FR2959514A1 - Preparing an industrial yeast strain Saccharomyces cerevisiae, comprises selecting strain, integrating an expression cassette/deletion of a cassette and inducing expression of a gene and deleting at least two copies of open reading frame - Google Patents

Abstract

Preparing an industrial yeast strain Saccharomyces cerevisiae, comprises: (i) selecting and obtaining the strain; (ii) integrating an expression cassette or deletion, in the genome of the yeast, of a cassette e.g. (a) combination of open reading frame (ORF) of the gene of Pichia stipitisXRm encoding for the mutated reductase xylose enzyme; (iii) inducing the expression of at least one gene from each step of the non-oxidative part of pentose phosphate pathway; and (iv) deleting at least two copies of ORF of Saccharomyces cerevisiaeGRE3 gene. Process for preparing an industrial yeast strain Saccharomyces cerevisiaeto produce ethanol from a medium comprising at least one pentose, comprises: (i) selecting and obtaining an industrial yeast strain of Saccharomyces cerevisiaecapable of producing high concentrations of ethanol, at least 14.5% (v/v), preferably at least 16% (v/v) on hydrolyzate of cereals, in conditions of simultaneous saccharification and fermentation (SSF) and at a temperature of 35[deg] C; (ii) integrating at least one expression cassette or deletion, in the genome of the yeast of step (i), at least one cassette such as (a) the combination of open reading frame (ORF) of the gene of Pichia stipitisXRm encoding for the mutated reductase xylose enzyme using the NADH, H+ as a cofactor preferably in place and instead of NADPH, H+/promoter and terminator of Saccharomyces cerevisiae, where the cassette is flanked upstream and downstream of recombinogenic regions allowing its integration targeted in the genome, (b) the combination of open reading frame (ORF) of Pichia stipitisXDH gene coding for the xylitol dehydrogenase enzyme/promoter and terminator of Saccharomyces cerevisiae, where the cassette is flanked upstream and downstream of recombinogenic regions allowing its targeted integration into the genome, (c) combination of open reading frame (ORF) of the gene of Saccharomyces cerevisiaeXKS1 encoding the xylulokinase enzyme/promoter and terminator of Saccharomyces cerevisiae, where the cassette is flanked upstream and downstream of recombinogenic regions allowing its integration targeted into the genome; (iii) inducing the expression of at least one gene from each step of the non-oxidative part of pentose phosphate pathway by placing it under the control of a promoter of a gene of the glycolysis strongly expressed during alcoholic fermentation; and (iv) deleting at least two copies of the open reading frame (ORF) of Saccharomyces cerevisiaeGRE3 gene encoding aldose dehydrogenase. Independent claims are included for: (1) yeast strain Saccharomyces cerevisiaeEG3 deposited under the NO: l-4295 at the CNCM; (2) yeast strain Saccharomyces cerevisiaeEG2 deposited under the NO: l-4294 at the CNCM and the strain resulting from the evolution directed strain EG3; (3) yeast strain Saccharomyces cerevisiaeEG1 deposited under the NO: l-4293 at the CNCM, and strain resulting from the directed evolution of the strain EG3; (4) yeast strain Saccharomyces cerevisiaeEG9 deposited under the NO: l-4450 at the CNCM; and #a process for producing ethanol, from a medium comprising at least one pentose, comprising fermenting the yeast.

Description

"Process for the preparation of industrial yeast, industrial yeast and application to the production of ethanol from at least one pentose"

The present invention relates to the field of processes for obtaining ethanol-producing yeast strains, the yeasts thus produced, and the industrial production of ethanol from said yeasts. More particularly, the present invention relates in its most general aspect to a process for the preparation of yeasts from so-called industrial Saccharomyces cerevisiae strains, said yeasts and their application to the industrial production of ethanol from industrial media containing at least a pentose.

The common point of the approaches of the prior art of the field consists of processes aimed at improving so-called laboratory strains with known and / or constructed genetic inheritance and whose ethanol production abilities are generally studied in under standardized and optimal laboratory conditions and conditions. Indeed, the scientific literature as well as the patent documents analyzed by the Applicant teach, most often, processes for obtaining haploid or diploid strains, weakly tolerant to stress, especially at high concentrations of ethanol and / or at high temperatures. and / or fermentation inhibitors. In addition, these methods for the most part require, for these strains, the use of auxotrophy markers and / or antibiotic resistance markers which can disqualify them for later use in an industrial setting for obvious reasons of cost and sometimes public health. The growth properties of previously developed strains are generally insufficient and these strains have never been confronted with the requirements of biomass production on an industrial scale, namely to name but three: high growth rate, drying, stability in storage. If so-called fermentative performances (aptitude for the anaerobic production of ethanol) are obtained in synthetic or defined laboratory media with these prior strains, they can not generally be transposed in industrial environments containing complex mixtures, for example from treatment residues of cellulosic or lignocellulosic materials which contain toxic compounds which can inhibit at different levels the cellular machinery of yeast, in particular furfural, HMF, phenolic derivatives, acetic acid. In addition, the ability to "scale up" or scale up these prior ethanol production processes is rarely documented. The Applicant has thus found that there is still the need for a process for the preparation of a so-called industrial yeast which takes into account both the constraints of the yeast and at the same time those of an end user in his applications. especially in terms of industrial production of ethanol at low cost and high yield. It is precisely to the satisfaction of this dual need that the present invention aims. Also, the present invention firstly relates to a process for the preparation of an industrial yeast strain Saccharomyces cerevisiae capable of producing ethanol from a medium comprising at least one pentose and which comprises the following steps: i) Selecting and providing a so-called industrial strain of yeast Saccharomyces cerevisiae capable of producing high concentrations of ethanol, of at least 14.5% (v / v) and preferably at least 16% on a cereal hydrolyzate, under conditions of Saccharification and Simultaneous Fermentation (SSF) and at a temperature of 35 ° C, integrate at least one expression or deletion cassette into the genome of the yeast of step (i), the so-called at least one cassette being selected from the group consisting of: a. The open reading frame (ORF) association of the Pichia stipitis XRm gene encoding the mutated xylose reductase enzyme using NADH, H + as a preferential co-factor in place of NADPH, H + / Saccharomyces promoter and terminator cerevisiae, said cassette being flanked upstream and downstream of recombinogenic regions allowing its targeted integration into the genome, b. The open reading frame (ORF) association of the Pichia stipitis XDH gene encoding the enzyme xylitol dehydrogenase / Saccharomyces cerevisiae promoter and terminator, said cassette being flanked upstream and downstream of recombinogenic regions allowing its integration. targeted in the genome, c. The open frame reading (ORF) association of the Saccharomyces cerevisiae XKS1 gene encoding the xylulokinase / Saccharomyces cerevisiae promoter and terminator enzyme, said cassette being flanked upstream and downstream of recombinogenic regions allowing its targeted integration. in the genome, (iii) inducing the expression of at least one gene from each step of the non-oxidative portion of the pentose phosphate pathway by placing it under the control of a promoter of a gene of the glycolysis strongly expressed during an alcoholic fermentation, and (iv) Deleting at least two copies of the open reading frame (ORF) of the Saccharomyces cerevisiae GRE3 gene encoding an aldose dehydrogenase.

The method according to the invention has, in particular, the following advantages: For the yeast manufacturer, it allows: the construction of a yeast strain Saccharomyces cerevisiae aneu / polyploid, prototrophic to allow the production of biomass on sources simple carbon, nitrogen, phosphorus in inexpensive media such as by-products of the sugar industry such as molasses for example, - to have a yeast strain Saccharomyces cerevisiae having a maximum growth rate (p max ) between 0.37 h-1 and 0.5 h-1 - to have a yeast strain Saccharomyces cerevisiae which, when it is produced according to a process as described in the reference book "Yeast Technology" (2nd edition, 1991, G. Reed and TW Nagodawithana, published by Van Nostrand Reinhold, ISBN 0-442-31892-8), provides a biomass production yield of at least 45 g yeast solids per 100 g d equivalent - The availability of a yeast strain Saccharomyces cerevisiae resistant to the drying process as described in EP 511108 and US 5 741 695, the loss of fermentative activity after drying should not exceed 30. %. - the production under industrial conditions (particularly inexpensive medium, good biomass yield, ready-to-use dry yeast) of a fresh or dry yeast from a yeast strain Saccharomyces cerevisiae genetically stable, robust because including tolerant at high concentrations of ethanol and capable of producing, for example from hemicellulosic biomasses, ethanol at least 80 g / L and at an elevated temperature of the order of 30 to 40 ° C.

Moreover, for the ethanol producer, the advantage of the process according to the invention is to have an active yeast (fresh - liquid or compressed - or dry), obtained according to a production method as described in US Pat. "Yeast Technology" work, from a yeast strain Saccharomyces cerevisiae as defined in the preceding paragraph which is: 15 - capable, under the conditions of SSF described in the patent document WO 2004/046333, to ferment at 35 ° C a cereal hydrolyzate to a minimum ethanol concentration of 14.5% (v / v) - capable, under the SSF conditions described in WO 2004/046333, of fermenting a hydrolyzate at 35 ° C cereals up to a minimum ethanol concentration of 16% (v / v).

The results of the process according to the invention are all the more remarkable since they were obtained from a so-called industrial strain, aneu / polyploid prototroph and in fact having a genetic material much more complex than that of a strain. so-called laboratory, making the consequences of modifications of said industrial strain at least unpredictable. This complex genetic background, which is specific to industrial strains, makes it all the more difficult to obtain genetically modified strains that are ultimately free of markers of resistance to antibiotics, particularly when many genetic targets need to be modified. Strains that are free of antibiotic resistance markers are clearly preferable for health and environmental reasons.

The prototrophic strains according to the invention have the advantage of growing on simple sources of carbon, nitrogen and phosphorus. But this characteristic makes the transformation vectors available in the scientific community (vectors using auxotrophic markers) inoperative. It is therefore necessary to have available tools / vectors using antibiotic resistance markers, said tools / vectors being advantageously constructed to ultimately allow the excision of these markers. The construction of the yeasts according to the invention required, for example, the use of 4 different positive markers (geneticin, phleomycin, hygromycin and blasticidine).

The strains according to the invention are aneu / polyploid is a characteristic generally encountered in industrial yeasts that are from the natural environment. The phylogenetic past of these strains is at the origin of this peculiarity. But it is an additional difficulty encountered when it is desired to disrupt / inactivate all copies of a given gene. However, this aneupolyploidy character is generally at the origin of many properties of interest of industrial yeasts (growth rate, resistance to different stresses, phenotypic stability).

In addition, the present inventors after long researches found with surprise that with the method according to the invention, implemented from the so-called industrial strain that they had selected: - the introduction of expression cassettes. and deletion did not induce any genomic instability in the modified yeast, which saw its genetic heritage improved, - it was not obligatory to regulate its XK activity. There is in fact a controversy in the prior art concerning the overexpression of XKS1 in laboratory strains, thus better defined, which suggests that the Xylulokinase activity must be finely regulated (Jin et al AEM 2003, 69,495-503 vs Ho et al., 1999, Advances in Biochemical Engineering / Biotechnology, Vol.65, pp. 163 - 192). In particular, the inventors have shown that with said industrial strain it is possible to carry out: the deletion of at least two copies of the S. cerevisiae GRE3 gene (the Gre3p enzyme being an aldose reductase which consumes NADPH H + which is produced largely via the oxidative part of the pentose pathway) in said industrial strain according to the invention has reduced by the same amount the consumption of NADPH, H + by said enzyme, the overexpression of XKS1 naturally, that is, step c) of the method according to the invention could be omitted, since XKS1 is an endogenous gene of S. cerevisiae. This overexpression could in particular be made possible after cyclic cultures when the method 3o comprises a subsequent step of directed evolution, as described below. In a preferred variant of this first object, the cassettes a), b) and c) of the step (ii) are all integrated. It is preferred in this variant that the XRm gene is a gene that has the following mutation, K270M, or a mutated XR gene that has a mutation (s) different (s) such as K270R described by Watanabe and al., Microbiol. 2007, 153, 3044-3054, so that this mutation confers on the encoded enzyme to use NADH; H + as a preferred co-factor instead of NADPH; H +.

Even more preferably, in this variant, the cloning of the mutated XR gene (XRm) is performed in a single copy. It is also preferred in this variant that said at least one gene of each step of the non-oxidative portion of the pentose phosphate pathway of step (iii) is selected from the group consisting of RPE1, RK11, TKL1 and TAL1 and that said promoter of a highly expressed glycolysis gene in alcoholic fermentation is selected from the group consisting of the promoter TDH3 for RPE1, RK11 and TKLI and PGK1 for TAL1.

According to complementary or alternative characteristics, in the process for preparing an industrial yeast strain Saccharomyces cerevisiae according to the invention: the promoter in step (ii) is chosen from the group consisting of ADH1, ADH2, PGK1, TDH3, PDC2 and GAL1 / 10, preferably ADH1, and the terminator is CYC1 or the proper terminator of the modified gene, such as the TAL1 terminator for the TAL1 gene. there is provided a subsequent step of directed evolution comprising the following successive steps of subjecting the yeast obtained to (i) a mutagenesis, (ii) a growth in cyclic cultures under 02 limited in a medium comprising said at least one pentose, and (iii) a selection by aerobic growth on a solid medium containing glycerol as the only carbon source, so as to provide non-deficient respiratory mutants of said yeast that exhibit anaerobic growth in the presence of a medium comprising said at least one pentose. In this variant, the mutagenesis of step (i) is preferably carried out under moderate conditions, namely moderate mutagenesis with 100 to 500 J / cm 2 and, more preferably, 300 J / cm 2 of ultraviolet rays. 254 nm. These conditions only result in a mortality of 10% of the population subjected to Ultra-violet. The inventors have thus surprisingly shown that with such a low controlled mortality, it is possible to reduce by 10 the duration of the cyclic culture directed evolution step necessary to obtain mutants capable of fermenting said at least one pentose. The survival rate is determined by plating on agar plates containing a nutrient medium an identical volume of the cell suspension before and after mutagenesis. The number of colonies is determined after 48 hours of growth. Preferably, the limitation at 02 of step (ii) of this variant is carried out thanks to a partial overpressure i0 in the equipment used (for example, flasks or fermenters) due to the CO2 produced. The cyclic cultures according to this variant, under the fermentation conditions of said at least one pentose, make it possible to enrich the population with mutants capable of fermenting said pentose and this in a time of 4 to 8 weeks and preferably 6 weeks which is relatively short and very interesting compared to what would be obtained by chemostat as described by Kuyper et al. (2004) 4, 655-664.

Although the "small" respiratory deficiency phenotype may be consistent with the fermentation criteria of the said at least one pentose, in this variant the present inventors have carried out a step of eliminating "small" yeasts because this phenotype is incompatible with the production processes. industrial yeasts within the meaning of the invention.

The present invention also relates to the industrial yeast strain Saccharomyces cerevisiae EG3 directly obtained by the process according to the invention before the step of directed evolution and which consists of the yeast strain deposited on April 14, 2010 at the CNCM (collection microbial culture of the Institut Pasteur) under No. 1-4295 under the conditions of the Budapest Treaty. The subject of the present invention is also the industrial yeast strain Saccharomyces cerevisiae EG2 directly obtained by the process according to the invention after the step of directed evolution and which consists of the yeast strain deposited on April 14, 2010 at the CNCM (collection microbial culture of the Pasteur Institute) under No. 1-4294 under the terms of the Budapest Treaty.

The subject of the present invention is also the industrial yeast strain Saccharomyces cerevisiae EG1 directly obtained by the process according to the invention after the step of directed evolution and which consists of a variant incapable of sporulating the yeast strain EG2 and deposited on April 14, 2010 at the CNCM (national collection of microbial cultures of the Pasteur Institute) under No. 1-4293 under the conditions of the Budapest Treaty. A strain that is unable to sporulate has an advantage in terms of environmental protection because it eliminates the risk of dissemination of transgenes by conjugation with other yeasts of the surrounding medium. This characteristic is even more important when genetically modified microorganisms are used on a very large scale.

More preferably, the industrial yeast strain Saccharomyces cerevisiae obtained is practically or totally free of markers including resistance to antibiotics.

The Saccharomyces cerevisiae yeast strains prepared according to the present invention, according to the criteria defined above, have preserved, after introduction of the genetic modifications and other mutations generated during the directed evolution step, their genotypic and phenotypic characteristics after a complete industrial process of production. In particular, the yeasts produced exhibit alcohol production kinetics, xylose consumption kinetics and a maximum amount of alcohol produced strictly identical to the yeast strain prior to the application of a complete industrial process.

Furthermore, the industrial characteristics of the strain selected beforehand as described above (growth rate, production yield, drying ability) remain unchanged.

The subject of the present invention is also a process for the production of ethanol from a medium containing at least one pentose by fermenting using a yeast according to the invention, mentioned above, or such as obtained by a process according to the invention as just described. Preferably, the ethanol production process has the following alternative and / or additional characteristics: it comprises a step of saccharification and simultaneous fermentation (SSF) in the presence of hexose polymers, mainly consisting of glucose, and at least one enzyme capable of hydrolyzing them, said at least one pentose is xylose, said medium is chosen from the group consisting of hydrolysates of lignin, cellulose, hemicellulose and dextrins. the average release rates of hexoses, mainly glucose, are of the order of 2.8 to 5.6 g / L / h with an extracellular concentration in hexose, predominantly in glucose, zero. The present inventors have implemented the ethanol production process according to the invention under the actual conditions of SSF (Fermentation and Simultaneous Saccharification), as practiced in the industry for the production of ethanol, in particular in the USA. .

The concentrations of sugars used (70 g / kg of Xylose and 130 g / kg of Glucose equivalent) are known to the Applicant, the maximum concentrations that can be encountered in the field. All published tests referring to the fermentation of xylose were carried out with significantly lower concentrations of total sugars.

Other features and advantages of the invention will emerge even more clearly on reading the detailed description which will follow, including exemplary embodiments with result tables which are given for purely illustrative and non-limiting purposes, and for the understanding of which Reference is made to the accompanying drawings in which: Figure 1 illustrates an XDH overexpression vector of Pichia stipitis, Figure 2 is a graph showing glucose released by enzymatic hydrolysis as a function of time under three initial release conditions (A). ): 2.8 g / L / h, (B): 3.9 g / L / h and (C): 5.6 g / L / h, Figures 3 to 5 show for a strain conforming to EG3 invention, the evolution of concentrations of glucose, xylose, ethanol, xylitol, glycerol over time, Figure 3 corresponds to the dose of enzyme A, Figure 4 to the dose of enzyme B and Figure 5 at the dose of enzyme C, Figures 6 to 8 show for yet another according to the invention EG1, the evolution of glucose, xylose, ethanol, xylitol, glycerol concentrations over time, FIG. 6 corresponds to the dose of enzyme A, FIG. 7 to the dose of enzyme B and FIG. 8 at the dose of enzyme C, FIG. 9 shows the evolution of the moving averages of the xylose consumption rates by each of the two strains EG1 and EG3 during the three tests carried out as a function of the moving average of the glucose concentrations in the medium over the same time interval, FIG. 10 is a graph illustrating the specific rate of production of xylitol (g / L / h) as a function of the specific rate of xylose consumption in the medium (g / L / h) for the two EG1 and EG3 strains, FIG. 11 is a graph illustrating the mass loss as a function of the fermentation time in the presence of xylose (70 g / L) by the two EG3 and EG2 evolutions (the ethanol strain). RedTM is the starting strain).

EXAMPLES EXAMPLE 1 The selection of the industrial strain is as described in the description above. All the DNA sequences that were used for the various transformations aimed at overexpressing a gene were obtained from a known standard vector (pUC type of E. Coli) in which were declined: the targets integration; selected promoters / terminators per gene of interest and resistance markers that will be removed later (see below). An example of a vector used for the overexpression of the XDH of Pichia stipitis is illustrated in FIG. 1. For the disruption of the copies of the GRE3 gene of the selected industrial strain, the inventors used PCR amplifiers from a plasmid of the type pUG6 (Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH, Nucleic Acids Res 1996 Jul 1, 24 (13): 2519-2524).

The yeast transformation step was carried out according to Gietz, R.D. and R.A. Woods. (2002) Transformation of yeast by the Liac / SS Carrier DNA / PEG method. Methods in Enzymology 350: 87-96. The yeast strains according to the invention, respectively EG1, EG2 and EG3 were deposited at the CNCM on April 14, 2010 and were assigned to them Numbers 1-4293, 1-4294 and I-4295 respectively. Strains according to the invention have the following genotype: Ethanol RedTM, BUD5 :: ADH1 p-PsXRm (K270M) -CYCI t; HO :: ADH1 p-PsXDH-CYC 1 t; BUD5 :: ADH1 p-XKS 1-CYC 1 t; RPEI :: TDH3p-RPEI -CYC 1 t; RKI1 :: TDH3p-RK1-CYC 1 t; TKL I :: TDH3p-TKL 1-CYC 1 t; TAL 1 :: PGK 1 p-TAL 1-CYC 1 t; AGRE3

EXAMPLE 2 The mutagenesis of the strains obtained in the preceding Example was carried out moderately, namely 100 to 500 J / cm 2 and preferably 300 J / cm 2 of ultraviolet at 254 nm. After one week of culture at 32 ° C. in a YE (Yeast Extract 0.5%) type medium containing 7% of Xylose with stirring, without aeration - the 02 limitation being achieved thanks to a partial overpressure in the flasks due to CO2 produced during fermentation - one ml of the culture is used to re-seed the same medium. This operation is repeated 6 times. The cells are finally spread on YE glucose agar medium at 20 g / l. Isolated colonies are collected and then successively cultured on: YE Glycerol at 20 g / L and aerobically to remove "small" i.e. respiratory-impaired mutants; 5 YE Glucose to check their growth rate; YE Xylose to identify the most interesting clones.

EXAMPLE 3 The inventors first tested in anaerobic batch culture the strains genetically modified so as to be able to convert xylose to ethanol as obtained in Example 2. They were able to measure the apparent Km for xylose in vitro. measuring the rate of production of CO2 as a function of the xylose concentration and that during the fermentation of xylose as sole source of carbon: it is 6.16 M. Among the strains tested, three are selected under SSF conditions in order to evaluate their ability to metabolize xylose at the same time as glucose. SSF assays were performed with low enzyme doses ranging from 4.3 to 8.6 pKat so that the glucose release rate was low and the residual glucose concentration during fermentation be zero.

The strains tested were the strain EG3 and the strain EG1 respectively obtained before and after the step of directed evolution. The cells of strain EG1 are incapable of sporulating. The ability to sporulate these cells is determined by microscopic observation of tetrads or asci obtained by culturing the cells for 48 hours on a poor medium type SAA (0.8% sodium acetate, 1.5% agar).

Test conditions. The tests were carried out at 32 ° C., pH 5. The seeding was 0.5 g yeast solids per kg of initial must. Progressive enzymatic release of glucose was achieved by using dextrins and adding glucoamylase. The glucoamylase doses used were low (between 4.3 pkat and 8.6 pkat) in order to simulate the kinetics of hydrolysis of the cellulose by cellulases taking place in 72 hours. The initial glucose release rates tested were (A): 2.8 g / L / h, (B): 3.9 g / L / h and (C): 5.6 g / L / h, respectively. .

As generally observed, the kinetics of hydrolysis decreases when 60-70% of the dextrins have been hydrolysed and the average glucose release rates are then of the order of 0.4-0.45 g / L / h for the three conditions with a slightly faster speed with condition A. (Figure 2).

In practice, the medium used is a synthetic medium containing yeast extract (5 g / kg), urea (2.5 g / kg), dipotassium phosphate (1 g / kg), a citrate buffer. 12 mM as well as minerals and vitamins.

Results obtained EG3 strain. Figures 3 to 5 show the evolution of glucose, xylose, ethanol, xylitol, and glycerol concentrations over time. These figures show that in 72 h: the strain EG3 consumed between 30 and 33 g of xylose according to the tests whereas it had hardly consumed in batch xylose. 17 to 20 g of xylitol were produced on the 30-33 g of xylose consumed is a ratio of 0.5 g / g for condition A and a ratio of 0.6 g / g for conditions B and C. - The amounts of glycerol produced were small, lower than the quantities expected in this type of test. Overall, the three trials yielded equivalent results.

TABLE 1: Molecules produced and consumed by strain EG3 (in g per kg of initial medium at 72 h) Dose Glucose xylose Biomass Xylitol glycerol Ethanol Carbon dioxide balance A 103.3 32.8 10 17.0 1.1 51.7 101% B 114.4 33.2 10 19.6 1.6 57.7 103% C 122.2 30.9 10 17.9 2.5 60.6 103% Strain EG1 Figures 4 to 6 give the evolution of glucose, xylose, ethanol, xylitol, and glycerol concentrations over time. These figures show that in 72 h: the EG1 strain consumed between 45 and 60 g of xylose according to the tests is almost twice as much as the strain EG3. 13 g of xylitol were produced on the 45-60 g of xylose consumed or a ratio of 0.2 g / g for the three tests the amounts of glycerol product were low but higher than the strain EG3.

Overall, the three trials yielded equivalent results.

TABLE 2 Molecules produced and consumed by strain EG1 (in g per kg of initial medium at 72 h) Glucose xylose dose Biomass Xylitol glycerol Ethanol Carbon dioxide balance A 102.5 60.61 10 12.74 2.86 65.49 100% B 115.6 51.65 10 11.90 4.17 69.34 102% C 120.9 45.96 10 9.86 5.33 70.18 103% Comments / assumptions on the results achieved, in particular relating to at the glucose concentration allowing xylose uptake.

The results obtained on glucose-xylose batch show a loss of mass loss slope which seems to indicate that the glucose was first consumed then the xylose was then consumed at a much slower rate.

The SSF test as carried out in the Example makes it possible to evaluate whether the uptake of the xylose would be greater in flow entering non-zero glucose but zero glucose concentration.

Figure 9 shows the evolution of moving averages of xylose consumption rates by each of the two strains in the three tests performed as a function of the moving average glucose concentration in the medium over the same time interval. The results show: - that there is xylose consumption by the strains tested at 5 g / kg of glucose in solution, that the rate of consumption of xylose observed with the strain EG3 is 2 times lower than those observed with strain EG1.

In a preferred variant of the ethanol production method according to the invention, as shown in the Example, the slow and controlled release of glucose allows the cells not to undergo a strong change in osmotic pressure and to avoid engorgement of fermental pathways (Glycolysis, Pentoses Phosphate, and Carriers of sugars) which would limit the use of xylose.

Surprisingly and remarkably, with the process according to the invention, the cells are capable of metabolizing 62 g / l of xylose in 50 hours in a medium rich in carbon sources of the order of 200 g / l (Example with 130 g / L 5 'of glucose equivalent and 70 g / L of Xylose). These conditions as drastic have, to our knowledge, never been described. According to the SSF tests carried out: the specific speed of the EG1 strain is 0.5 g xylose / g MS yeast / h. 12.74 g of xylitol were formed for 60.61 g of xylose consumed, ie a ratio of 21 g / 100 g. 15 20 25 30

Claims (12)

REVENDICATIONS1. Process for the preparation of an industrial yeast strain Saccharomyces cerevisiae capable of producing ethanol from a medium comprising at least one pentose and which comprises the following steps: (i) Selecting and providing a Saccharomyces yeast strain so-called industrial cerevisiae capable of producing high concentrations of ethanol of at least 14.5% (v / v) and preferably at least 16% (v / v) on a cereal hydrolyzate, under conditions of Saccharification and Simultaneous Fermentation (SSF) and at a temperature of 35 ° C. (ii) Integrating at least one expression or deletion cassette into the yeast genome of step (i), said at least one cassette being selected from the group consisting of: a. The open reading frame (ORF) association of the Pichia stipitis XRm gene encoding the mutated xylose reductase enzyme using NADH, H + as a preferential co-factor in place of NADPH, H + / Saccharomyces promoter and terminator cerevisiae, said cassette being flanked upstream and downstream of recombinogenic regions allowing its targeted integration into the genome, b. The open reading frame (ORF) association of the Pichia stipitis XDH gene encoding the enzyme xylitol dehydrogenase / promoter and terminator of Saccharomyces cerevisiae, said cassette being flanked upstream and downstream of recombinogenic regions allowing its targeted integration in the genome, i0 15c. The open reading frame (ORF) association of the Saccharomyces cerevisiae XKS1 gene encoding the xylulokinase enzyme / Saccharomyces cerevisiae promoter and terminator, said cassette being flanked upstream and downstream of recombinogenic regions allowing its targeted integration into the genome, (iii) inducing the expression of at least one gene from each step of the non-oxidative portion of the pentose phosphate pathway by placing it under the control of a promoter of a highly expressed glycolysis gene alcoholic fermentation, and (iv) Delineate at least two copies of the open reading frame (ORF) of the Saccharomyces cerevisiae GRE3 gene encoding an aldose dehydrogenase. 2. Method according to claim 1, characterized in that the Saccharomyces cerevisiae promoter is chosen from the group consisting of ADH1, ADH2, PGK1, TDH3, PDC2 and GAL1 / 10, preferably ADH1, and that the terminator 20 of Saccharomyces cerevisiae is constituted by CYC1 or by the proper terminator of the modified gene. 3. Method according to any one of claims 1 to 2, characterized in that it comprises a subsequent step of directed evolution comprising the following successive steps of subjecting the obtained yeast to (i) mutagenesis, (ii) growth in 02-restricted cyclic cultures in a medium comprising said at least one pentose, and (iii) aerobic growth selection on a solid medium containing glycerol as sole carbon source, so as to provide non-deficient respiratory mutants. of said yeast which show growth in anaerobiosis in the presence of a medium comprising said at least one pentose. 4. Yeast strain Saccharomyces cerevisiae EG3 deposited under No. I-4295 at the CNCM. 5. Yeast strain Saccharomyces cerevisiae EG2 deposited under No. I-4294 at the CNCM. Strain resulting from the directed evolution of the strain EG3. 6. Yeast strain Saccharomyces cerevisiae EG1 deposited under No. I-4293 at the CNCM. Strain resulting from the directed evolution of the strain EG3. Yeast as obtained by a process according to any one of claims 1 to 3, characterized in that it is free of markers of resistance to antibiotics. 8. A method for producing ethanol, from a medium comprising at least one pentose, by fermentation of a yeast according to any one of claims 4 to 7, or as obtained by a process according to any one of any of claims 1 to 3. 9. Method according to the preceding claim characterized in that it comprises a step of Saccharification and Simultaneous Fermentation (SSF) in the presence of hexose polymers, mainly consisting of glucose, and at least one enzyme capable of hydrolyzing them. 10. Method according to any one of claims 8 to 9 characterized in that said at least one pentose is xylose. 11. Process according to any one of claims 8 to 10, characterized in that said medium is chosen from the group formed by the hydrolysates of lignin, cellulose, hemicellulose and dextrins. 12. Method according to the preceding claim taken in combination with claim 9, characterized in that the average release rates of hexoses, mainly glucose, are of the order of 2.8 to 5.6 g / L / h with an extracellular concentration in hexose, mainly in glucose, null.