WO2003008563A9 - Improved cytochrome p450 oxygenases - Google Patents
Improved cytochrome p450 oxygenasesInfo
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- WO2003008563A9 WO2003008563A9 PCT/US2002/023340 US0223340W WO03008563A9 WO 2003008563 A9 WO2003008563 A9 WO 2003008563A9 US 0223340 W US0223340 W US 0223340W WO 03008563 A9 WO03008563 A9 WO 03008563A9
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- amino acid
- isolated nucleic
- nucleic acid
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Definitions
- the invention relates to novel uses for cytochrome P450 BM-3.
- the invention relates to novel variants of cytochrome P450 enzymes which are more resistant to organic solvents and/or are capable of oxidizing alkanes or alkenes.
- paraffin processes for controlled, stereo- and regioselective oxidation of hydrocarbon feed stocks to more valuable and useful products such as alcohols, ketones, acids, and peroxides would have a major impact on the chemical and pharmaceutical industries.
- selective oxyfunctionalization of hydrocarbons thus remains one of the great challenges for contemporary chemistry.
- Monooxygenases have unique properties that distinguish them from most chemical catalysts. Most impressive is their ability to catalyze the specific hydroxylation of non-activated C-H; one of the most useful biotransformation reactions, which is often difficult to achieve by chemical means, especially in water, at room temperature under atmospheric pressure.
- organic solvents not aqueous solutions, are generally used. The use of organic solvents has many advantages, most importantly are a) higher solubility of often in aqueous solutions poor soluble nonpolar compounds; b) suppression of water-dependent side reactions; c) alteration in enantioselectivity; and d) elimination of microbial contaminations (Dordick, 1992).
- the cytochrome P450 monooxygenases (“P450s”) is a group of widely-distributed heme enzymes that inserts one oxygen atom of O 2 into a diverse range of hydrophobic substrates, often with high regio- and stereoselectivity. The second oxygen atom is reduced to H2O.
- the active sites of all cytochrome P450s contain an iron protoporphryin
- cytochrome P450 BM-3 from Bacillus megaterium (EC 1.14.14.1) also known as CYP102, is a water-soluble, catalytically self-sufficient P450 containing a monooxygenase domain (64 kD) and a reductase domain (54 kD) in a single polypeptide chain (Narhi and Fulco, 1986 and 1987; Miura and Fulco, 1975;
- P450 BM-3 The minimum requirements for activity are substrate, dioxygen and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH).
- NADPH cofactor nicotinamide adenine dinucleotide phosphate
- Nucleotide and amino acid sequences for P450 BM-3 can be found in, and are hereby inco ⁇ orated by reference from, the GenBank database under the accession Nos. J04832 (SEQ ID NO:l) and P14779 (SEQ ID NO:2), respectively.
- P450 BM-3 hydroxylates fatty acids of chain length between C12 and C18 at subterminal positions, and the regioselectivity of oxygen insertion depends on the chain length (Miura and Fulco, 1975; Boddupalli et al., 1990).
- the optimal chain length of saturated fatty acids for P450 BM-3 is 14-16 carbons, and the enzyme was initially believed to have no activity towards fatty acids smaller than C12 (Miura and Fulco, 1975).
- P450 BM-3 is also known to hydroxylate the corresponding fatty acid amides and alcohols and forms epoxides from unsaturated fatty acids (Miura and Fulco, 1975; Capdevila et al., 1996; Graham-Lorence et al., 1997; Ruettinger and Fulco, 1981).
- the enzyme was reported to be inactive towards alkanes and methyl esters lacking the polar functionality of the natural substrates (Miura and Fulco, 1975).
- P450 BM-3 is also known to hydroxylate the corresponding fatty acid amides and alcohols and forms epoxides from unsaturated fatty acids (Miura and Fulco, 1975; Capdevila et al., 1996; Graham-Lorence et al., 1997; Ruettinger and Fulco, 1981).
- the enzyme was reported to be inactive towards alkanes and methyl esters lacking the polar functionality of the natural substrates (Miura and Fulco, 1975).
- BM-3 could accept shorter-chain alkanes, although with very low activity (Munro et al., 1993).
- Powerful techniques for creating enzymes with modified or improved properties are now available, such as directed evolution (Arnold, 1998), in which iterative cycles of random mutagenesis, recombination and functional screening for improved enzymes accumulate the mutations that confer the desired properties.
- Mutant P450 BM-3 enzymes with modified activity have now been reported in the literature. For example, an F87A
- P450 BM-3 mutants that could oxidize polycyclic aromatic hydrocarbons ("PAHs") such as phenanthrene, fluoranthene, and pyrene.
- PAHs polycyclic aromatic hydrocarbons
- the Schmid group recently reported mutants of P450 BM-3 that can hydroxylate a variety of nonnatural substrates, including octane, several aromatic compounds and heterocyclic compounds (Appel et al., 2001).
- P450 BM-3 mutants for epoxidation of substrates such as long-chain unsaturated fatty acids (Miura and Fulco,
- P450 BM-3 substrates fatty acids, alcohols, amides, C>12 alkanes, polyaromatic hydrocarbons, heterocycles, etc.
- P450 BM-3 substrates fatty acids, alcohols, amides, C>12 alkanes, polyaromatic hydrocarbons, heterocycles, etc.
- organic solvent resistance of P450 BM-3 in water miscible co-solvents is low and insufficient for industrial applications.
- no P450 BM-3 mutants with improved or altered solvent resistance have been identified.
- the present invention is based on the discovery of P450 BM-3 variants that have improved alkane oxidation activity, alkene oxidation activity, and/or improved stability in organic co-solvents.
- the invention provides an isolated nucleic acid encoding a cytochrome P450 variant which has a higher capability than the corresponding wild-type cytochrome P450 to oxidize at least one substrate selected from an alkane comprising a carbon-chain of no more than 8 carbons and an alkene comprising a carbon-chain of no more than 8 carbons, wherein the wild-type cytochrome P450 comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 2 (i.e., the wild-type sequence of P450 BM-3), and the cytochrome P450 variant comprises an amino acid substitution at a residue corresponding to a residue of SEQ ID NO:2 selected from V78, H236, and E252.
- the amino acid sequence has at least 80% sequence identity to SEQ ID NO:2.
- the amino acid sequence is SEQ ID NO:2.
- the higher capability is a higher maximum turnover rate of the substrate into an oxidized product, and the maximum turnover rate of the variant is at least
- the capability to oxidize is preferably the capability to hydroxylate, and the alkane can be selected from one or more of octane, hexane, cyclohexane, pentane, butane, and propane.
- the capability to oxidize can be either or both of the capability to epoxidate or hydroxylate, and the alkene can be selected from one or more of 1-hexene, 2-hexene, 3-hexene, cyclohexene, isoprene, allyl chloride, propene, and styrene.
- the cytochrome P450 variant comprises amino acid substitutions at residues corresponding to at least two residues of SEQ ID NO:2 selected from V78, H236, and E252, preferably at all of these residues, and, even more preferably, the amino acid substitution is selected from V78A, H236Q, and E252G.
- the cytochrome P450 variant can comprise amino acid substitutions at two residues selected from V78, H236, and E252, preferably all of these residues, and, even more preferably, the amino acid substitutions are V78A, H236Q, and E252G.
- the cytochrome P450 variant may comprise at least one further amino acid substitution at a residue selected from H138, T175, V178, A184, N186, F205, D217, S226, R255, A290, A295, L353, and G396.
- the amino acid substitution at these residues is selected from one or more of H138Y, T175I, VI 781, A184V, N186D,
- the cytochrome P450 variant comprises the amino acid substitutions V78A, H138Y, T175I, V178I, A184V, H236Q, E252G, R255S, A290V, A295T, and L353V, or the amino acid substitutions V78A, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, and L353V.
- the invention also provides an isolated nucleic acid encoding a cytochrome P450 variant which has a higher organic solvent-resistance than the corresponding wild-type cytochrome P450, wherein the wild-type cytochrome P450 comprises an amino acid sequence has at least 60% sequence identity to SEQ ID NO:2, and the cytochrome P450 variant comprises an amino acid substitution at a residue corresponding to a residue of
- the amino acid sequence has at least 80% sequence identity to SEQ ID NO:2.
- the amino acid sequence is SEQ ID NO:2.
- the organic solvent-resistance may be, for example, a higher maximum turnover rate of a substrate into an oxidized product in a solution comprising an organic solvent, and the oxidized product may be a hydroxylated product.
- the organic solvent is selected from THF, DMSO, methanol, ethanol, propanol, dioxane, and dimethylformamide.
- the hydroxylation activity of the cytochrome P450 BM-3 variant can be at least twice the hydroxylation activity of wild-type cytochrome P450 BM-3.
- the hydroxylation activity of the cytochrome P450 BM-3 variant can be at least twice the hydroxylation activity of wild-type cytochrome P450 BM-3.
- the cytochrome P450 variant may comprise amino acid substitutions at residues corresponding to at least two residues of SEQ ID NO:2 selected from T235, R471, E494, and S1024; preferably at least three residues, an the amino acid substitutions at residues may correspond to T235, R471, E494, and S1024 of SEQ ID NO:2.
- the cytochrome P450 variant may comprise amino acid substitutions at least two residues selected from T235, R471, E494, and S1024, preferably three, and, even more preferably, the cytochrome P450 variant comprises amino acid substitutions at T235, R471, E494, and S1024.
- the amino acid substitutions can be T235A, R471A, E494K, and S1024E.
- the cytochrome P450 variant further may further comprise an amino acid substitution at residue F87, for example, F87A.
- the cytochrome P450 variant comprises the mutations F87A, T235A, R471A, E494K, and
- the invention also provides for an isolated nucleic acid encoding a variant of a parent cytochrome P450 oxygenase, the variant having (i) a higher ability than the parent to oxidize a substrate selected from an alkane comprising a carbon-chain of no more than 8 carbons or alkene comprising a carbon-chain of less than 8 carbons; and (ii) at least one amino acid substitution in a secondary structural element of the cytochrome P450 heme domain selected from the helix B' domain, the helix H domain, and the helix I domain, wherein the parent comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO:2.
- the amino acid sequence of the parent has at least 80% sequence identity to SEQ ID NO:2.
- the amino acid sequence of the parent is SEQ ID NO:2.
- the amino acid substitution can, for example, be at a residue corresponding to a residue of SEQ ID NO:2 selected from V78, H236, and E252, and correspond to V78A, H236Q, or E252G.
- the variant further comprises at least one amino acid substitution at a residue corresponding to a residue of SEQ ID NO:2 selected from H138, T175, V178, A184, F205, S226, R255, A290, A295, and L353, such as, for example, V78A, H138Y, T175I, V178I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A295T, and L353V.
- the amino acid substitutions are at residues corresponding to amino acid residues V78, H236, and E252 of SEQ ID NO:2, such as, for example, V78A, H236Q, and E252G.
- the invention also provides for an isolated nucleic acid encoding a variant of a parent cytochrome P450 oxygenase, the variant having: (i) a higher organic solvent resistance than the parent; and (ii) at least one amino acid substitution in a secondary structural element of the selected from the helix H domain of the cytochrome P450 heme domain and the helix of the flavin domain; wherein the parent comprises an amino acid sequence having at least 60% sequence identity to SEQ ID NO:2.
- the amino acid sequence of the parent has at least 80% sequence identity to SEQ ID NO:2.
- the amino acid sequence of the parent is SEQ ID NO:2.
- the amino acid substitution can be at a residue corresponding to a residue of SEQ ID NO:2 selected from T235 and E494, and the cytochrome P450 variant may optionally comprise a further amino acid substitution at a residue corresponding to a residue of SEQ ID NO:2 selected from R471 and S1024. If so, the amino acid substitution can be selected from T235A and E494K, and the further amino acid substitution can be selected from R471A and S1024E. In a preferred embodiment, the amino acid substitution is selected from T235A and E494K, and the further amino acid substitution is selected from R471A and S1024E.
- the variant further comprises an amino acid substitution at a residue corresponding to residue F87 of SEQ ID NO:2, such as, for example, F87A, F87G, F87V, F87I, F87F, F87W, F87D, F87N, F87H, F87K, or F87R.
- a preferred variant comprises amino acid substitutions at residues corresponding to amino acid residues T235, R471, E494, and S1024 of SEQ ID NO:2, and the amino acid substitutions are preferably
- T235A, R471A, E494K, and S1024E with or without the amino acid substitution F87A.
- the invention provides for an isolated nucleic acid encoding a cytochrome P450 variant, the cytochrome P450 variant comprising the amino acid substitutions V78A, H236Q, and E252G of SEQ ID NO:2, wherein the variant may further comprise the amino acid substitutions H138Y, T175I, V178I, A184V, N186D, D217V, S226I, R255S, A290V,
- A295T, L353V, and G396M or the amino acid substitutions T175I, A184V, F205C, S226R, R255S, A290V, L353V.
- the invention also provides for an isolated nucleic acid encoding a cytochrome P450 variant, the cytochrome P450 variant comprising the amino acid substitutions T235A, R471A, E494K, and S1024E of SEQ ID NO:2, optionally comprising the amino acid substitution F87A.
- the invention also provides for amino acid sequences comprising the above-mentioned amino acid substitutions.
- FIGURE 1 Gas chromatogram of the oxidation products of octane, catalyzed by wild-type P450 BM-3.
- FIGURE 2. The screening assay for alkane oxidation activity uses the substrate analog 8-pnpane (p-nitrophenoxyoctane). Terminal hydroxylation generates the unstable hemiacetal, which decomposes to the aldehyde and p-nitrophenolate, which is monitored at 410 nm.
- FIGURE 4 Progression of activity for P450 BM-3, as measured on 8-pnpane.
- FIGURE 5 Initial rates of NADPH oxidation vs alkane concentration for purified P450 BM-3 mutant 1X139-3.
- FIGURE 6 Gas chromatogram of the oxidation products of hexane, catalyzed by IX79_1.
- FIGURE 7 Gas chromatogram of the oxidation products of cyclohexane, catalyzed by IX79_1.
- FIGURE 8 Gas chromatogram of the oxidation products of butane, catalyzed by 1X139-3.
- FIGURE 9 Alternative representation of the pNCA assay principle (see also FIG. 2).
- FIGURE 10 Re-screen results of 1st mutant generation, investigating organic solvent resistance in 1% (v/v) (see Example 2).
- FIGURES 11A and B Mutagenic pathways for parent F87A and the co-solvents DMSO (A) and THF (B) (see Example 2), showing increased residual activity versus enzyme generation.
- FIGURES 12A to C Organic solvent resistance profile of the parent, showing organic solvent resistance against percentage of solvent (see Example 2).
- A DMSO as solvent.
- B THF as solvent.
- C Purified wild-type, F87A, and F87ASB3, using DMSO as solvent.
- FIGURES 13A to F Organic solvent resistance profile of back-mutated BM-3 variants.
- FIGURE 14 Maximum turnover rates (mole substrate/min mole enzyme) for P450 BM-3 wildtype (black bars) and 1X139-3 (shaded bars) on alkane and fatty acid substrates.
- FIGURE 15. Maximum rates reported for alkane hydroxylation by alkane monooxygenases CYP4B111, CYP52A38, P450cam9, AlkB18, and sMMOlO. Rates for P450 BM-3 wildtype and mutant 1X139-3 were determined in this work.
- FIGURES 16A and B Optical spectra for 1X139-3 and wildtype P450 BM-3.
- A Mutant 1X139-3 (0.5 mM) in potassium phosphate buffer (0.1 M, pH 8) (-A-); with laurate (0.5 mM, solid line) in 1% methanol; hexane (1.0 mM, - ⁇ -) in 1% methanol; propane (saturated solution, -•-).
- FIGURE 17 Wildtype P450 BM-3 in potassium phosphate buffer (0.1 M, pH 8) (- A-); with laurate (0.5 mM, solid line) in 1% methanol; hexane (1.0 mM, 1.0 mM, - ⁇ -) in 1% methanol; propane (saturated solution, -•-).
- FIGURE 17 Initial rates of NADPH consumption (mole substrate/min/mole enzyme) in the presence of alkenes for wild-type (black bars) and 1X139-3 (shaded bars). "ND" indicates that NAPDH consumption was not detectable over background.
- FIGURE 18 Gas chromatogram of the oxidation products of styrene (A) and propene (B) catalyzed by 1X139-3.
- FIGURES 19A and B. (A) Abso ⁇ tion spectra in the presence of 1X139-3 after the 4-NBP assay with isoprene and NADPH (o), NADPH without isoprene (D), and isoprene without NADPH ( ⁇ ).
- B Abso ⁇ tion spectra in the presence of 1X139-3 after the NBP assay with styrene and NADPH (o), NADPH without styrene (D), and styrene without NADPH ( ⁇ ).
- FIGURES 21 A to F Representative topology diagrams of the heme domain of P450 variants of the invention, based on P450BM-P; the heme domain of P450 BM-3.
- A topology of P450BM-P; the topology is depicted with helices represented by black bars, and the length of each of the bars is in approximate proportion to the length of the helix.
- the strands of ⁇ -sheets are shown with arrows.
- the strands are grouped by the secondary structural elements which they comprise.
- the structural elements are grouped into the ⁇ - helical-rich domain and the ⁇ -sheet-rich domain.
- the heme is shown by the square at the NH 2 -terminal end of the L-helix.
- this topology diagram could be used for other P450s (Peterson et al., 1995).
- B Topology of P450 BM-3, showing location of the heme group.
- C Location of residues H138 and A78 relative to the heme group.
- D Location of residues T175, V178, A184, N186, and D217 relative to the heme.
- E Location of residues H236, R255, and E252 relative to the heme.
- F Location of residues L353, G396, A290, and A295 relative to the heme.
- Those sections labeled A, betal, beta3, beta4, D, E, F, G, I, K, and J denote secondary structural elements conserved in P450s.
- FIGURE 22 Positions of the amino acid substitutions in P450 BM-3 mutant
- the variant contains 11 amino acid substitutions, which are represented as spheres on the crystal structure of the substrate-bound enzyme (PDB: 1FAG). Five are clustered on the highly flexible F-G helix-loop-helix structure and the I helix along which it slides during substrate binding and release.
- the invention provides novel variants or mutants of P450 BM-3 which are capable of oxidizing alkanes, alkane derivatives, and or saturated or unsaturated hydrocarbons. Accordingly, the invention provides variant P450 BM-3 enzymes which have a higher oxidation activity towards at least one alkane, alkane derivative, alkene, or alkene derivative than wild-type P450 BM-3.
- the variant P4590 BM-3 has a higher oxidation activity towards a saturated hydrocarbon such as octane, hexane, cyclohexane, propane, ethane, and/or butane, or towards an unsaturated hydrocarbon such as propene, hexene, cyclohexene, isoprene, allyl chloride, and/or styrene, or derivatives thereof, than wild-type P450 BM-3.
- a saturated hydrocarbon such as octane, hexane, cyclohexane, propane, ethane, and/or butane
- an unsaturated hydrocarbon such as propene, hexene, cyclohexene, isoprene, allyl chloride, and/or styrene, or derivatives thereof.
- the P450 BM-3 variants comprise mutations at one or more of the following residues of SEQ ID NO:2, counting the starting methionine as position 0 ("zero"): V78, H138, T175, V178, A184, N186, F205, D217, S226, H236, E252, R255, A290, A295, L353, and G396.
- the mutation or mutations are selected from V78A, H138Y, T175I, V178I, A184V, N186D, F205C, D217V, S226I, S226R, H236Q, E252G, R255S, A290V, A295T, L353V, and G396M.
- the P450 BM-3 variants can comprise at least one, preferably at least three, and more preferably at least 5, and even more preferably at least all of these amino acid mutations.
- the P450 BM-3 variant comprises the mutations V78A, H236Q, and the E252G mutations. See also Table 1A.
- the invention provides P450 BM-3 variants with a higher organic solvent resistance in at least one water-miscible co-solvent than wild-type P450 BM-3.
- the P450 BM-3 variants of the invention have a higher organic solvent resistance towards water-miscible co-solvents such as, but not limited to, DMSO, THF, methanol, ethanol, propanol, dioxane, and dimethylfonamide.
- water-miscible co-solvents such as, but not limited to, DMSO, THF, methanol, ethanol, propanol, dioxane, and dimethylfonamide.
- the P450 BM-3 variant comprises mutations at one or more of the following residues of SEQ ID NO:2, counting the starting methionine as position 0 ("zero"): F87, T235, R471, E494, and S1024.
- the mutation at F87 can be, for example, F87A, F87G, F87V, F87I, F87F, F87W, F87D, F87N, F87H, F87K, F87R.
- the mutations at T235, R471, and E494 can be, for example, T235A, R471C, R471A, and E494K.
- the mutation at S1024 can be, for example, S1024R, S1024T, S1024K, and S1024E.
- the novel P450 BM-3 variants comprise mutations in at least one, preferably at least three of amino acid residues F87, T235, R471, E494, and S1024. Most preferably, the variant comprises the mutations T235A, R471A, E494K, and S1024E, with or without the mutation F87A. See also Table 1 A.
- the invention provides for P450 BM-3 mutants having specific nucleic acid and amino acid sequences.
- the nucleic acid sequences include those which encode for the P450 BM-3 variants in Table IB.
- the amino acid sequences include those which have the combinations of amino acid mutations in Table IB, where all mutations refer to SEQ ID NO:2, counting the starting methionine residue as position 0 ("zero").
- the invention provides for novel variants of P450 enzymes other than P450 BM-3, which have a higher activity for alkane oxidation and/or higher solvent resistance than the corresponding wild-type enzyme. These novel variants can be identified when aligned with the respective non-BM-3 amino acid sequence with that of
- P450 BM-3 amino acid positions in the non-BM-3 sequence that are aligned with one or more of the following amino acid residues in SEQ ID NO:2 are identified: V78, F87, H138, T175, V178, A184, N186, F205, D217, S226, T235, H236, E252, R255, A290, A295, L353, G396M, R471, E494, and S1024. Mutations in amino acid residues of the non-BM-3 enzyme which are aligned with and identical to the aforementioned BM-3 amino acid residues results in novel P450 variants according to the invention. See FIG. 20.
- the mutation in the non-BM-3 sequence results in one or more of the following amino acid substitutions: V78'A, F87'A, F87'G, F87'V, F87T, F87'F, F87'W, F87'D, F87'N, F87'H, F87'K, F87'R, H138 ⁇ , T175T, V178T, A184'V, N186'D, F205'C, D217'V, S226'I, S226'R, T235 ⁇ , H236'Q, E252O, R255'S, A290'V, A295'T, L353'V, G396'M, R471 'C, R471 ⁇ , E494'K, S1024'R, S1024'T, S1024'K, and S1024 ⁇ , where the amino acid residue is the amino acid residue aligned with the corresponding P450- BM-3 residue (denoted by prime (') sign).
- the invention provides for variants of non-BM-3 enzymes, wherein the wild-type sequences are at least 30, preferably at least 50, more preferably at least 70, even more preferably at least 90%, and optimally at least 95% identical to SEQ ID NO: 2.
- the oxidase activity of a P450 variant for one or more alkane- or alkene- substrates is at least five, more preferably at least 10, and even more preferably at least 15 times that of the co ⁇ esponding wild-type cytochrome P450.
- the organic solvent resistance of a P450 variant is at least two, preferably at least 3, and even more preferably at least five times that of the corresponding wild-type cytochrome P450.
- non-BM-3 P450 may be a P450 BM-3 variant, which has one or more mutations as compared to wild-type P450 BM-3.
- Such variants including variants displaying more than 60% sequence identity to SEQ ID NO:2, are described in, e.g., PCT application PCT US02/11954, filed April 16, 2002.
- Crystal structures of wildtype P450 BM-3 with and without substrate reveal large conformational changes upon substrate binding at the active site (Haines et al., 2001; Li and Poulos, 1997; Paulsen and Ornstein, 1995; and Chang and Loew, 2000).
- the substrate free structure displays an open access channel with 17 to 21 ordered water molecules.
- Substrate recognition serves as a conformational trigger to close the channel, which dehydrates the active site, increases the redox potential, and allows dioxygen to bind to the heme.
- P450 BM-3 may be compared to naturally-occurring enzyme that hydroxylates linear alkanes.
- Pseudomonas oleovorans is able to oxidize n-alkanes using hydroxylase machinery comprising an integral membrane oxygenase ( ⁇ -hydroxylase), a soluble NADH-dependent reductase and a soluble metalloprotein (rubredoxin) which transfers electrons from the reductase to the hydroxylase (Staijen et al., 2000).
- ⁇ -hydroxylase has been cloned from P. oleovorans into Escherichia coli, where it has been expressed and purified (Shanklin et al., 1997).
- a tyrosine (Tyr51) at the entrance to the substrate-binding pocket makes a hydrogen bond to the carboxylate group of the substrate in the crystal structure of the enzyme bound with palmitoleic acid (Li and Poulos, 1997).
- Arg 47 also at the entrance to the binding pocket, may form an ionic interaction as well.
- Nonpolar alkane substrates must rely solely on hydrophobic partitioning into the enzyme's extended substrate channel, and poor substrate recognition may contribute to P450 BM-3's sluggish activity on octane and other alkanes or alkenes.
- the present invention provides evolved enzymes which oxidize alkanes to a higher degree, or which have a higher resistance to organic solvents and/or co-solvents, than the corresponding wild-type enzyme(s).
- a P450 BM-3 variant according to the invention has been generated which su ⁇ asses the activity of the alkane hydroxylase from P. oleovorans on octane.
- the mutant also showed similar high activity on hexane, cyclohexane, and pentane, which was not shown to be a substrate for P450 BM-3 before, and is also efficient on butane and propane.
- the cytochrome P450 variants of the invention show that it is possible to reach activities on unreactive nonnatural substrates that are close to the activity of the native enzyme, e.g., P450 BM-3, on its best natural substrates (for P450 BM-3; long chain fatty acids), which are about 1000 times higher than that of eukaryotic P450s and one of the highest activities of P450s known so far.
- the native enzyme e.g., P450 BM-3
- P450 BM-3 long chain fatty acids
- the strategy described here also provides improvements of the P450 BM-3 activity on a number of substrates, including shorter chain alkenes such as propene, allyl chloride, isoprene, 1-hexene and styrene. It should also be possible to target other key properties such as regioselectivity, enantioselectivity and catalyst stability.
- a preferred technique to improve the alkane-oxidation and co-solvent resistance of wild-type or parent cytochrome P450 enzymes, including P450 BM-3, is directed evolution.
- General methods for generating libraries and isolating and identifying improved proteins according to the invention using directed evolution are described briefly below. More extensive descriptions can be found in, for example, Arnold (1998); U.S. Patent Nos. 5,741,691; 5,811,238; 5,605,793 and 5,830,721; and International Applications WO 98/42832, WO 95/22625, WO 97/20078, WO 95/41653 and WO 98/27230.
- the basic steps in directed evolution are (1) the generation of mutant libraries of polynucleotides from a parent or wild-type sequence; (2) (optional) expression of the mutant polynucleotides to create a mutant polypeptide library; (3) screening/selecting the polynucleotide or polypeptide library for a desired property of a polynucleotide or polypeptide; and (4) selecting mutants which possess a higher level of the desired property; and (5) repeating steps (1) to (5) using the selected mutant(s) as parent(s) until one or more mutants displaying a sufficient level of the desired activity have been obtained.
- the property can be, but is not limited to, alkane oxidation capability and solvent-resistance.
- the parent protein or enzyme to be evolved can be a wild-type protein or enzyme, or a variant or mutant.
- the parent polynucleotide can be retrieved from any suitable commercial or non-commercial source.
- the parent polynucleotide can correspond to a full-length gene or a partial gene, and may be of various lengths.
- Preferably the parent polynucleotide is from 50 to 50,000 base pairs. It is contemplated that entire vectors containing the nucleic acid encoding the parent protein of interest may be used in the methods of this invention.
- Any method for generating mutations in the parent polynucleotide sequence to provide a library of evolved polynucleotides including e ⁇ or-prone polymerase chain reaction, cassette mutagenesis (in which the specific region optimized is replaced with a synthetically mutagenized oligonucleotide), oligonucleotide-directed mutagenesis, parallel PCR (which uses a large number of different PCR reactions that occur in parallel in the same vessel, such that the product of one reaction primes the product of another reaction), random mutagenesis (e.g., by random fragmentation and reassembly of the fragments by mutual priming); site-specific mutations (introduced into long sequences by random fragmentation of the template followed by reassembly of the fragments in the presence of mutagenic oligonucleotides); parallel PCR (e.g., recombination on a pool of DNA sequences); sexual PCR; and chemical mutagenesis (e.g.
- nucleotide precursor by sodium bisulfite, nitrous acid, hydroxylamine, hydrazine, formic acid, or by adding nitrosoguanidine, 5-bromouracil, 2-aminopurine, and acridine to the PCR reaction in place of the nucleotide precursor; or by adding intercalating agents such as proflavine, acriflavine, quinacrine); i ⁇ adiation (X-rays or ultraviolet light, and/or subjecting the polynucleotide to propagation in a host cell that is deficient in normal DNA damage repair function); or DNA shuffling (e.g., in vitro or in vivo homologous recombination of pools of nucleic acid fragments or polynucleotides).
- intercalating agents such as proflavine, acriflavine, quinacrine
- i ⁇ adiation X-rays or ultraviolet light, and/or subjecting the polynucleotide to propagation in a host cell
- any one of these techniques can also be employed under low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence, or to mutagenize a mixture of fragments of unknown sequence.
- the evolved polynucleotide molecules can be cloned into a suitable vector selected by the skilled artisan according to methods well known in the art. If a mixed population of the specific nucleic acid sequence is cloned into a vector it can be clonally amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector and/or express the mutant or variant protein or enzyme sequence. Any one of the well-known procedures for inserting expression vectors into a cell for expression of a given peptide or protein may be utilized.
- Suitable vectors include plasmids and viruses, particularly those known to be compatible with host cells that express oxidation enzymes or oxygenases.
- E. coli is one exemplary prefe ⁇ ed host cell.
- Other exemplary cells include other bacterial cells such as Bacillus and Pseudomonas, archaebacteria, yeast cells such as Saccharomyces cerevisiae, insect cells and filamentous fungi such as any species of Aspergillus cells.
- plant, human, mammalian or other animal cells may be preferred.
- Suitable host cells may be transformed, transfected or infected as appropriate by any suitable method including electroporation, CaCl 2 mediated DNA uptake, fungal infection, microinjection, microprojectile transformation, viral infection, or other established methods.
- the mixed population of polynucleotides or proteins may then be tested or screened to identify the recombinant polynucleotide or protein having a higher level of the desired activity or property.
- the mutation screening steps can then be repeated until the selected mutant(s) display a sufficient level of the desired activity or property. Briefly, after the sufficient level has been achieved, each selected protein or enzyme can be readily isolated and purified from the expression system, or media, if secreted. It can then be subjected to assays designed to further test functional activity of the particular protein or enzyme. Such experiments for various proteins are well known in the art, and are described below and in the Examples below.
- the evolved enzymes can be used in biocatalytic processes for, e.g., alkane hydroxylation and alkene epoxidation, or for improving yield of reactions involving oxidation of substrates with low solubility in aqueous solutions.
- the enzyme variants of the invention can be used in biocatalytic processes for production of chemicals from hydrocarbons, particularly alkanes and alkenes, in soluble or immobilized form.
- the enzyme variants can be used in live cells or in dead cells, or it can be partially purified from the cells.
- One prefe ⁇ ed process would be to use the enzyme variants in any of these forms (except live cells) in an organic solvent, in liquid or even gas phase, or for example in a super-critical fluid like CO 2 .
- the organic solvent would dissolve high concentrations of the non-polar substrate, so that the enzyme could work efficiently on that substrate.
- the enzyme can be used in living cells, and the cofactor recycling can be accomplished by feeding the cells the appropriate substrate(s).
- the NADPH and oxygen can also be replaced by a peroxide, for example hydrogen peroxide, butyl peroxide or cumene peroxide, or by another oxidant.
- Mutations that enhance the efficiency of peroxide-based oxidation by BM-3 or other cytochrome P450 enzymes can serve to enhance the peroxide shunt activity of the enzyme variants described here.
- the mutations described here can be combined with such mutations, for example, and tested for their contributions to peroxide-driven alkane and alkene oxidation.
- recombinant nucleic acid which encode cytochrome P450 enzymes with improved alkane-oxidation capability and/or solvent-resistance can be screened for alkane- oxidation activity or for activity or stability in a solvent/co-solvent mixture.
- Such tests are well known in the art. Examples of suitable tests are provided in the Examples and discussed below.
- a screening method to detect oxidation comprises combining, in any order, substrate, oxygen donor, and test oxidation enzyme.
- the assay components can be placed in or on any suitable medium, carrier or support, and are combined under predetermined conditions. The conditions are chosen to facilitate, suit, promote, investigate or test the oxidation of the substrate by the oxygen donor in the presence of the test enzyme, and may be modified during the assay.
- the amount of oxidation product, i.e., oxidized substrate is thereafter detected using a suitable method.
- a screening method can comprise a coupling enzyme such as horseradish peroxidase to enable or enhance the detection of successful oxidation.
- one or more cofactors, coenzymes and additional or ancillary proteins may be used to promote or enhance activity of the test oxidation enzyme, coupling enzyme, or both.
- substrate, oxygen donor, and other components of the screening assay are supplied to the transformed host cells or to the growth media or support for the cells.
- the test enzyme is expressed and retained inside the host cell, and the substrate, oxygen donor, and other components are added to the solution or plate containing the cells and cross the cell membrane and enter the cell.
- the host cells can be lysed so that all intracellular components, including any recombinantly expressed intracellular enzyme variant, can be in direct contact with any added substrate, oxygen donor, and other components.
- the oxidation product may be a colored, luminescent, or fluorescent compound, so that transformed host cells that produce more active oxidation enzymes "light up” in the assay and can be readily identified, and can be distinguished or separated from cells which do not "light up” as much and which produce inactive enzymes, less active enzymes, or no enzymes.
- a fluorescent reaction product can be achieved, for example, by using a coupling enzyme, such as laccase or horseradish peroxidase, which forms fluorescent polymers from the oxidation product.
- a chemiluminescent agent, such as luminol can also be used to enhance the detectability of the luminescent reaction product, such as the fluorescent polymers.
- Detectable reaction products also include color changes, such as colored materials that absorb measurable visible or UV light.
- an alkane analog such as 8-pnpane (see FIG. 2 and Example 1), can be prepared that generates yellow color upon hydroxylation.
- This "su ⁇ ogate" substrate with a C8 backbone and a p-nitrophenyl moiety is an analog of octane, and allows use of a colorimetric assay to conveniently screen large numbers of P450 BM-3 or other cytochrome P450 variants mutants for increased hydroxylation activity in microtiter plates (Schwaneberg et al., 1999;
- a substrate such as 12-pNCA can be added together with an organic co-solvent (e.g., tetrahydrofurane (THF), DMSO, ethanol, methanol, acetone, etc.) and 12-pNCA conversion initiated by adding a Isocitric co-factor regeneration solution (e.g., Isocitric acid 20 mM; dH 2 O, NADP+ 3 mM, Isocitric dehydrogenase 0.8 U/ml).
- organic co-solvent e.g., tetrahydrofurane (THF), DMSO, ethanol, methanol, acetone, etc.
- Isocitric co-factor regeneration solution e.g., Isocitric acid 20 mM; dH 2 O, NADP+ 3 mM, Isocitric dehydrogenase 0.8 U/ml.
- the reaction can be stopped by adding UT-buster (NaOH 1.5 M, 1.5 M Urea, 50 % (v/v) DMSO), and abso ⁇ tion at 410 nm recorded.
- UT-buster NaOH 1.5 M, 1.5 M Urea, 50 % (v/v) DMSO
- Enzyme variants displaying improved levels of the desired activity or property in the screening assay(s) can then be expressed in higher amounts, retrieved, optionally purified, and further tested for the activity or property of interest.
- cytochrome P450 variants created by directed evolution and selected for a desired property or activity can be further evaluated by any suitable test or tests known in the art to be useful to assess the property or activity.
- the enzyme variants can be evaluated for their alkane-oxidation capability, alkene-oxidation capability, and/or organic-solvent resistance.
- An assay for alkane-oxidation capability essentially comprises contacting the cytochrome P450 variant with a specific amount of alkane substrate, or a substrate which is an alkane analog such as 8-pnpane, in the presence of an oxygen donor, and any other components (e.g., NADPH) that are necessary or desirable to include in the reaction mixture, such as NADPH and buffering agents. After a sufficient incubation time, the amount of oxidation product formed, or, alternatively, the amount of intact non-oxidized substrate remaining, is estimated.
- the amount of oxidation product and/or substrate could be evaluated chromatographically, e.g., by mass spectroscopy (MS) coupled to high-pressure liquid chromatography (HPLC) or gas chromatography (GC) columns, or spectrophotometrically, by measuring the absorbance of either compound at a suitable wavelength.
- MS mass spectroscopy
- HPLC high-pressure liquid chromatography
- GC gas chromatography
- V max maximum catalytic rate
- Prefe ⁇ ed substrates include, but are not limited to, methane, ethane, propane, butane, pentane, hexane, heptane, octane, and cyclohexane.
- HPLC and GC techniques particularly when coupled to MS, can be used to dete ⁇ nine not only the amount of oxidized product, but also the identity of the product.
- octane can be oxidized to octanol where the hydroxyl group is positioned on any of the carbon atoms in the octanol molecule.
- Alkene-oxidation can be evaluated by methods similar to those described for alkanes, simply by replacing an alkane with the co ⁇ esponding alkene, and designing an assay which promotes and detects epoxide formation of the alkene.
- an assay which detects NADPH consumption may be used.
- Preferred alkene substrates include ethene, propene, butene, pentene, hexene, heptene, and octene.
- Organic solvent resistance of a cytochrome P450 variant is advantageously evaluated by conducting an oxidation reaction in the presence of a certain amount of organic solvent or co-solvent.
- This amount can be varied from, e.g., about 0.1% to about 99.9% (v/v) organic solvent or co-solvent, more preferably from about 0.5% to about 50% (v/v) organic solvent or co-solvent, and, most preferably, from about 1% to about 10% (v/v) organic solvent or co-solvent, of the total reaction volume.
- the amount of oxidation product is then detected as a measure of the organic-solvent resistance of the enzyme variant.
- Such assays can be conducted using various amounts of solvent or co-solvent, and on enzyme variants stored for various periods of time in solutions comprising a certain amount of organic solvent or co-solvent.
- Preferred organic co-solvents include THF, DMSO, acetone, acetonitrile, and ethanol.
- a P450 BM-3 variant of the invention can comprise at least one of these mutations, optionally in combination with another mutations selected from the ones described in Table 1A, a mutation not described in Table 1A, or no other mutation.
- the variant P450 BM-3 enzymes of the invention can have a higher oxidation activity towards a saturated hydrocarbon, e.g., octane, hexane, cyclohexane, propane, ethane, and/or butane, than wild-type P450 BM-3.
- Prefe ⁇ ed amino acid mutations are those listed in Table 1A.
- P450 BM-3 variants including variants comprising truncated, deleted, and inserted amino acid sequences, that comprise one or more of these mutations and that show enhanced alkane- oxidation activity in a suitable assay as compared to wild-type P450 BM-3.
- the particularly active P450 BM-3 mutants 1X139-3 and "J" comprised 11 and 10 amino acid mutations, respectively; V78A, H138Y, T175I, V178I, A184V, H236Q, E252G, R255S, A290V, A295T, and L353V for 1X139-3; and V78A, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, and L353V for 'J".
- wildtype P450 BM-3 as the parent, a clone with approximately 2-fold increased activity for 8-pnpane was isolated.
- a P450 BM-3 mutant comprising at least one, preferably at least two, and most preferably all three of these mutations, or a nucleic acid encoding such mutants, is a prefe ⁇ ed embodiment of the invention.
- a P450 BM-3 variant with improved organic solvent-resistance according to the invention comprises at least one of the mutations in Table 1A, optionally in combination with other mutations selected from the ones described in Table 1A, a mutation not described in Table 1 A, or no other mutation.
- P450 BM-3 variants having particularly improved solvent resistance comprised R471A, E494K, and S1024E mutations, optionally with the mutation F87A.
- a P450 BM-3 mutant comprising at least one, preferably at least two, and most preferably all three of these mutations, with or without the F87A mutation, or a nucleic acid encoding such mutants, is a preferred embodiment of the invention.
- a skilled artisan could easily identify P450 BM-3 variants, including variants comprising truncated, deleted, and inserted amino acid sequences, that comprise one or more of the mutations in Table 1A and that show improved organic solvent-resistance in a suitable assay as compared to wild-type P450 BM-3.
- Directed evolution techniques can thus significantly improve the organic solvent resistance of P450 BM-3 and its mutant P450 BM-3 F87A toward DMSO and THF, or any other organic solvents or co-solvents. It was also identified herein that position F87 - located at the end of the substrate access channel directly above the heme - plays a size-dependent key role in modulating the monooxygenase activity in organic co-solvents. Because the mutations are located at the interface between the reductase domain and the P450 domain, it is believed, while not being limited to any theory, that the mutations create tighter domain bonding, which support electron transfer from the reductase to the heme in the presence of cosolvent.
- mutation 1024 which is not in the crystallized reductase fragment
- all of the mutations improving organic solvent resistance are located at the interface between heme domain and reducase. It is therefore possible that the mutations "stabilize" the orientation of heme to reducase allowing an electron transfer from the reducase to the heme. Potentially, and without being bound to any theory, this electron- transfer could be essential, or at least important, for activity.
- the P450 BM-3 variants of the invention have an at least two-fold improvement in the capability to oxidize a chosen alkane (e.g., octane, hexane, pentane, butane, cyclohexane, or propane), and/or an at least two-fold improvement in the organic solvent-resistance, as compared to wild-type P450 BM-3.
- a chosen alkane e.g., octane, hexane, pentane, butane, cyclohexane, or propane
- the improvement for either or both of these properties is at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 15-fold.
- the P450 mutations of the present invention are wholly unexpected.
- Schmid and coworkers recently described the engineering of P450 BM-3 to hydroxylate short chain (C8-C10) fatty acids not accepted by the wildtype enzyme (Li et al., 2001).
- They focused on eight individual amino acids within the binding pocket to perform saturation mutagenesis.
- This approach successfully discovered mutations (Val26Thr, Arg47Phe, Ala74Gly, Leul88Lys, Phe87Ala) that altered the enzyme's substrate specificity.
- the invention also provides for novel non-P450 BM-3 cytochrome P450 oxygenases in which one or more of the amino acid residues listed in
- sequence alignment software such as SIM (alignment of two protein sequences), LALIGN (finds multiple matching subsegments in two sequences), Dotlet (a Java applet for sequence comparisons using the dot matrix method); CLUSTALW (available via the
- ALIGN at Genestream (IGH)
- DIALIGN multiple sequence alignment based on segment-to-segment comparison, at University of Bielefeld, Germany), Match-Box (at University of Namur, Belgium), MSA (at Washington University), Multalin (at INRA or at PBIL), MUSCA (multiple sequence alignment using pattern discovery, at IBM), and AMAS (Analyse Multiply Aligned Sequences).
- a person of skill can choose suitable settings, or simply use standard default settings, in these programs to align P450 BM-3 with another cytochrome P450 enzyme. See FIG. 20 for representative sequence alignments, and Table 2 for representative non-P450 BM-3 mutations.
- P450 enzymes can be taken from the literature, and amino acid residues co ⁇ esponding to the mutated amino acid residues of the invention identified. For example, such information can be derived from de Montellano (1995) (see, especially, Figure 1 on page
- FIG. 21 shows a topological view of a cytochrome P450 enzyme, including the various domains of cytochrome P450 enzymes and the mutations contemplated by the present invention in each of those domains. While the topological view presented in FIG. 21 is that of P450 BM - P , with only minor modifications, this topology diagram may be used for other P450s. Briefly, FIG. 10 shows where mutations disclosed herein were made and these are summarized in Table 3 below.
- Heme domain helix B' V78
- Heme domain loop connecting helices B' and C F87
- Heme domain helix F T175
- Heme domain helix F VI 78
- Heme domain helix F Al 84
- Heme domain helix F Nl 86 Heme domain: helix G D217
- Heme domain helix G S226
- Heme domain helix H H236
- Heme domain helix I R255
- Heme domain helix J A290
- Heme domain loop connecting helices K' and L G396 R471
- a P450 variant may be prepared by making one or more mutations in one or more of the domains of P450 identified in Table 3 above. Further, the topological view of FIG. 10 allows one to compare BM-3 variants with other P450 enzymes and identify those residues of non-BM-3 enzymes that could be mutated according to the secondary and tertiary structural motifs within the enzyme(s).
- the invention provides novel non-P450 BM-3 cytochrome P450 oxygenases in which one or more of the amino acid residues listed in Table 1 A have been conserved. Conservation of an amino acid residue can show that the residue has an important function for the oxygenase activity and/or stability of the P450 enzyme.
- the P450 BM-3 mutations identified herein to improve utilization of hydrogen peroxide as oxygen source and/or thermostability can simply be translated onto such non-P450 BM-3 enzymes to yield improved properties according to the invention.
- P450 BM-3 variants were created and identified by directed evolution techniques, specifically iterative cycles of random mutagenesis and recombination, and functional screening.
- the PCR product was digested with BamRl and EcoRI.
- the P450 BM-3 gene was ligated into expression vector pCWOri (+) (Bames, 1996) (p BM-3 WT18-6), which is under the control of double Ptac promoter and contains an ampicillin resistance coding region.
- a silent mutation was introduced to construct a S ⁇ cl site 130 bases upstream of the end of the heme domain.
- the QuikChange (Stratagene) protocol was followed and the primers were as follows: 5'-CATACAAACTACGAGCTCGATATTAAAGAAAC-3' (S ⁇ cl site underlined;
- the P450 BM-3 gene which includes a silent mutation to introduce a S ⁇ cl site 130 bp upstream of the end of the heme domain, was cloned behind the double tac promoter of the expression vector pCWori (p BM-3_WT18 6) (Farinas et al., 2001). This plasmid was used for production of wildtype protein and as a starting clone for directed evolution.
- TB media 500 ml
- trace elements 125 ⁇ L: 0.5 g MgCl 2 , 30.0 g FeCl 2 6H 2 O, 1.0 g ZnCl 2 4H 2 0, 0.2 g CoCl 2 6H20, 1.0 g Na 2 MoO 4 2H 2 O, 0.5 g CaCl 2 2H 2 O, 1.0 g CuCl 2 and 0.2 g H 2 BO 3 in IL HCl solution (90 % v/v distilled water: concentrated HCl)) (Joo et al., 1999) was inoculated with 500 ⁇ l of an overnight culture of E. coli BL21 containing the expression plasmid. After shaking for
- aminolevulinic acid hydrochloride (ALA) (0.5 mM) was added, and expression was induced by addition of IPTG (ImM) and cells were harvested by centrifugation after a total cultivation time of 30 hours.
- ALA aminolevulinic acid hydrochloride
- mutagenic PCR was performed on the heme domain in a 100 mL reaction as described in Zhao et al. (1999) with some modifications.
- the mutated P450 BM 3 fragment was 1291 base pairs.
- the reaction contained MgCl 2 (7 mM) and the following forward and reverse primers (40 pmol each):
- the reaction also contained p BM-3 WT18-6 (10 ng), dNTPs (0.2mM dGTP, 0.2mM dATP, ImM dCTP, 1 mM dTTP), and Taq polymerase (5 units, Roche), KC1 ( 50 mM), and Tris-HCI (lOmM, pH 8.3, 20 °C).
- MnC12 0.0, 0.05, and 0.1 mM was added to the PCR mixture to alter the error rate of the polymerase.
- PCR was performed in a thermocycler (PTC200, MJ Research, Waltham, MA) for 30 cycles (95 °C, 45 s; 50 °C, 30 s; 72 °C, 2 min).
- the PCR product was restricted with B ⁇ mT ⁇ l and S ⁇ cl and ligated into expression vector pCWOri (+).
- the resulting plasmid was transformed into E. coli strain
- DH5 ⁇ and the colonies were selected on agar plates containing ampicillin (100 mg/ml).
- the PCR product was restricted with BamHI and Sad and ligated into expression vector pCWOri (+).
- the resulting plasmid was transformed into E. coli strain DH5 ⁇ and the colonies were selected on agar plates containing ampicillin (100 mg/ml).
- e ⁇ or prone PCR was also performed using the GeneMo ⁇ h PCR Mutagenesis Kit (Stratagene) applying conditions of high e ⁇ or rate (1-10 ng template DNA).
- the PCR product was restricted with B ⁇ m ⁇ T and S ⁇ cl and ligated into expression vector pCWOri (+).
- the resulting plasmid was transformed into E. coli strain DH5 ⁇ and the colonies were selected on agar plates containing ampicillin (100 mg/ml). Recombination was done in 50 mL reactions as described (Zhao et al., 1999).
- Each reaction contained buffer (Qiagen l xPCR buffer), template (10 ng each), forward and reverse primer (final 0.15 mM), dNTPs (200 ⁇ M each) and Taq DNA polymerase (2.5 units, Qiagen).
- PCR was performed in a thermocycler (PTC200, MJ Research, Waltham, MA) (1. 95 °C, 2 min; 2. 95 °C 30 s; 3. 50 °C, 10 s; repeat steps 2 and 3 100x).
- a robot (Qpix, Genetix) picked and inoculated colonies into 1 ml deep-well plates containing LB media (400 ml) and ampicillin (100 mg/ml). The plates were incubated at 37 °C, 270 RPM, and 80 % relative humidity. After 24 hours, the culture liquid (50 ml) was added to TB (450 ml) containing, ampicillin (100 mg ampicillin/ml), thiamine (5 mg/ml), and trace elements (0.25 ml/ml).
- the screening procedure was modified to the following procedure.
- the plates with the picked colonies were incubated in LB (containing 100 ⁇ g /L ampicillin) at 30 °C, 270 RPM, and 80 % relative humidity.
- TB 500 ml
- ampicillin 100 mg ampicillin ml
- thiamine 5 mg/ml
- trace elements 0.25 ml/ml
- ⁇ -aminolevulinic acid hydrochloride (1 mM) and 10 ⁇ M isopropyl-thiogalactopyranoside
- the plates were centrifuged and supernatants were discarded.
- Cell pellets were washed with Tris-HCI (350 ml, pH 8.3), frozen at -20 °C for at least 8 hours and then resuspended in 400 ⁇ l Tris-HCI (350 ml, pH 8.3) containing lysozyme (0.5 mg/ml), deoxyribonuclease I (0.1 mg/ml) and MgCl 2 (10 mM). After incubation at 37 °C for 45 minutes, the plates were centrifuged and the lysate (150 ml) was transfe ⁇ ed to a 96-well plate.
- the frozen cell pellets were resuspended in phosphate buffer (1 mL, 0.1 M, pH 8.0) containing lysozyme (0.5 mg/mLO, Dnasel (0.1 ⁇ L/mL) and MgCl 2 (10 mM).
- lysozyme 0.5 mg/mLO, Dnasel (0.1 ⁇ L/mL) and MgCl 2 (10 mM).
- the lysates were centrifuged and the supernatants were diluted for activity measurements in 96 well microtiter plates.
- mutant libraries were screened using 8-pnpane as described above. Also, a cofactor (NADPH) depletion assay was used to determine the turnover rates.
- the lysates were diluted into 96 well microtiter plates containing phosphate buffer (200 ⁇ L, 0.1 M, pH 8.0), alkane substrate (0.5-1.0 mM), and DMSO (1%).
- the liquid alkanes were added to the buffer using alkane stock solutions in DMSO, whereas gaseous alkanes were bubbled into buffer for ⁇ 45 minutes to obtain saturated solutions.
- the reaction was initiated by addition of NADPH (200 ⁇ M), and the oxidation of NADPH was monitored at 340 nm. Only the mutants active in both screens were isolated and recharacterized.
- the most active clones from the primary screen were streaked out on agar plates to get single colonies. Four to 8 single colonies were recultured in deep well plates and rescreened as described above. In the rescreen all clones were also assayed for hydroxylation of the target substrate octane. The same dilutions of lysates in buffer were used as in the 8-pnpane assay. A stock octane substrate solution (225 ⁇ M) in DMSO (1%) was added to the lysates.
- the cells were already lysed in a buffer, where the 0.1 M Tris-HCI from the lysis buffer described above was replaced by 30 mM phosphate buffer pH 7.4 to avoid organic substances (which could conceivably become substrates for the enzyme) in the buffer.
- Phosphate buffer was saturated with propane by bubbling propane into the buffer for 1 hour.
- Thirty ⁇ l of the bacterial lysates were pipeted into 96 well microtiter plates and 120 ⁇ l of the propane saturated buffer were added. The reaction was started again by addition of 50 ⁇ l of 3 mM NADPH and followed at 340 nm for 3 min.
- Agar plate colony development Cells were grown on LB agar plates containing 100 ⁇ g/ml ampicillin, 1 mM aminolevulinic acid hydrochloride and 10 ⁇ M isopropyl thiogalactopyranoside. The latter two substances are not necessary if the activity resulting from a leaky promoter system is high enough.
- a substrate solution was prepared containing 30 mM phosphate buffer pH 7.4, 100 ⁇ M polymyxin B sulfate as a cell permeabilizer, 2 mM NADPH and 5 mM alkane substrate. The substrate solution was sonicated before use to emulsify most of the substrate in the buffer system.
- a nitrocellulose membrane was soaked with this substrate solution and placed on the colonies on the agar plate.
- the optimal reaction time was estimated by testing the assay on control agar plates with colonies of clones with different hydroxylation activity before starting the main screen. After the reaction time the color reagent (0.5 mg/ml NBT in 30 mM phosphate buffer pH 7.4 and some crystals of the catalyst PMS) was pipeted directly onto the nitrocellulose membrane. Colonies located under white spots on the membrane were picked with a toothpick and streaked out on fresh agar plates to get single colonies for the re-screen.
- the enzymes were purified and quantified as described above.
- the substrate concentration co ⁇ esponding to the maximum turnover rate was determined by monitoring NADPH consumption with a plate reader in the presence of enzyme in phosphate buffer (0.1 M pH 8.0) and varying amounts of substrate in methanol (1%). After identifying the concentration of substrate that coincides with the maximum rate, the rate was measured using an UV-Vis spectrophotometer and 1 cm path length quartz cuvettes.
- a typical reaction solution contained enzyme (700 ⁇ L, 0.35 - 3.5 ⁇ M) in potassium phosphate buffer (0.1 M, pH 8.0) and substrate in methanol (1%). The reaction was initiated by the addition of NADPH (300 ⁇ L, 200 ⁇ M), and the abso ⁇ tion at 340 nm was monitored.
- the amount of H 2 O 2 was determined using 2,2'-azino-di-[3-ethyl-benzothiazidine- 6-sulfonic acid/horseradish peroxidase assay by following published procedures (Yeom,
- the enzymes were purified and quantified as described above.
- the oxidation was performed with solutions containing octane in DMSO (1 mM octane; 1% DMSO), P450 BM-3 (2-3 ⁇ M), and NADPH (1-5 mM) in Tris-HCI (50 mM) containing NaCI (340 mM).
- Catalytic activity of P450 BM-3 was measured spectrophotometrically by monitoring the rate of NADPH oxidation, as described (Matson et al. (1977).
- the assay solution contained 0.1 nmole P450 BM-3, octane in DMSO, and 0.8 mM NADPH, 50 mM NaCI in 0.1 M Tris-HCI, pH 8.2.
- the enzymes were purified and quantified as described above.
- the substrate concentration co ⁇ esponding to the maximum turnover rate was determined by monitoring NADPH consumption with a plate reader in the presence of enzyme in phosphate buffer (0.1 M pH 8.0) and varying amounts of substrate in methanol (1%). After identifying the concentration of substrate that coincides with the maximum rate, the rate was measured using an UV-Vis spectrophotometer and 1 cm path length quartz cuvettes.
- a typical reaction solution contained enzyme (700 ⁇ L, 0.35 - 3.5 ⁇ M) in potassium phosphate buffer (0.1 M, pH 8.0) and substrate in methanol (1%).
- the reaction was initiated by the addition of NADPH (300 ⁇ L, 200 ⁇ M), and the abso ⁇ tion at 340 nm was monitored.
- the substrates examined were pentane, hexane, cyclohexane, and octane.
- the amount of H 2 O 2 was determined using 2,2'-azino-di-[3-ethyl-benzothiazidine-
- 6-sulfonic acid/horseradish peroxidase assay by following published procedures (Yeom & Sligar, 1997).
- a typical reaction contained alkane saturated buffer (700 ⁇ L) and enzyme (0.5 ⁇ M). Addition of NADPH (300 ⁇ L, 200 ⁇ M) in buffer initiated the reaction, and the rate of NADPH oxidation was follow at 340 nm.
- Biocatalytic oxidations were performed under oxygen limited conditions in sealed vials.
- the solution contained alkane (1 mM) in DMSO (1% DMSO) and enzyme (1 ⁇ M) in potassium phosphate buffer (100 mM, pH 8.0).
- the solution was sti ⁇ ed at room temperature for 5 minutes, and the reaction was initiated by the addition of NADPH ( 1 mM).
- reactions with gaseous alkanes were carried out in a sealed 20 mL vial containing enzyme (1.0 ⁇ M) in potassium phosphate buffer (5 mL, 100 mM, pH 8.0). The headspace was filled with either propane or butane. The reaction was initiated with the addition of NADPH (1 mM). The reaction mixture was analyzed directly by GC/MS using an Hewlett Packard 5890 Series II gas chromatograph coupled with an Hewlett Packard 5972
- the GC was fitted with HP FFAP column (crosslinked FFAP, 30m ⁇ 0.25 mm ⁇ ⁇ .25 mm film thickness).
- the condition for octane is as follows: Isothermic at 120 °C for 6 minutes. The condition for hexane and cyclohexane: (1) 100 °C for 5 minutes to 50 °C. (2) 100 °C to 200 °C at 25 °C/min. (3) Isothermic at 200 °C for 2 minutes. The condition for propane and butane: (1) 30 °C for 3 minutes. (2) 30 °C to
- the products were identified with GC/MS using a Hewlett Packard 5890 Series II gas chromatograph coupled with a Hewlett Packard 5989A mass spectrometer.
- the GC was fitted with an HP 1 column (crosslinked methyl silicone gum, 12 m 0.2 mm 0.33 mm).
- HP 1 column crosslinked methyl silicone gum, 12 m 0.2 mm 0.33 mm.
- the temperature gradient is as follows: 1) 40 to 50 °C at 15 °C , 2) 50 to 75 °C at 10 °C /min, 3) 75 to 160 °C/min.
- Authentic standards were used to identify the retention times of the products.
- the products were further verified by matching the fragmentation distributions with a database in the software provided by the instrument manufacturer.
- Wildtype P450 BM-3 is active towards octane
- ketones A possible mechanism for the formation of the ketones is hydroxylation of the alcohol to generate a gem-diol which dehydrates to the co ⁇ esponding ketone (Boddupalli et al., 1992; March, 1992a). Another possible mechanism is via the pinacol rearrangement (March, 1992b). However, it is also possible that traces of protein impurities were responsible for this oxidation, and further tests were therefore performed to validate the results. It was tested whether P450 BM-3 catalyzes the oxidation of 3-octanol to
- the P450 BM-3 enzyme has a kcat 120-fold less and a Km that is 15 times larger (kcat/Km ⁇ 2000 times less) than on its preferred C 16 fatty acid substrate.
- P450BM-3 catalyzes hydroxylation of octane without uncoupling
- Hydrogen peroxide production during catalysis by P450 BM-3 was also monitored in order to determine whether the rate of NADPH oxidation was affected by uncoupling (Oliver et al., 1997; Fruetel et al., 1994). H 2 O 2 was not detected under the experimental conditions, which indicates that the P450 catalyzes the hydroxylation of octane without significant uncoupling. This was consistent with previous reports that indicated efficient coupling for P450 BM-3 acting on unnatural substrates such as styrene or alkyl trimethylammonium compounds (Oliver et al., 1997; Fruetel et al., 1994).
- Directed evolution improves the activity of cytochrome P450 BM-3 towards octane and other substrates
- Mutant library construction was focused on the P450 BM-3 heme domain, which contains the substrate binding site and the rnonooxygenase activity.
- the libraries were generated by e ⁇ or prone PCR (Zhao et al., 1999), using MnCl 2 concentrations of 0.0, 0.05, and 0.1 mM, which yielded 70, 60, and 50% active transformants, respectively.
- the 2 nd generation library was also created with e ⁇ or-prone PCR using VIII118 2C9 as the template, and 9 mutants were isolated that were between 1.8 and 2.5 times more active than VIII 118 2C9. IX18_18 was found to be 2.38 times more active than VIII 118 2C9, and it was used for comparison for the 3 rd generation library.
- the 3 rd generation was produced by StEP recombination (Zhao et al., 1999), using 9 variants from the 2 nd generation, and the most active mutant (1X35 4) was found to be 1.74 times more active than 1X118_18. 1X35_4 was used for comparison for the 4 th generation.
- the 4 th generation was constructed by e ⁇ or-prone PCR using the 1X35_4 as the template.
- the GeneMo ⁇ h PCR Mutagenesis Kit (Stratagene) was used to create the libraries.
- the most active variant (IX 79_1) was 1.35 times as active compared to IX35_4. 1X79 1 was used for comparison for the 5 th generation library.
- the screening procedure was redesigned after generation four, as described under "Materials and Methods.”
- the 5 th generation was produced using IX79_1 as the template. Again, Genemo ⁇ h was used to create the libraries.
- Using a modified screening procedure in generation five resulted in highly improved variants for alkane hydroxylation.
- the best three clones are between 1.24-1.86 times more active than IX79_1.
- the best clone from the 5 th generation (1X139-3) was 23 times more active than wildtype.
- the same library of the 5 th generation was used for a second screen using mutant 1X139-3 for comparison.
- Selected clones including the three best clones from the first screen (1X139-3, 1X139-37 and 1X139-43), were recombined by StEP to produce the library of the 6 th generation.
- Several hundred clones of this new library have been analyzed using the microtiter plate assays, and results indicate further improvement of the alkane hydroxylating activity.
- the mutant "J" V78A, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, and L353V was shown to be 2.0 and 1.6 fold more active for NADPH consumption for propane and butane, respectively.
- step ix35 4 Val78Ala GTA ⁇ GCA His236Gln CAT ⁇ CAG Glu252Gly GAG ⁇ GGG
- Evolved P450 BM-3 variants hydroxylate various substrates
- the chosen screen was sensitive to hydroxylation of the methylene adjacent to the oxygen atom of the su ⁇ ogate the p-nitrophenoxyoctane substrate.
- Activity towards octane, hexane, and cyclohexane was therefore measured, by measuring the rate of NADPH oxidation, which was assumed to be fully coupled to alkane oxidation.
- relative activities determined using NADPH oxidation were assumed to equal the relative activities on the different substrates.
- the kc at for 1X139-3 improved 50 times, while the K m increased 45-fold (FIG. 5).
- the wildtype kinetic values for hexane and cyclohexane were of the same magnitude as for octane.
- IX79_1 in the presence of cyclohexane and NADPH produced cyclohexanol, and cyclohexanone was not detected (FIG. 7). Again, no products were detected with IX79_1 and cyclohexane alone.
- Results also showed that 1X139-3 was able to oxidize propane and butane, as verified with GC/MS.
- the reaction mixture contained enzyme, propane or butane, and in the presence of NADPH produced 2-propanol and 2-butanol (FIG. 8), respectively. Terminal hydroxylation was not detected under these reaction conditions. Since the activity towards medium chain alkanes had already reached a significant level in generation five and 1X139-3 displayed detectable activity for propane oxidation, it was decided to include propane into the screening program in the sixth generation. NADPH consumption assays of the sixth generation showed that mutant 1X139-3 and some new variants also oxidized propane.
- This assay was based on the formation of the pu ⁇ le dye formazan upon reaction of NADPH with Nitro Blue Tetrazolium (NBT) salt in presence of the catalyst, phenazine methosulfate (oxidized, PMS) This color reaction is known as a photometric standard assay for dehydrogenase activity resulting in reduction of their cofactor NADP+ to NADPH.
- the total activity of P450 BM-3 in 96-well plates is relatively low, especially in the presence of an organic solvent that further reduces the fraction of active enzymes.
- the mutant F87A converts the 12-pNCA substrate 4-5fold faster than the wild-type, the K m value is 1.5-fold lower, and the chromophore from 2-pNCA substrate is released completely and not only to 33 % (Farinas, 2001; Schwaneberg, 1999a). Therefore, the mutant F87A converts the 12-pNCA substrate 4-5fold faster than the wild-type, the K m value is 1.5-fold lower, and the chromophore from 2-pNCA substrate is released completely and not only to 33 % (Farinas, 2001; Schwaneberg, 1999a). Therefore, the mutant F87A converts the 12-pNCA substrate 4-5fold faster than the wild-type, the K m value is 1.5-fold lower, and the chromophore from 2-pNCA substrate is released completely and not only to 33 % (F
- thermostability was under selective pressure by using the temperature inducible PRPL-promoter system, and only clones that showed a high activity and a high organic solvent resistance were used as parents for further generations.
- Enyzmes were of highest available purity grade. Enyzmes were purchased from New England Biolabs, Stratagene, and Boehringer Mannheim.
- the P450 BM-3 and P450 BM-3 F87A genes are under the control of the strong temperature inducible PRPL-promoter. Mutated BM-3 F87A variants were cloned into the pUSCI BM-3 vector by using BamHI//EcoRI or Age//EcoRl restriction sites. Transformed clones grown on LBamp plates (Genetix) were transfe ⁇ ed via a colony picker (QPix; Genetix) into 96 well plates (flat bottom; Rainin) containing 120 ⁇ l LB culture supplemented with 12 ⁇ g ampicillin per well.
- TBamp solution was supplemented with 75 ⁇ l trace element solution (0.5 g CaCl 2 2H 2 0, 0.18 g ZnSO 4 ⁇ 7H 2 0, 0.10 g MnS0 4 , H 2 O, 20.1 g Na 2 -EDTA, 16.7 g FeCl 3 x 6H 2 O, 0.16 g CuSO 4 x 5H 2 0, 0.18 g CoCl 2 6H2 ⁇ , add IL H 2 O and autoclave) and 2 mg aminolaevulinic acid per 50 ml TB.
- E. coli cells were grown in this medium for 6 h at 37°C then induced for 14 h at 42°C. All deep-well plates were covered with a taped lid. The original LBamp plates were stored until further use at 80°C after adding 100 ⁇ l glycerol (sterile, 50% (v/v)).
- the 12-pNCA conversion is initiated by adding 20 ⁇ l of a Isocitric co-factor regeneration solution (Isocitric acid 20 mM; dH 2 0, NADP + 3 mM, Isocitric dehydrogenase 0.8 U/ml).
- Isocitric acid 20 mM; dH 2 0, NADP + 3 mM, Isocitric dehydrogenase 0.8 U/ml The reaction was stopped after visible color development by the addition of 50 ⁇ l NaOH (1.5 M).
- the abso ⁇ tion at 410 nm of the clear solution was recorded.
- the P450 BM-3 variants revealing a high activity and a high organic solvent resistance were used for rescreening.
- Tris/HCl buffer (25 mM) and 5 ⁇ l 12-pNCA (15 mM, dissolved in DMSO) were pipetted using the Multimek96.
- 15 ⁇ l THF solution (15 % (v/v); ddH 2 0) were additionally added to each well of the assay plate.
- the 12-pNCA conversion is initiated by adding 20 ⁇ l of a Isocitric co-factor regeneration solution (Isocitric acid 20 mM; dH 2 0, NADP+ 3 mM, Isocitric dehydrogenase 0.8 U/ml).
- the reaction was stopped after visible color development by the addition of 100 ⁇ l UT-buster (NaOH 1.5 M, 1.5 M Urea, 50 % (v/v) DMSO). After 8-12 h incubation and removal of the bubbles using the Bunsen burner the abso ⁇ tion at 410 mn of the clear solution was recorded.
- the P450 BM-3 variants revealing a high activity and a high organic solvent resistance were cultured and expressed in shaking flasks for further characterization.
- the filtrate was loaded on a SuperQ650M anion exchanger column (TosoHaas) and purified as previously described (Schwaneberg, 1999b).
- BM-3 F87A concentrations were measured by CO-difference spectra, as reported by
- the principle of the p-nitrophenoxycarboxylic acid (pNCA) assay system is described in FIG. 9. ⁇ -Hydroxylation of pNCA by P450 BM-3 leads to an unstable hemiacetal intermediate, which spontaneously dissociates into the ⁇ -oxycarboxylic acid and the yellow chromophore p-nitrophenolate.
- This assay system allows a continuous photometric detection of the P450 BM-3 activity, as measured by the maximum turnover rate, i.e., the number of product molecules generated per minute(Schwaneberg et al., 1999a). TABLE 5
- N means that any nucleotide (A, T, G, or C) can be used.
- thermocycler PTC 200 MJ Reseach
- Protocol 1 First mutant generation.
- Buffer 10X (Provided in kits) 5 dNTP mix (Provided in kits) 1
- Protocol 3 Site directed and saturation mutagenesis.
- Annealing temperature 55 °C for pT235, pT471 and pT102
- Random mutations were introduced by PCR into the BM-3 F87A gene coding for 1049 amino acids and a His6 tag at the C-terminal end, under conditions designed to generate an average of one to two amino acid substitutions per gene (protocol 1).
- the mutant library was screened for clones with improved organic solvent resistance by comparing the, activity in the presence and absence of a co-solvent. Approximately 6,520 clones were tested in 96 well plates using the 12-pNCA assay in presence and absence of an organic solvent. The candidates with high total activity and high activity ratios (activity in presence divided by activity in absence of an organic solvent) were selected for re-screening (protocol 1). Positive results of these assays were verified after expressing 39 clones of the first generation in 500 ml scale.
- FIG. 12A Sequencing revealed an exchange of R(CGC) ⁇ 471A(GCT).
- the SM of the position A235 of clone SB3 resulted in no further improvements. All 5 sequenced clones contained an alanine at aa-position 235.
- SM at position 81024 revealed two more active clones, F87ABCIFIO and F87ABCIB6.
- F87ABCIF10 contains a R(AGA)1024T(ACG) substitution and BC1B6 a R(AGA)1024K(AAA) substitution.
- F87ABCIFIO revealed increased total activity of 4.4 fold at 10 % (v/v) DMSO and of 7.9- fold at 2 (v/v) THF compared to the grandparent F87A (Table 7).
- F87ASF5 revealed a higher organic solvent resistance for DMSO and THF than any previous clones.
- F87A5F5 a compared to F87A, increased total activity of 5.5- fold at 10 % (v/v) DMSO and of 10-fold at 2 % (v/v) THF (Table 7) was discovered.
- FIGS. 12A-G show that the evolved mutants, including F87A5F5, exhibit an increased resistance to these organic solvents.
- Purification F87A, F87ASB3 and the wild-type were simultaneously expressed and one by one purified (FIG. 12C).
- a comparison of the organic solvent resistance between lysed crude extracts and purified monoxygenase revealed very similar resistance toward DMSO (FIG. 12C and 13A).
- the resistance of the purified enzyme was reduced between 3-9 %. This reduction might be correlated to hydrophobic impurities in crude protein extracts and relatively small amounts of THF present in the reaction solution.
- FIGs. 12B and 13B show that the activity profile of the fraction of in organic solvent active clones is especially for DMSO shifted to higher co-solvent concentrations.
- the back-mutated clone W5F5 has an increased total activity of 5.9-fold at 25 % (v/v) DMSO and of 3.4 fold at 2 (v/v) THF compared to the wild-type (Table 7).
- improvements are generally for the wild-type mutants, especially in the case of THF, lower compared to the factors for
- the evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for selective oxidation of hydrocarbons.
- alkane hydroxylases among the best known of which are the large, membrane-associated complexes of methane monooxygenase (MMO) and AlkB
- MMO methane monooxygenase
- AlkB the evolved enzyme is water soluble and does not require additional proteins for catalysis.
- the evolved alkane hydroxylase was found to have even higher activity on fatty acids, the presumed biological substrates for P450
- BM-3 which was already one of the most efficient P450s known.
- a broad range of substrates that includes the gaseous alkane propane induces the low to high spin shift, which activates the enzyme.
- the first soluble catalyst for alkane hydroxylation at room temperature, this laboratory-evolved P450 opens new opportunities for clean, selective, hydrocarbon activation for chemical synthesis and bioremediation.
- mutagenic PCR of the heme domain was performed as described (Farinas et al., 2001), using the following primers together with Taq polymerase (Roche): Bamfor 5'-ACAGGATCCATCGATGCTTAGGAGGTCATATG-3' (SEQ ID NO:
- the PCR product was cloned by replacing the BamHI/SacI fragment of p BM-
- clones from this pre-culture were inoculated using a 96 replicator pin into 2 ml deep well plates containing Terrific broth media (TB, 400 ⁇ L), ampicillin (100 mg/L), isopropy- ⁇ -D- thiogalactoside (IPTG, 10 ⁇ M), and ALA (0.5 mM).
- the clones were cultivated at 30 °C for 24-30 hours.
- Cell pellets were frozen at -20 °C and resuspended in phosphate buffer (1 mL, 0.1 M, pH 8.0) containing lysozyme (0.5 mg/mL), DNase I (0.1 ⁇ g/mL) and MgCl 2 (10 mM). After 60 min at 37 °C, the lysates were centrifuged and the supernatant was diluted for activity measurements in 96 well microtiter plates. High throughput determination of enzymatic activity
- Mutant libraries were screened on 8-pnpane as described (See Example 1 and Farinas et al., 2001).
- a cofactor (NADPH) depletion assay was used to determine turnover rates.
- E. coli lysates of the mutants were diluted into 96 well microtiter plates containing phosphate buffer (200 ⁇ l, 0.1 M, pH 8.0), alkane substrate (0.5-lmM), and
- a solid phase NADPH depletion assay was used preselection of the fifth-generation mutant library.
- Cells were grown on LB agar plates containing ampicillin (100 ⁇ g/ml), ALA (1 mM) and isopropyl- ⁇ -d-thiogalactopyranoside (10 ⁇ M).
- the assay solution contained phosphate buffer (0.1 M, pH 8.0), polymyxin B sulfate (100 ⁇ M) as a cell permeabilizer, NADPH (2 mM), and substrate (5 mM) and was sonicated before use .
- a nitrocellulose membrane soaked with this substrate solution was placed directly onto the colonies on the agar plate.
- the sensitivity of the assay was regulated by the NADPH concentration.
- nitro blue tetrazolium 0.5 mg/ml
- phosphate buffer 0.1 M ,pH 8.0
- some crystals of phenazine methosulfate were pipeted directly onto the membrane. Active colonies, which deplete NADPH, were identified as white spots on the pu ⁇ le membrane. Positive colonies were picked with a toothpick and streaked out on fresh agar plates to obtain single colonies for rescreening.
- Dissociation constants for octane, hexane, and lauric acid were determined at 25 °C as described (Modi et al., 1995, hereby inco ⁇ orated by reference in its entirety) from the change in abso ⁇ tion at 418 nm upon substrate binding.
- an enzyme solution 3-5 mM
- buffer 0.1 M potassium phosphate pH 8.0
- Methanol (1%) added to an enzyme solution does not induce a spin state shift.
- reaction solution contained enzyme (3-5 mM) and laurate (ImM) in buffer (20 mM MOPS, 100 mM KCl, pH 7.4). Aliquots of the enzyme/substrate solution were removed and replaced with an equal volume of an enzyme solution.
- Table 8 shows the relative amounts of different products obtained for alkane oxidation, comparing wild-type cytochrome P450 BM-3 to the evolved mutant enzyme 1X139-3.
- the most active BM-3 variant acquired sufficient activity to enable us to screen mutant libraries for activity directly on an alkane (octane) using a high throughput NADPH consumption assay (see Example 1).
- NADPH oxidation alone may not provide an accurate measure of catalytic activity since reducing equivalents from NADPH can be diverted into forming reduced oxygen intermediates (H 2 O or H 2 O 2 ). Therefore all subsequent generations were screened using a combination of the 8-pnpane assay, sensitive to product formation, and NADPH consumption in the presence of octane (see Experimental).
- the P450 enzyme was expressed we also determined that the enzyme had become toxic to the E.
- P450 BM-3 1X139-3 is a better catalyst than known, naturally-occurring alkane monooxygenases acting on their most prefe ⁇ ed substrates (FIG. 15).
- the prefe ⁇ ed substrate for the non-heme iron alkane monooxygenase (AlkB) from Pseudomonas oleovorans is octane; its reported maximal turnover rate is 210 min-1 (Shanklin et al., 1997).
- AlkB is membrane-bound and requires two additional proteins, NADPH-rubredoxin reductase and rubredoxin, for catalytic activity.
- Soluble (sMMO) and particulate methane monooxygenase (pMMO) are large (about 300 kDa), multimeric iron (and in the case of pMMO, iron-copper) enzymes with turnover rates of 200-250 min "1 for gaseous alkanes (methane, 222 min "1 ) (Fox et al., 1990; Fox et al., 1989; Tonge et al., 1977). Rates on alkanes larger than C4 are much lower
- the P450 resting state contains an iron protopo ⁇ hyin IX as a low-spin six-coordinate ferric species with a dissociable water ligated trans to the proximal cysteinate (Ortiz de Montellano, 1995). Substrate binding displaces water and generates a high-spin five-coordinate species.
- the spin state shift causes the redox potential of the heme to increase, which activates the protein for hydroxylation.
- the heme's abso ⁇ tion maximum at 419 nm corresponds to the low-spin species; a shift to 390 nm is characteristic of the high-spin form. This spectral shift is induced in 1X139-3 by all the substrates (FIG.
- Crystal structures of wildtype P450 BM-3 with and without substrate reveal large conformational changes upon substrate binding at the active site (Haines, D.C., Tomchick, D.R., Machius, M. & Peterson, J.A. Pivotal role of water in the mechanism of P450 BM-3. Biochemistry 40, 13456-13465 (2001); Li, H.Y. & Poulos, T.L.
- the substrate free structure displays an open access channel with 17 to 21 ordered water molecules.
- Substrate recognition serves as a conformational trigger to close the channel, which dehydrates the active site, increases the redox potential, and allows dioxygen to bind to the heme.
- Five of the 11 amino acid substitutions in 1X139-3 occur in the region which undergoes the largest conformational change, the F and G helices and the loop connecting them, as well as the I helix across which the F and G helices must slide ( Figure 4).
- the F and G helices serve as a lid which closes over the substrate access channel upon substrate binding. Attempts to engineer catalyst specificity are often limited to altering amino acids directly involved in substrate recognition and binding.
- This mutation may facilitate conformational changes that permit alkanes to bind more favorably.
- Rational engineering of the substrate binding pocket of P450 BM-3 produced a triple mutant (F87V, L188Q, A74G) with increased activity for octane (Appel et al., 2001).
- Directed evolution to produce mutant LX139-3 did not find any beneficial mutations at these sites. The fact that a few amino acid substitutions can produce a significant increase in
- P450 BM-3's activity on fatty acids indicates that natural selection does not place an advantage on maximizing activity, possibly because such a broadly active enzyme is also toxic to the host organism, as it is to E. coli.
- P450 BM-3 mutant 1X139-3 is the fastest alkane hydroxylase known. Easily over-expressed in E. coli, soluble and requiring no additional electron transfer proteins for catalysis, this enzyme should prove an attractive catalyst for selective hydrocarbon oxidation.
- Epoxidation of alkenes is an important reaction in organic synthesis since they are important chemical building blocks.
- the oxirane ring is subject to ring opening by various nucleophiles (oxygen, sulfur, nitrogen, carbon), which yield bifunctional compounds (Carey and Sundberg, 1990).
- epoxides are used in the production of polymers (polyether polyols), as well as glycols, polyglycols, and alkanolamines.
- Optically pure epoxides are useful intermediates in the synthesis of pharmaceuticals, agrochemicals, perfumes, and liquid crystals where chirality plays a critical role in function.
- the mutant 1X139-3 was shown to have broad substrate specificity for alkanes with varying chain length (C8-C3). Furthermore, the variant was also shown to be more active on fatty acids. The activity for alkenes was also investigated, and the mutant is also more efficient in alkene oxidation. 1X139-3 may prove to be a general catalyst for hydrocarbon oxidation, and it may find uses in the fine chemical industry as well as in bioremediation.
- the enzyme was purified and quantified as described above.
- a typical reaction solution contained enzyme (1.0 ml, 1 ⁇ M), alkene (10 ⁇ L, 1.0 mM), and methanol (1%) in potassium phosphate buffer (0.1 M, pH 8.0).
- the solution was incubated at room temperature, and the reaction was initiated by the addition of NADPH (200 ⁇ L, 200 ⁇ M).
- the rate of NADPH oxidation was monitored at 340 nm.
- a typical reaction contained purified enzyme (1.0 ml, 1 ⁇ M), alkene (10 ⁇ L, 1.0 mM), and methanol (1%) in phosphate buffer (0.1 M, pH 8.0). The solution was incubated at room temperature for 5 minutes, and the reaction was initiated by the addition of
- NADPH (200 ⁇ L, 200 ⁇ M). The solution was allowed to stir aerobically at room temperature for 30 minutes.
- the reaction contained enzyme (3.0 ml, 1.0 ⁇ M) in potassium phosphate buffer (0.1 M, pH 8.0), and the resulting solution was sealed in a 20 ml vial with a septum. The head-space was filled with propene and the reaction was initiated by the addition of NADPH (100 ⁇ l, 0.5 mM). The reaction was sti ⁇ ed at room temperature for 1.5 hours. The products were analyzed by gas chromatography/mass spectrometry using an
- the GC was fitted with a HP FFAP column (crosslinked FFAP, 30 m x 0.25 mm x 0.25 ⁇ m film thickness).
- the condition for propene is as follows: (1) 35 °C for 1.7 minutes. (2) 35 to 200 °C at 20 °C/min. (3) 200 °C for 1 minute.
- the condition for styrene, cyclohexene, and 1-hexene is as follows: (1) 100 °C for 4.5 minutes. (2) 100 to 200 °C at 20 °C/min. (3) 200 for 7.0 minutes.
- Authentic standards were used to identify the products by retention time. Products were further verified by matching the fragmentation distributions with a database in the software provided by the instrument manufacturer.
- a typical reaction contained enz zyymi e (1.0 ml, 1 x 10 "6 M) in potassium phosphate buffer (0.1 M, pH 8.0), alkene (1.0 x 10 "3 M), and methanol (1 %) in a vial.
- the reaction was initiated with NADPH (50 ⁇ l, 1.0 x 10 "3 M), and allowed to stir aerobically for 5 minutes.
- 300 ⁇ l of a stock solution of 4-(4-nitrobenzyl)pyridine (5% w/w in acetone) was added to the reaction, and the vial was sealed. The reaction was incubated at 80 °C for 20 minutes and chilled on ice.
- P450 BM-3 is known to form epoxides from various substrates that vary in size and structure (Martinez and Stewart, 2000; Fruetel et al., 1994; Capdevila et al., 1996; Ruettinger and Fulco, 1981; Schneider et al., 1999).
- P450 BM-3 oxidizes arachidonic acid to 18(R)-hydroxyeicosatetraenoic acid and 14(S),15(R)- epoxyeicosatrienoic acid (80 and 20% of total products, respectively), and eicosapentaenoic acid to 17(S),18(R)-epoxyeicosatetraenoic acid (99% total products) (Capdevila et al., 1996).
- the products for styrene, propene, cyclohexene, and 1-hexene were characterized by gas chromatography/mass spectrometry.
- the sole product for stryene was styrene oxide, which is similar to what is found for the wildtype (Martinez et al., 2000).
- the major product from propene oxidation is propene oxide and allyl alcohol is only a minor species.
- the products for styrene, propene, cyclohexene, and 1-hexene were characterized by gas chromatography/mass spectrometry.
- the sole product for stryene was styrene oxide, which is similar to what is found for the wildtype (Fruetel et al., 1994).
- the products from propene oxidation is propene oxide and allyl alcohol. Propene oxidation has not been previously reported by P450 BM-3.
- GC/MS analysis of cyclohexene oxidation by 1X139-3 revealed two products, cyclohexene oxide and 1,2-cyclohexane diol.
- the sole product of the bioconversion was most likely cyclohexene oxide, and 1,2-cyclohexane diol probably occured via base- catalyzed hydrolysis of cyclohexene oxide (see reaction conditions) (March, 1992).
- Cyclohexene oxide was converted to 1 ,2-cyclohexane diol in potassium phosphate buffer (0.1 M, pH 8.0), as verified using GC/MS.
- P450 BM-3 is known to form the corresponding epoxide from cis-9- hexadecenoic acid as well as hydroxylated products (Ruettinger and Fulco, 1981), which demonstrates that there is no mechanistic restraint. Hence, a steric restraint exist that hinders terminal epoxidation. This is further supported by the fact terminal oxidation of fatty acids and alkanes are not observed.
- the 4-(nitrobenzyl)pyridine (4-NBP) (Kim and Thomas, 1992) assay was used to determine epoxide formation for allyl chloride, isoprene, 2-hexene and 3- hexene since the co ⁇ esponding epoxides were not available as standards.
- Nucleophilic attack of the oxirane ring by 4-NBP results alkylation, and the product results in a pu ⁇ le colored
- 1X139-3 in the presence of alkenes induces small spin state shifts. Whereas, the wildtype in presence does not produce a spin state shift. This indicates that the substrate binding properties have been altered. 1X139-3 is very active for alkane hydroxylation, fatty acid oxidation and alkene epoxidation, and the substrate specificity is broad.
- This mutant may be useful a general catalyst for hydrocarbon oxidation. The mutant may be useful for fine chemical synthesis when only one product is detected such as styrene oxide. Alternatively, the mutant may find applications in bioremediation when more than one product is generated.
- Cytochrome P450 monooxygenase or "P450 enzyme” means an enzyme in the superfamily of P450 haem-thiolate proteins, which are widely distributed in bacteria, fungi, plants and animals. The enzymes are involved in metabolism of a plethora of both exogenous and endogenous compounds. Usually, they act as oxidases in multicomponent electron transfer chains, called here P450-containing monooxygenase systems.
- the unique feature which defines whether an enzyme is a cytochrome P450 enzyme is the reduced form of the enzyme which binds carbon monoxide and results in a characteristic abso ⁇ tion maximum at 450 nm..
- Reactions catalyzed by cytochrome P450 enzymes include epoxidation, N-dealkylation, 0-dealkylation, S-oxidation and hydroxylation.
- the most common reaction catalyzed by P450 enzymes is the monooxygenase reaction, i.e., insertion of one atom of oxygen into a substrate while the other oxygen atom is reduced to water.
- P450 BM-3 GenBank Accession Nos.
- J04832 (SEQ ID NO:l) and P14779 (SEQ ID NO:2)); CYP 2C3 (GenBank P00182, SEQ ID NO:3); CYP 2C9 (GenBank PI 1712; SEQ ID NO:4), CYP 2Dlv (GenBank P10633; SEQ ID NO:5), and CYP 108 (GenBank P33006; SEQ ID NO:6).
- An “oxidation”, “oxidation reaction”, or “oxygenation reaction”, as used herein, is a chemical or biochemical reaction involving the addition of oxygen to a substrate, to form an oxygenated or oxidized substrate or product.
- An oxidation reaction is typically accompanied by a reduction reaction (hence the term “redox” reaction, for oxidation and reduction).
- a compound is “oxidized” when it receives oxygen or loses electrons.
- a compound is “reduced” when it loses oxygen or gains electrons.
- An oxidation reaction can also be called an “electron transfer reaction” and encompass the loss or gain of electrons (e.g., oxygen) or protons (e.g., hydrogen) from a substance.
- a "co-solvent” or “indirect solvent” herein means a non-solvent that becomes an acceptable solvent or co-solvent when a small amount of active solvent is added.
- water is a non-solvent for various hydrophobic substances, but the addition of a water-miscible solvent such as DMSO, tetrahydrofuran (THF), methanol, ethanol, propanol, dioxane, or dimethylformamide (DMF), or other solvents known in the art, increases the solubility of hydrophobic compounds in the mixture.
- a water-miscible solvent such as DMSO, tetrahydrofuran (THF), methanol, ethanol, propanol, dioxane, or dimethylformamide (DMF), or other solvents known in the art
- organic solvent resistance of an enzyme means its ability to function, optionally function over time, in an organic solvent or in a co-solvent.
- One way to evaluate organic solvent resistance is to assess the ability of the enzyme to resist a loss of activity over time, in one or more co-solvent systems .
- a more "organic-solvent resistant" enzyme can be one that is more resistant to loss of structure (unfolding) or function (enzyme activity) when exposed to an organic solvent or co-solvent.
- Prefe ⁇ ed systems for testing organic solvent resistance include water/DMSO and water/THF mixtures, for example, 10% (v/v) DMSO and 2% (v/v/) THF.
- Alkane-oxidation capability herein means the capability of a cytochrome P450 enzyme to oxidize at least one alkane.
- the alkane-oxidation capability of an enzyme can be evaluated, for example, by mixing the enzyme with an alkane in the presence of any necessary co-factors, and evaluate whether the alkane is oxidized.
- the capability of a cytochrome P450 variant to oxidize an alkane can be related to the capability of the co ⁇ esponding wild-type P450 to oxidize the same alkane, e.g., by comparing maximum turnover rate, total amount of product formed, or any other suitable measure of enzyme activity.
- Many examples of alternative assays are provided herein.
- Prefe ⁇ ed alkanes for which alkane-oxidation capability can be evaluated include 8- pnpane, octane, hexane, cyclohexane, propane, ethane, and/or butane.
- Alkene-oxidation capability herein means the capability of a cytochrome P450 enzyme to oxidize at least one alkene.
- the alkene-oxidation capability of an enzyme can be evaluated, for example, by mixing the enzyme with an alkene in the presence of any necessary co-factors, and evaluate whether the alkene is oxidized to form an epoxide or hydroxylated product.
- the capability of a cytochrome P450 variant to oxidize an alkene can be related to the capability of the co ⁇ esponding wild-type P450 to oxidize the same alkene, e.g., by comparing maximum turnover rate, total amount of product formed, or any other suitable measure of enzyme activity.
- Prefe ⁇ ed alkenes for which alkane-oxidation capability can be evaluated include octene, hexene, propene, ethene, and/or butene.
- a “protein” or “polypeptide”, which terms are used interchangeably herein, comprises one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds.
- an “enzyme” means any substance, preferably composed wholly or largely of protein, that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions.
- the term “enzyme” can also refer to a catalytic polynucleotide (e.g. RNA or DNA).
- a “native” or “wild-type” protein, enzyme, polynucleotide, gene, or cell means a protein, enzyme, polynucleotide, gene, or cell that occurs in nature.
- a "parent" protein, enzyme, polynucleotide, gene, or cell is any protein, enzyme, polynucleotide, gene, or cell, from which any other protein, enzyme, polynucleotide, gene, or cell, is derived or made, using any methods, tools or techniques, and whether or not the parent is itself native or mutant.
- a parent polynucleotide or gene encodes for a parent protein or enzyme.
- a “mutant”, “variant” or “modified” protein, enzyme, polynucleotide, gene, or cell means a protein, enzyme, polynucleotide, gene, or cell, that has been altered or derived, or is in some way different or changed, from a parent protein, enzyme, polynucleotide, gene, or cell.
- a mutant or modified protein or enzyme is usually, although not necessarily, expressed from a mutant polynucleotide or gene.
- a “mutation” means any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, gene, or cell.
- a mutation occurs in a polynucleotide or gene sequence, by point mutations, deletions, or insertions of single or multiple nucleotide residues.
- a mutation includes polynucleotide alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences.
- a mutation in a gene can be "silent", i.e., not reflected in an amino acid alteration upon expression, leading to a "sequence-conservative" variant of the gene. This generally arises when one amino acid corresponds to more than one codon. Table 9 outlines which amino acids correspond to which codon(s).
- Threonine T ACT, ACC, ACA, ACG Aliphatic hydroxyl
- “Function-conservative variants” are proteins or enzymes in which a given amino acid residue has been changed without altering overall conformation and function of the protein or enzyme, including, but not limited to, replacement of an amino acid with one having similar properties, including polar or non-polar character, size, shape and charge
- amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and can be, for example, at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, as determined according to an alignment scheme.
- sequence similarity means the extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and/or conservation.
- Sequence identity herein means the extent to which two nucleotide or amino acid sequences are invariant.
- Sequence alignment means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the pu ⁇ ose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity/identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA. When using all of these programs, the prefe ⁇ ed settings are those that results in the highest sequence similarity.
- the "activity" of an enzyme is a measure of its ability to catalyze a reaction, i.e., to
- enzyme activity can be represented as the amount of product produced per unit of time or per unit of enzyme (e.g., concentration or weight), or in terms of affinity or dissociation constants.
- the "stability" or “resistance” of an enzyme means its ability to function, over time, in a particular environment or under particular conditions.
- One way to evaluate stability or resistance is to assess its ability to resist a loss of activity over time, under given conditions.
- Enzyme stability can also be evaluated in other ways, for example, by determining the relative degree to which the enzyme is in a folded or unfolded state.
- one enzyme has improved stability or resistance over another enzyme when it is more resistant than the other enzyme to a loss of activity under the same conditions, is more resistant to unfolding, or is more durable by any suitable measure.
- a more "organic-solvent" resistant enzyme is one that is more resistant to loss of structure (unfolding) or function (enzyme activity) when exposed to an organic solvent or co- solvent.
- substrate means any substance or compound that is converted or meant to be converted into another compound by the action of an enzyme catalyst.
- the term includes aromatic and aliphatic compounds, and includes not only a single compound, but also combinations of compounds, such as solutions, mixtures and other materials which contain at least one substrate.
- Prefe ⁇ ed substrates for hydroxylation using the cytochrome P450 enzymes of the invention include alkanes such as propane, ethane, butane, pentane, hexane, cyclohexane, and octane, and alkane derivatives such as alkanes substituted with one or more chemical group such as nitro-, sulphur-, halogen- and oxygen-containing groups, as well as aromatic groups, e.g., p-nitrophenoxyoctane (8-pnpane).
- alkanes such as propane, ethane, butane, pentane, hexane, cyclohexane, and octane
- alkane derivatives such as alkanes substituted with one or more chemical group such as nitro-, sulphur-, halogen- and oxygen-containing groups, as well as aromatic groups, e.g., p-nitrophenoxyoctane (8
- Preferred substrates for epoxidation include alkenes such as propene, ethene, butene, pentene, hexene, cyclohexene, octene, as well as alkene derivatives, which are alkanes substituted with or linked to chemical substituents such as nitro-, sulphur-, halogen- and oxygen- containing groups, and/or aromatic groups.
- alkenes such as propene, ethene, butene, pentene, hexene, cyclohexene, octene
- alkene derivatives which are alkanes substituted with or linked to chemical substituents such as nitro-, sulphur-, halogen- and oxygen- containing groups, and/or aromatic groups.
- cofactor means any non-protein substance that is necessary or beneficial to the activity of an enzyme.
- a "coenzyme” means a cofactor that interacts directly with and serves to promote a reaction catalyzed by an enzyme. Many coenzymes serve as carriers. For example, NAD+ and NADP+ carry hydrogen atoms from one enzyme to another.
- An "ancillary protein” means any protein substance that is necessary or beneficial to the activity of an enzyme.
- oxygen donor means a substance, molecule or compound which donates oxygen to a substrate in an oxidation reaction.
- oxygen donor is reduced (accepts electrons).
- oxygen donors which are not limiting, include molecular oxygen or dioxygen (O 2 ) and peroxides, including alkyl peroxides such as t-butyl peroxide, and most preferably hydrogen peroxide (H 2 O 2 ).
- a peroxide is any compound having two oxygen atoms bound to each other by a single bond, i. e. , dioxygen (O 2 ) has a double bond between the oxygen atoms.
- an “oxidation enzyme” is an enzyme that catalyzes one or more oxidation reactions, typically by adding, inserting, contributing or transferring oxygen from a source or donor to a substrate. Such enzymes are also called oxidoreductases or redox enzymes, and encompasses oxygenases, hydrogenases or reductases, oxidases and peroxidases.
- An “oxidase” is an oxidation enzyme that catalyzes a reaction in which molecular oxygen
- dioxygen or O 2 is reduced, for example by donating electrons to (or receiving protons from) hydrogen.
- a “luminescent” substance means any substance which produces detectable electromagnetic radiation, or a change in electromagnetic radiation, most notably visible light, by any mechanism, including color change, UV absorbance, fluorescence and phosphorescence.
- a luminescent substance according to the invention produces a detectable color, fluorescence or UV absorbance.
- chemiluminescent agent means any substance which enhances the detectability of a luminescent (e.g., fluorescent) signal, for example by increasing the strength or lifetime of the signal.
- a “polynucleotide” or “nucleotide sequence” is a series of nucleotide bases (also called “nucleotides”) in DNA and RNA, and means any chain of two or more nucleotides.
- a nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide (although only sense stands are being represented herein).
- PNA protein nucleic acids
- This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.
- the polynucleotides herein may be flanked by natural regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.
- the nucleic acids may also be modified by many means known in the art.
- Non-limiting examples of such modifications include methylation, "caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
- uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
- charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
- a “coding sequence” or a sequence “encoding” a polypeptide, protein or enzyme is a nucleotide sequence that, when expressed, results in the production of that polypeptide, protein or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme.
- a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
- the coding sequence is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.
- the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
- a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
- gene also called a "structural gene” means a DNA sequence that codes for or co ⁇ esponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription.
- a gene encoding a protein of the invention for use in an expression system, whether genomic DNA or cDNA, can be isolated from any source, particularly from a human cDNA or genomic library. Methods for obtaining genes are well known in the art, e.g., Sambrook et al (supra).
- a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For pu ⁇ oses of defining this invention, the promoter sequence is bounded at its
- Polynucleotides are "hybridizable" to each other when at least one strand of one polynucleotide can anneal to another polynucleotide under defined stringency conditions.
- Stringency of hybridization is determined, e.g., by (a) the temperature at which hybridization and/or washing is performed, and (b) the ionic strength and polarity (e.g., formamide) of the hybridization and washing solutions, as well as other parameters.
- Hybridization requires that the two polynucleotides contain substantially complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated.
- hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of 0.5 ⁇ SSC at 65°C) requires that the sequences exhibit some high degree of complementarity over their entire sequence.
- Conditions of intermediate stringency such as, for example, an aqueous solution of 2xSSC at 65°C
- low stringency such as, for example, an aqueous solution of 2xSSC at 55°C
- lxSSC is 0.15 M NaCI, 0.015 M Na citrate.
- Polynucleotides that hybridize include those which anneal under suitable stringency conditions and which encode polypeptides or enzymes having the same function, such as the ability to catalyze an oxidation, oxygenase, or coupling reaction of the invention.
- expression system means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
- Common expression systems include bacteria (e.g., E. coli and B. subtilis) or yeast (e.g., S. cerevisiae) host cells and plasmid vectors, and insect host cells and Baculovirus vectors.
- a "facile expression system” means any expression system that is foreign or heterologous to a selected polynucleotide or polypeptide, and which employs host cells that can be grown or maintained more advantageously than cells that are native or heterologous to the selected polynucleotide or polypeptide, or which can produce the polypeptide more efficiently or in higher yield.
- the use of robust prokaryotic cells to express a protein of eukaryotic origin would be a facile expression system.
- Prefe ⁇ ed facile expression systems include E. coli, B. subtilis and S. cerevisiae host cells and any suitable vector.
- transformation means the introduction of a foreign (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence.
- the introduced gene or sequence may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by the genetic machinery of the cell.
- a host cell that receives and expresses introduced DNA or RNA has been "transformed” and is a "transformant" or a
- the DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.
- vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
- Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology.
- a common type of vector is a "plasmid”, which generally is a self-contained molecule of double-stranded DNA, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
- Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
- Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
- Prefe ⁇ ed vectors are described in the Examples, and include without limitations pcWori, pET-26b(+), pXTDl4, pYEX-Sl, pMAL, and pET22-b(+).
- Other vectors may be employed as desired by one skilled in the art. Routine experimentation in biotechnology can be used to determine which vectors are best suited for used with the invention, if different than as described in the Examples. In general, the choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of this invention.
- express and expression mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a co ⁇ esponding gene or DNA sequence.
- a DNA sequence is expressed in or by a cell to form an "expression product" such as a protein.
- the expression product itself e.g. the resulting protein, may also be said to be “expressed” by the cell.
- a polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.
- a polynucleotide or polypeptide is "over-expressed" when it is expressed or produced in an amount or yield that is substantially higher than a given base-line yield, e.g. a yield that occurs in nature.
- a polypeptide is over-expressed when the yield is substantially greater than the normal, average or base-line yield of the native polypolypeptide in native host cells under given conditions, for example conditions suitable to the life cycle of the native host cells.
- Isolation or purification of a polypeptide or enzyme refers to the derivation of the polypeptide by removing it from its original environment (for example, from its natural environment if it is naturally occurring, or form the host cell if it is produced by recombinant DNA methods).
- Methods for polypeptide purification are well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercu ⁇ ent distribution.
- polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence.
- the polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix.
- antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents.
- Other purification methods are possible.
- a purified polynucleotide or polypeptide may contain less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated.
- a "substantially pure" enzyme indicates the highest degree of purity which can be achieved using conventional purification techniques known in the art.
- Cytochrome P450 Structure, Mechanism, and Biochemistry (Ed.: Ortiz de Montellano, P. R. ), Plenum Press, New York, NY., 1995, pp. 3-48.
- Zhao HM et al. Nature Biotechnol 1998;16:258-261.
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