WO2009115660A2

WO2009115660A2 - In vivo selection

Info

Publication number: WO2009115660A2
Application number: PCT/FR2009/000112
Authority: WO
Inventors: Julien Sylvestre
Original assignee: Julien Sylvestre
Priority date: 2008-01-31
Filing date: 2009-02-02
Publication date: 2009-09-24
Also published as: FR2927090A1; WO2009115660A3

Abstract

The invention relates to a method for the in vivo selection of live cells of microorganisms on the basis of the enzyme activity expressed by said cells outside the cytoplasm of their cytoplasm. The invention uses a partition system for the selection, and can particularly be used for cellulases.

Description

SELECTION IN VIVO

DESCRIPTION

The invention relates to the field of directed evolution and cell selection in vivo. In particular, the invention relates to the selection of cells on the basis of an enzymatic activity located outside the cytoplasm of said cells.

Throughout the document, "cell" refers to a living cell of microorganism: bacterium, yeast, microalgae or protozoan.

The invention is concerned with the selection of cells based on the enzymatic activity of one or more enzymes produced by these cells and located outside the cytoplasm of said cells. These enzymes may be free outside the cells - "extracellular enzymes" - or be attached to the outer, non-cytoplasmic, cell - "peri-cellular enzymes".

These extracellular and peri-cellular enzymes constitute a large part of the enzymes used in the industry, in particular, without this list being considered exhaustive: lipases, proteases, amylases, phytases, laccases and cellulases.

The object of the invention is to select, within a diverse population of cells, cells having a given level of extracellular or peri-cellular enzymatic activity.

The said diverse population of cells can be directly isolated in the wild, for example by taking samples in special natural environments such as hot springs, contaminated soils, the ruminant rumen. Alternatively, the diverse population of cells may be artificially formed, for example by heterologous expression of genes that have been synthesized or mutated or recombined or by metagenome techniques.

By selection is meant a method of molecular biology inspired by natural evolution that enriches a diverse population of cells into positive cells relative to a given criterion. The selection operates directly on a population ("en masse") and is opposed in this sense to screening at low or high rate, which is another method in which the cells are evaluated one by one against the criterion of interest. . When possible, the selection, which by definition operates in a massively parallel manner, makes it possible to sort quickly and simply a number of cells much larger than the screening. This increases the probability of isolating positive cells relative to the criterion of interest. The advantageous characteristics of each of the selected cells can be validated subsequently by a screening step.

The selection can be absolute and only the cells adapted to a given selection criterion are recovered at the end of the process, the others being eliminated. More frequently, selection brings relative enrichment: most or all of the cells in the initial population divide but some, better adapted to the chosen selection criterion, divide faster than others and the proportion of these cells in the total population thus increases mechanically at course of time. When several selection cycles with relative enrichment are performed consecutively, a population comprising an increased fraction of improved cells is ultimately obtained.

The selection of extracellular or peri-cellular enzymatic activities is made complicated by a phenomenon called "cross-feeding": the selective advantages provided by the enzymes of a cell are shared with the neighboring cells, by diffusion, and these advantages Selective measures are therefore "diluted", which limits or cancels the possibility of selection.

For example, one volume of medium contains cellulose as the sole source of carbon and two different cells, one "positive" producing efficient cellulases and the other "negative" producing no cellulases. In the presence of cellulose in the culture medium, the positive cell produces and exports cellulases which degrade cellulose into oligomers and carbohydrate monomers which can then be imported into the cytoplasm to obtain the carbon necessary for its growth. However, the same oligomeric and carbohydrate monomers derived from the activity of the positive cell can also diffuse and benefit the negative cell which, although it did not need to produce any cellulase, can thus grow using the degradation products of cellulose as a carbon source. Thus cross-feeding by eliminating or weakening the link between activity of a cell and benefit or disadvantage for said cell complicates or prevents selection.

A solution was recently proposed by Zhiliang Fan and al. ("Theoretical analysis of selection-based strain improvement for microorganisms with growth depend on extracytoplasmic enzymes" Biotechnology and Bioengineering

(2005) 92 (1): 35-44). According to these authors, in the case of peri-cellular enzymes, when placed under sufficient dilution conditions, diffusion and therefore the phenomenon of cross-feeding are limited because of the proximity between each cell, its enzymes, which are exposed on its surface, and the substrate. The authors propose to solve the problem of extracellular enzymes, secreted in the volume of culture and not bound to the cell, making them, artificially, pericellular.

This solution, however, has several major disadvantages and limitations.

First of all, even in the case of enzymes which are natively pericellular, there is necessarily a non-zero diffusion of the products and therefore a non-zero level of cross-feeding, which limits the efficiency of the selection. In addition there is a bias, which concerns the shape of the cells: in the case of an insoluble substrate of size equivalent to the size of the cells, cells having a flattened shape will be advantageously compared to cells having a more rounded shape.

Above all, the solution of Zhiliang Fan et al. proposes modifying extracellular enzymes to render them peri-cellular and benefit from the relative decrease in cross-feeding for these peri-cellular enzymes. We will see that this modification, by attaching initially extra-cellular enzymes to the surface of the cell to make them peri-cellular, is not always possible, it is not never simple and that it very often has negative consequences on the selection.

The methods used to expose enzymes or other proteins on the cell surface are called "display" methods. A first method, the "cell surface display" uses a fusion of the enzyme of interest with a protein of the cellular periphery, for example the protein Aga2p in yeast.

Another method, "phage display", uses the expression in a bacteriophage.

In the case of the phage display, as in the case of the cell-display surface, the molecular biology procedures used are complex and therefore do not make it possible to quickly select, in routine, diversified populations of cells.

The cell surface display is limited to relatively small population sizes (approximately 10 ⁵ different cells), which does not make it possible to isolate rare cells.

The phage display has the disadvantage of being limited to prokaryotic proteins, which excludes a whole class of proteins that express themselves poorly or in an inactive form in prokaryotes.

Two additional problems are, on the one hand, the difficulty of expressing multi-enzymatic complexes and, on the other hand, the risk of bias due to the presence of a fusion protein. In addition, in some cases, problems of accessibility of the substrate, related to the presence of the cell attached to the enzyme, can arise.

The method according to the invention provides a robust and elegant solution to these drawbacks and limitations of current methods of selection applied to extracellular and peri - cellular enzymes.

SUMMARY DESCRIPTION OF THE INVENTION

The method according to the invention is a method of selecting cells on the basis of the activity of extracellular or peri-cellular enzymes produced by these cells characterized by the use of a selective liquid medium sealed in a sealed manner. The process isolates each cell in an isolated compartment, impervious to the products of the enzymatic activity of said cell.

This tightness makes it possible to eliminate any cross-feeding phenomenon without any manipulation of molecular biology being necessary.

The process according to the invention starts from a diversified population of living cells of microorganisms in a selective medium. This population is dispersed in the compartments of an emulsion, under conditions such that the growth and cell growth rate in the compartments depends on their adaptation to the selection criterion introduced by the selective medium. The oily phase is chosen so as to limit the exchanges between the compartments.

In a preferred embodiment, the method according to The invention comprises the following four steps:

a) preparing an aqueous phase containing a diverse population of cells expressing extracellular or peri-cellular enzymes and a substrate of these enzymes allowing a selection of said cells on the basis of the activity of these enzymes;

b) an oily phase containing oil and surfactants is prepared separately - this oily phase is formulated so that it is tight to the products resulting from the activity of the enzymes of step a) on the substrate of the step a);

c) is formed from the aqueous phase of step a) and the oily phase of step b), an inverse emulsion consisting of droplets of the aqueous phase dispersed in the oily continuous phase;

d) the cells are allowed to grow within these droplets of emulsions.

In a particular embodiment, at the end of step d), the two aqueous and oily phases are separated, the cells are recovered from the aqueous phase and dispersed in a new selective medium, optionally a medium formulated to exert a selection pressure stronger than the initial selection pressure, thus obtaining a new aqueous phase. An oily phase identical to that prepared in step b) is prepared separately and steps c) and d) are iterated from these two new aqueous and oily phases. It is thus possible to carry out several selection cycles, typically around ten, leading to successive enrichments. After one or more cycles of selection in emulsion, the emulsions can be directly spread on a solid support, selective or not. Alternatively, the aqueous phase can be separated from the oily phase before spreading.

In a preferred embodiment, to ensure a good stability of emulsion I ¹ and a total absence of exchange between the aqueous compartments is used a so-called oily phase crystallizable. This crystallizable oily phase is solid at the growth temperature but liquid at the emulsification temperature. In addition, a suitable combination of oils and surfactants is chosen to keep the cells alive at the emulsification temperature. The following formula can be used in particular: decane, x% v SpanβO, x% v Cholesterol where x is between 1 and 6, preferably 2 and 4.

Preferably, in step c), emulsification conditions (initial concentration of the cells, drop size of the prepared emulsion and aqueous volume fraction) are used, which are such that, on average, one cell per unit is used. drop.

DETAILED DESCRIPTION OF THE INVENTION

The method according to the invention is a method of selecting cells on the basis of the activity of extracellular or peri-cellular enzymes produced by these cells characterized by the use of a selective liquid medium sealed in a sealed manner. The process isolates each cell in an isolated micro-compartment, sealed to the products of the enzymatic activity of said cell. This tightness makes it possible to eliminate any cross-feeding phenomenon without any manipulation of molecular biology being necessary.

c) from the aqueous phase of step a) and the oily phase of step b), an inverse emulsion is formed; consisting of droplets of the aqueous phase dispersed in the oily continuous phase;

d) the cells are allowed to grow within these droplets of emulsions.

In step a), the selection factor may be a substrate that provides a source of carbon (C) or a source of nitrogen (N), without which the cell is unable to grow and divide .

In step b), the use of a suitable combination of one or more oils and one or more surfactants ensures the stability of the emulsion for several days or up to several weeks.

In step c), the emulsions can be created directly by manual shear, using a stirring device such as a magnet stir bar, a mixer or ultrasound. Alternatively, in step b), the emulsion can be created using a microfluidic type device.

In step d), the cells are allowed to grow under suitable temperature conditions for 6 hours to 15 days and preferably for 12 hours to 4 days.

The empirical distribution of the number of cells per drop can be approximated by a Poisson distribution, which has a variance equal to the mean. To limit the frequency of the drops containing two or more cells, it is possible to set an average number of cells per drop of less than 1, for example 0.1 or 0.01. A good monodispersity of the emulsions is preferable because it improves the efficiency of the selection. However, it is obvious that in view of the large number of cells, the selective value is continuously distributed, and the large number of drops, irrespective of the polydispersity of the drops, a relative enrichment of the most suitable cells is observed .

Creating drops of a suitable diameter, which are stable for the duration of the experiment, at the temperature of the experiment and in which the cells can grow, is a non-trivial problem. In a preferred embodiment, in order to ensure a very good stability of the emulsion and a total absence of exchanges between the aqueous compartments, the invention solves this problem by using a so-called crystallizable oily phase. This crystallizable oily phase is solid at the growth temperature but liquid at the emulsification temperature. In addition, a suitable combination of oils and surfactants is chosen to keep the cells alive at the emulsification temperature.

The crystallizable inverse emulsion formula used depends on the growth temperature of the cells, the size desired for the drops, the stability required for the emulsion, the type of emulsification and the method used to separate the phases after pushes cells. This formula may include, but may not be considered exhaustive: octane, decane, docecane, kerozene, mineral oil, suppocire, silicone oil, fluorocarbon oil, edible vegetable oil, unpurified triglycerides and one or more surfactants of the following list: PGPR, SpanβO, Span80, Arlacel P135, Tween, cholesterol. The following formula can be used: decane, x% v SpanβO, x% v cholesterol where x is between 1 and 6, preferably 2 and 4.

In a particular embodiment, at the end of step d), the two aqueous and oily phases are separated. For phase separation, it is possible in particular to use a physical process such as centrifugation or a chemical process such as the addition of surfactants destabilizing the inverse emulsions. The cells are then recovered from the aqueous phase and dispersed in a new selective medium, optionally a medium formulated to exert a selection pressure higher than the initial selection pressure, thus obtaining a new aqueous phase. An oily phase identical to that prepared in step b) is prepared separately and steps c) and d) are iterated from these two new aqueous and oily phases. It is thus possible to carry out several selection cycles, typically around ten, leading to successive enrichments. After one or more cycles of selection in emulsion, the emulsions can be directly spread on a solid support, selective or not. Alternatively, the aqueous phase can be separated from the oily phase before spreading.

Depending on the oil used, and whether the emulsion is crystallized or not, it is possible to allow certain gases (especially oxygen, which allows the growth of aerobic cells) to diffuse into the continuous phase. which can help renew the environment). Preferably, the oil is crystallized and does not allow any diffusion of gases or molecules and the cells are selected anaerobically. The culture of Anaerobic microorganisms are industrially advantageous because they avoid having to stir the cultures to oxygenate the medium, which significantly increases the cost of the process.

Cellulose is the most abundant biopolymer on earth. Having enzymes capable of degrading cellulose effectively could replace a significant portion of liquid fuels and petroleum products with substitutable products derived from cellulose. Cellulases therefore represent an enzymatic activity which has a major industrial interest. These enzymes are necessarily extracellular or peri-cellular because their substrate, cellulose, can not enter the cell. Degrading cellulose to its monomers (C5 and C6 sugars) involves several enzymes. These enzymes can be produced by cells and then separated from said cells and added after expression to a reactor containing the cellulosic biomass. Alternatively, living cells can be used directly within a bioreactor containing the cellulose to produce the necessary enzymes in situ.

The use of cell-surface display to select cellulases has been proposed by many authors for several years without significant results being obtained on real biomass.

The use of phage display for selecting cellulases has also been described in principle by Anzhou M. et al (World Journal of Microbiology & Biotechnology (2008) 24 (10): 2003-2009) however this article exposure of a non mutant cellulase on the surface of a phage, without any selection being made.

To our knowledge, and despite the strong competition in this area of major interest, no author has, to date, shown significant improvement in the activity of cellulases or microorganisms on an insoluble substrate using methods of the phage display or cell-surface display type.

It is an object of the present invention to provide a new selection method capable of improving the efficiency of cellulases and other enzymes acting on insoluble substrates, isolated or provided by living cells. Examples 1 and 2 describe two variants of this method applied to cellulases.

Another variant of the present invention which allows selection of microorganism consortium is described in Example 3.

Example 1 Selection of Cellulolytic Bacteria

This first example is a native strategy, which starts from microorganisms with good properties of substrate use, anaerobic cellulolytic bacteria.

We start from a 90OmL liquid sample of a natural medium known to contain cellulolytic bacteria, for example the rumen of a ruminant, compost, a hot spring, sewage or a paper mill. In general, the cellulases of these bacteria are expressed as cellulosomes attached to the cell wall. To this liquid is added 10OmL of a source of cellulosic biomass, for example sugarcane bagasse or corn cake, said biomass having previously undergone a physical and / or chemical pre-treatment, for example a pretreatment of AFEX type, dilute acid, basic or ionic liquid. The characteristic dimensions of the biomass particles obtained after the pre-treatment are of the order of 50 microns. The final volume of the aqueous phase is therefore 1 liter.

2L of an oily phase of the following composition is independently prepared: decane, 3% v SpanβO, 3% v cholesterol. It is placed at 45 ⁰ C and is introduced gradually by volumes of 10mL vigorously stirring with a broad spatula the aqueous phase in the oily phase so as to obtain a homogeneous inverse emulsion. The quality of the emulsion is checked using a high magnification optical microscope. The emulsion is cooled to a temperature of 30 ^° C., the oily continuous phase crystallizes and is incubated for 24 hours. The emulsion is recovered, heated to 45 ° C. and the aqueous and oily phases are separated by centrifuging for 5 minutes at 4000 g. The aqueous phase is recovered and 10OmL of this phase is spread on a large rectangular petri dish (30 × 20 cm) containing as sole carbon source of the cellulose. A few hours are allowed to grow and among the first colonies that appear, a significant proportion are clones of effective cellulolytic bacteria.

EXAMPLE 2 Heterologous Expression in Yeast of Extra Cellular Cellulose Fungi This second example is based on a recombinant strategy, starting from microorganisms having good enzyme production properties and supporting relatively high product (ethanol) titers. Mutation mutants of endobetaglucanase, cellobiohydrolase and betaglucosidase of Trichoderma Resei are carried out by mutagenesis methods known to those skilled in the art. These three genes are heterologously expressed in the yeast Saccharomyces cerevisiae. The corresponding enzymes are secreted by the yeast cell. The application of the four emulsion selection steps of the invention then makes it possible to enrich the population with cells that correctly express effective cellulases.

Example 3 Selection of a Consortium of Two Cell Types with Complementary Metabolic Activities

This third example uses a coculture of two yeast populations of the Pichia pastoris species, one of which expresses a scaffold protein and the other expresses a scaffold binding cellulase. After introduction of the genes encoding the scaffold protein and the cellulase respectively, the two yeast populations are separately mutated by methods known to those skilled in the art to obtain two diverse populations. The numbers of cells are adjusted by measuring the optical density at 600 nm and the volumes are adjusted to obtain identical optical densities in the two populations, and then identical volumes of these populations are mixed. This results in a co-culture of two types of cells that produce synergistic molecular components. The application of the four emulsion selection steps of the invention, by fixing, in step c), a number of cells per drop equal to 2, then allows to enrich the population in pairs of cells that together correctly express effective cellulases. It has been shown that the use of such co-cultures may, in some cases, lead to better efficacy than the simultaneous expression of all the necessary components) metabolic activity by a single cell. The compartmentalization provided by the process according to the invention is, to our knowledge, the first method allowing such direct selection of consortia.

Claims

1- A method of selecting cells characterized in that a diverse population of living cells of microorganisms is transferred into a selective medium and then immediately dispersed in the compartments of an emulsion, in that the growth and the growth rate of the cells in the compartments depends on their adaptation to the selection criterion introduced by the selective medium and in that no exchange takes place between said compartments.

2- Method according to claim 1) comprising the following four steps:

c) from the aqueous phase of step a) and the oily phase of step b), an inverse emulsion consisting of droplets of the dispersed aqueous phase is formed; in the oily continuous phase;

d) the cells are allowed to grow within these droplets of emulsions.

3- The method of claim 2) wherein the emulsion used is crystallizable and wherein the oily phase is solid at the temperature at which the cells grow during the selection.

4- The method of claim 3) wherein the oily phase used in the following formula: decane, x% v Span60, x% v cholesterol where x is between 1 and 6, preferably 2 and 4.

5. Method according to any one of the preceding claims wherein the average number of cells per drop is less than or equal to 3.

6. Process according to any one of claims 2) to 5) wherein the cells are incubated in the drops of the emulsion in step d) for a period of between 6 hours and 15 days, preferably between 12 hours and

4 days. 7- The method of claim 6) wherein, after step d), the selected cells are subjected to one or more new iterations of the method according to claim 6) and wherein the selection pressure is maintained or increased at thread selections cycles.

8- Process according to claim 7) at the end of which the aqueous phase is separated from the oily phase and spread on a solid support, selective or not.

9) Process according to claim 7) after which the emulsion is directly spread on a solid support, selective or not.

10- Process according to any one of the preceding claims, wherein the selective medium used comprises as sole carbon source of the cellulose and where the cells express on the outside or on their surface, free or in the form of cellulosomes, one or more enzymes. cellulolytic cells and where these cells are selected for their ability to degrade cellulose.

11- Cells obtained at the end of the process according to any one of the preceding claims.