solution was autoclaved. The pH
was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine * HCl 0.1 g, and antibiotics were added after sterilization and pH
adjustment.

1000X Modified Trace Metal Solution:

[0843] The 1000X modified trace metal solution was generated using the following components: citric acids * H2O 40 g, MnS04 * H2O 30 g, NaCl 10 g, FeS04 * 7H20 1 g, CoC12 * 6H20 1 g, ZnSO * 7H20 1 g, CuS04 * 5H20 100 mg, H3B03 100 mg, and NaMoO4 *

100 mg. Each component is dissolved one at a time in Di H2O, pH to 3.0 with HC1/NaOH, then q.s. to volume and filter sterilized with 0.22 micron filter.

[0844] Fermentation was performed in a 15-L bioreactor with ATCC 11303 E. coli cells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. This experiment was carried out to monitor isoprene formation from glucose at the desired fermentation pH
7.0 and temperature 30 C. An inoculum of E. coli strain taken from a frozen vial was streaked onto an LB broth agar plate (with antibiotics) and incubated at 37 C. A single colony was inoculated into tryptone-yeast extract medium. After the inoculum grew to OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

[0845] Glucose was fed at an exponential rate until cells reached the stationary phase. After this time the glucose feed was decreased to meet metabolic demands. The total amount of glucose delivered to the bioreactor during the 50 hour fermentation was 2.0 kg. Induction was achieved by adding IPTG. The IPTG concentration was brought to 25 M when the optical density at 550 nm (OD550) reached a value of 10. The IPTG concentration was raised to 50 M
when OD550 reached 190. The OD550 profile within the bioreactor over time is shown in Figure 99. The isoprene level in the off gas from the bioreactor was determined as described herein. The isoprene titer increased over the course of the fermentation to a final value of 1.4 g/L (Figure 100). The total amount of isoprene produced during the 50 hour fermentation was 10.0 g. The profile of the isoprene specific productivity over time within the bioreactor is shown in Figure 101. The molar yield of utilized carbon that contributed to producing isoprene during fermentation was 1.1%. The weight percent yield of isoprene from glucose was 0.5%.
Example 13: Flammability modeling and testing of isoprene 1. Summary of flammability modeling and testing of isoprene

[0846] Flammability modeling and experiments were performed for various hydrocarbon/oxygen/nitrogen/water/carbon dioxide mixtures. This modeling and experimental tested was aimed at defining isoprene and oxygen/nitrogen flammability curves under specified steam and carbon monoxide concentrations at a fixed pressure and temperature.
A matrix of the model conditions is shown in Table 9, and a matrix of the experiments performed is shown in Table 5.

Table 9. Summary of Modeled Isoprene Flammability Steam Carbon Isoprene Oxygen Temperature Pressure Dioxide Series Concentration Concentration Concentration ( C) (psig) (wt%) Concentration (vol. %) (vol. %) (wt. %) A 40 0 0 0 Varying Varying B 40 0 4 0 Varying Varying C 40 0 0 5 Varying Varying D 40 0 0 10 Varying Varying E 40 0 0 15 Varying Varying F 40 0 0 20 Varying Varying G 40 0 0 30 Varying Varying Table 10. Summary of Isoprene Flammability Tests Temperature Pressure Steam Isoprene Oxygen Series Number (oC) (psig) Concentration Concentration Concentration (vol. %) (vol. %) (vol. %) 1 40 0 0 Varying Varying 2 40 0 4 Varying Varying It Description of calculated adiabatic flame temperature (CAFT) model

[0847] Calculated adiabatic flame temperatures (CAFT) along with a selected limit flame temperature for combustion propagation were used to determine the flammability envelope for isoprene. The computer program used in this study to calculate the flame temperatures is the NASA Glenn Research Center CEA (Chemical Equilibrium with Applications) software.

[0848] There are five steps involved in determining the flammability envelope using an adiabatic flame temperature model for a homogeneous combustion mechanism (where both the fuel and oxidant are in the gaseous state): selection of the desired reactants, selection of the test condition, selection of the limit flame temperature, modification of the reactants, and construction of a flammability envelope from calculations.

[0849] In this first step, selection of desired reactants, a decision must be made as to the reactant species that will be present in the system and the quantities of each. In many cases the computer programs used for the calculations have a list of reactant and product species. If any of the data for the species to be studied are not found in the program, they may be obtained from other sources such as the JANAF tables or from the internet. In this current model data for water, nitrogen, oxygen and carbon dioxide were present in the program database. The program database did not have isoprene as a species; therefore the thermodynamic properties were incorporated manually.

[0850] The next step is to decide whether the initial pressure and temperature conditions that the combustion process is taking place in. In this model the pressure was 1 atmosphere (absolute) and the temperature was 40 C, the boiling point of isoprene.

[0851] The limit flame temperature for combustion can be either selected based on theoretical principles or determined experimentally. Each method has its own limitations.

[0852] Based on prior studies, the limit flame temperatures of hydrocarbons fall in the range of 1000 K to 1500 K. For this model, the value of 1500 K was selected. This is the temperature at which the reaction of carbon monoxide to carbon dioxide (a highly exothermic reaction and constitutes a significant proportion of the flame energy) becomes self sustaining.

[0853] Once the limit flame temperature has been decided upon, model calculations are performed on the given reactant mixture (species concentrations) and the adiabatic flame temperature is determined. Flame propagation is considered to have occurred only if the temperature is greater than the limit flame temperature. The reactant mixture composition is then modified to create data sets for propagation and non-propagation mixtures.

[0854] This type of model shows good agreement with the experimentally determined flammability limits. Regions outside the derived envelope are nonflammable and regions within it are flammable. The shape of the envelope forms a nose. The nose of the envelope is related to the limiting oxygen concentration (LOC) for gaseous fuels.

III. Results from calculated adiabatic flame temperature (CAFT) model

[0855] Plotted in Figs. 68 through 74 are the CAFT model results for Series A
to G, respectively. The figures plot the calculated adiabatic flame temperature (using the NASA CEA
program) as a function of fuel concentration (by weight) for several oxygen/nitrogen ratios (by weight). The parts of the curve that are above 1500 K, the selected limit flame temperature, contain fuel levels sufficient for flame propagation. The results may be difficult to interpret in the form presented in Figs. 68 through 74. Additionally, the current form is not conducive to comparison with experimental data which is generally presented in terms of volume percent.

[0856] Using Series A as an example the data in Figure 68 can be plotted in the form of a traditional flammability envelope. Using Figure 68 and reading across the 1500 K temperature line on the ordinate one can determine the fuel concentration for this limit flame temperature by dropping a tangent to the abscissa for each curve (oxygen to nitrogen ratio) that it intersects.
These values can then be tabulated as weight percent of fuel for a given weight percent of oxidizer (Figure 75A). Then knowing the composition of the fuel (100 wt.%
isoprene) and the composition of the oxidizer (relative content of water, oxygen and nitrogen) molar quantities can be established.

[0857] From these molar quantities percentage volume concentrations can be calculated. The concentrations in terms of volume percent can then be plotted to generate a flammability envelope (Figure 75B). The area bounded by the envelope is the explosible range and the area excluded is the non-explosible range. The "nose" of the envelope is the limiting oxygen concentration. Figs. 76A and 76B contain the calculated volume concentrations for the flammability envelope for Series B generated from data presented in Figure 69.
A similar approach can be used on data presented in Figs. 70-74.

IV. Flammability testing experimental equipment and procedure

[0858] Flammability testing was conducted in a 4 liter high pressure vessel.
The vessel was cylindrical in shape with an inner diameter of 6" and an internal height of 8.625". The temperature of the vessel (and the gases inside) was maintained using external heaters that were DEMANDE OU BREVET VOLUMINEUX

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Claims (19)

1. A method for producing a fuel constituent from a bioisoprene composition comprising chemically transforming a substantial portion of the isoprene in the bioisoprene composition to non-isoprene compounds by:
(a) subjecting the bioisoprene composition to heat or catalytic conditions suitable for isoprene dimerization to produce an isoprene dimer and then catalytically hydrogenating the isoprene dimer to form a saturated C10 fuel constituent; or (b) (i) partially hydrogenating the bioisoprene composition to produce an isoamylene, (ii) dimerizing the isoamylene with a mono-olefin selected from the group consisting of isoamylene, propylene and isobutene to form a dimate and (iii) completely hydrogenating the dimate to produce a fuel constituent.

2. The method of claim 1, wherein at least about 95% of isoprene in the bioisoprene composition is converted to non-isoprene compounds.

3. The method of claim 1, wherein the bioisoprene composition is heated to about 150 °C to about 250 °C to produce an unsaturated cyclic isoprene dimer and the unsaturated cyclic isoprene dimer is hydrogenated catalytically to produce a saturated cyclic isoprene dimer fuel constituent.

4. The method of claim 1, wherein the method comprises: (i) contacting the bioisoprene composition with a catalyst for catalyzing cyclo-dimerization of isoprene to produce an unsaturated cyclic isoprene dimer and the unsaturated cyclic isoprene dimer is hydrogenated catalytically to produce a saturated cyclic isoprene dimer fuel constituent.

5. The method of claim 4, wherein the catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of a nickel catalyst, iron catalysts and chromium catalysts.

6. The method of claim 1, wherein the step of partially hydrogenating the bioisoprene composition comprises contacting the bioisoprene composition with hydrogen gas and a catalyst for catalyzing partial hydrogenation of isoprene.

7. The method of claim 6, wherein the catalyst for catalyzing partial hydrogenation of isoprene comprises a palladium catalyst.

8. The method of claim 1, wherein the step of dimerizing the isoamylene with a mono-olefin comprises contacting the isoamylene with the mono-olefin in the presence of a catalyst for catalyzing dimerization of mono-olefin.

9. The method of claim 8, wherein the catalyst for catalyzing dimerization of mono-olefin comprises an acid catalyst.

10. The method of claim 1, further comprising purifying the isoprene from the bioisoprene composition prior to chemically transforming the bioisoprene composition to a fuel constituent.

11. A system for producing a fuel constituent from a bioisoprene composition, wherein a substantial portion of the isoprene in the bioisoprene composition is chemically converted to non-isoprene compounds, the system comprising a bioisoprene composition and :
(a) (i) one or more chemicals capable of dimerizing isoprene in the bioisoprene composition or a source of heat capable of dimerizing isoprene in the bioisoprene composition;
and (ii) a catalyst capable of hydrogenating the isoprene dimer to form a saturated C10 fuel constituent; or (b) (i) a chemical capable of partially hydrogenating isoprene in the bioisoprene composition to produce an isoamylene, (ii) a chemical capable of dimerizing the isoamylene with mono-olefins selected from the group consisting of isoamylene, propylene and isobutene to form a dimate and (iii) a chemical capable of completely hydrogenating the dimate to produce a fuel constituent.

12. The system of claim 11, wherein the bioisoprene composition comprising greater than about 2 mg of isoprene and comprising greater than or about 99.94% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.

13. The system of claim 11, wherein the one or more chemicals capable of dimerizing isoprene comprises catalyst for catalyzing cyclo-dimerization of isoprene comprising a catalyst selected from the group consisting of ruthenium catalysts, nickel catalysts, iron catalysts and chromium catalysts.

14. The system of claim 11, wherein the catalyst for hydrogenating the unsaturated isoprene dimers comprises a catalyst selected from the group consisting of palladium catalysts, nickel catalysts, ruthenium catalysts and rhodium catalysts.

15. The system of claim 11, wherein the chemical capable of partially hydrogenating isoprene comprises a palladium catalyst.

16. The system of claim 11, wherein the chemical capable of dimerizing the isoamylene with mono-olefins comprises an acid catalyst.

17. A fuel composition comprising a fuel constituent produced by the method of claim 1.

18. The fuel composition of claim 17, wherein the fuel composition is substantially free of isoprene.

19. The fuel composition of claim 17, wherein the fuel composition has .delta.13C value which is greater than -22%0 or within the range of -32%0 to -24%0.