DK2646544T3

DK2646544T3 - Fremgangsmåde til fremstilling af en industriel gær, industriel gær og anvendelse deraf ved fremstilling af ethanol fra mindst en pentose

Info

Publication number: DK2646544T3
Application number: DK11794083.3T
Authority: DK
Inventors: Thomas Desfougeres; Georges Pignede
Original assignee: Lesaffre & Cie
Priority date: 2010-12-03
Filing date: 2011-12-02
Publication date: 2017-01-16
Also published as: PL2646544T3; ES2608784T3; BR112013013696A2; AR084087A1; WO2012072793A1; CA2819323A1; US20120142067A1; CL2013001569A1; PT2646544T; BR112013013696B1; CA2819323C; EP2646544A1; AU2011334846A1; EP2646544B1; FR2968313B1; MY180908A; MX347050B; HRP20161752T1; AU2011334846B2; MX2013006209A

Description

A METHOD FOR PREPARING AN INDUSTIAL YEAST, INDUSTRIAL AND APPLICATION TO THE PRODUCTION OF ETHANOL FROM AT LEAST ONE

PENTOSE

TECHNICAL FIELD

The present description relates to the field of methods for obtaining yeast strains producing ethanol, to the yeasts thusproduced, and to the industrial production of ethanol from said yeasts. More particularly, the present description in its most general aspect relates to a method for preparing yeasts from so-called industrial strains of Saccharomyces cerevisiae, to said yeasts and their application to the industrial production of ethanol from industrial media containing at least one pentose, notably xylose.

TECHNICAL BACKGROUND

The point in common of most approaches of the prior art in the field consists in methods aiming at improving strains with known genetic heritage and/or constructed genetic heritage and which capabilities for producing ethanol are generally studied in media and under « ideal » laboratory conditions.

Indeed, scientific literature as well as patent documents analyzed by the Applicant most often teach methods for obtaining haploid or diploid strains that are little tolerant to stresses notably to strong concentrations of ethanol and/or to high temperatures and/or to fermentation inhibitors. Furthermore, these methods for the most part require resorting, for these strains, to the use of auxotrophy markers and/or markers of resistance to antibiotics which may disqualify them for subsequent use in an industrial medium for obvious reasons of cost or even sometimes of health or respect of the environment.

The growth properties of strains previously developed are generally insufficient and these strains have never been confronted with biomass production requirements on an industrial scale, i.e. to only mention three of them: strong growth rate, drying capacity, storage stability.

If so-called fermentative performances (anaerobic ethanol production capacity) are obtained in synthetic or defined media, so-called laboratory media, with these previous strains, they generally cannot be transposed to industrial media including, for example, complex mixtures stemming from celluloseprocessing residues which contain toxic compounds which can inhibit the yeast's cell mechanism at different levels, notably furfural, HMF, phenolic derivatives, acetic acid. Furthermore, the « scale up » or scale transposition capacity of these earlier ethanol production methods is seldom documented.

Document WO 2008/133665 teaches the production of alcohol from a yeast strain with a « genetic background » of the type: - Mutated STP15 gene (F117S, Y195H, K218R). - Exogenous genes encoding XI / XR / XDH or XK. "XI" designates xylose isomerase, "XR" designates xylose reductase, "XDH" designates xylitol dehydrogenase, and "XK" designates D-xylulokinase.

Document WO 2005/113774 describes a recombinant operon comprising two nucleic acid sequences respectively encoding an XI of E.coli and an XDH of Trichoderma reesei in the context of the production of xylitol.

The document: PloS Genetics of Gavin Sherlok et al., published on May 13th 2010, describes an XDH1 gene which is present in some specific Saccharomyces cerevisiae strains, which may encode a xylitol dehydrogenase.

The document in the name of David Brat, Eckard Boles and Beate Wiedemannn, in Appl. Environ. Microbiol., April 2009, Vol. 75, No.8, p. 2304-2311, describes the expression, in Saccharomyces cerevisiae, of the xylose isomerase gene from Clostridium phytofermentans.

It emerges from the review of these documents of the prior art, as well as from the work of the inventors of the present invention, that given the very different genetic backgrounds/heritages of the strains of Saccharomyces cerevisiae yeasts used with the purpose of growing on and/or fermenting xylose, the consequences, for example, of an overexpression and/or of thr deletion of native genes and/or of the introduction of one or more heterologous genes cannot be predicted.

SUMMARY OF THE INVENTION

Thus, the Applicant having studied many strains of the alcohol, brewery and bakery types, has surprisingly noticed that the introduction of certain expression or deletion cassettes in yeast strains so as to express therein a metabolic route XI-XDH made them particularly efficient at producing ethanol. Furthermore, the

Applicant noticed that the introduction of the nucleic acid encoding XI is not sufficient by itself for efficiently fermenting xylose.

In general, the Applicant noticed that the introduction of expression cassettes of a gene encoding an enzyme capable of transforming any carbohydrate (notably xylose) into xylulose (D-xylulose) and of a gene encoding an enzyme capable of transforming any pentol (notably xylitol) into xylulose in a single step, made all the strains thus modified particularly effectient at growing on and/or fermenting xylose.

By "enzyme capable of transforming xylose into xylulose", is meant a xylose isomerase enzyme.

By "enzyme capable of transforming xylitol into xylulose in a single step" is meant a xylitol dehydrogenase enzyme.

Indeed, the Applicant confirmed that, unlike the so-called fungal route associating XR and XDH, the so-called bacterial isomerization route (an example of which is that of C. phytofermentans), when it is applied, does not involve any co-substrates. Furthermore, this route offers the possibility of avoiding accumulation of xylitol which is a metabolic intermediate present in the fungal route and which may significantly reduce the ethanol production yield.

Very recently, in certain S. cerevisiae strains, notably those for winemaking, a gene XDH1 was identified as being essential for the metabolism of xylose of said strains (PLoS genetics 2010, 6, 1-17). Also, the Applicant noticed that even by suppressing the GRE3 gene of the modified strains, there are other parasitic activities that may transform xylose into xylitol (aldose reductase activity), which is detrimental for the XI activity thereby reducing the sought ethanol yield.

The work carried out by the Applicant shows that reinforcement of the xylitol dehydrogenase activity results in the absence of inhibition of XI and therefore in the stimulation of this route, which allows the production of ethanol, from a medium including at least xylose, with a good kinetic yield.

In other words, in the prior art, two different pathways were explored so as to make possible the fermentation of xylose by yeast: the so-called fungal pathway, which makes use of the XR and XDH enzymes; and the so-called bacterial pathway, which makes use of the XI enzyme. The present description combines the enzymes from both of these two pathways in an original manner, so as to obtain an improved result.

The invention is defined in the appended claims.

The description particularly relates to a yeast strain comprising at least one copy of an exogenous gene encoding a xylose isomerase, and one copy of an exogenous gene encoding a xylitol dehydrogenase.

By "exogenous" gene (as opposed to "endogenous") is meant a gene which is not naturally present in the yeast species of interest. The gene encoding a xylitol dehydrogenase may be a XYL2 gene, but in this case it is an XYL2 gene from another species than that of the strain of interest.

The description also relates to a method of preparing a yeast strain comprising at least a copy of an exogenous gene encoding a xylose isomerase, and a copy of an exogenous gene encoding a xylitol dehydrogenase.

The description also relates to a method of producing ethanol using the yeast strains according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS - Fig. 1 illustrates an overexpression vector of XDH from Pichia stipitis. - Fig. 2 illustrates an overexpression vector of XI from Clostridium phytofermentans. - Fig. 3 is a graph illustrating the production of ethanol versus the fermentation time at 32°C for two yeast strains according to the invention after directed evolution, and for the Ethanol red™ strain. The tested clones were inoculated with an amount of 5g of dry mass/L in a YF + 70 g/L xylose medium, - Fig. 4 is a graph illustrating the production of ethanol versus the fermentation time at 32°C for two yeast strains, one according to the invention after directed evolution, the other strain resulting from the first one but after substitution of the copy of the XDH gene by a marker of resistance to kanamycin (KanMX4). The tested clones were inoculated with an amount of 5g of dry mass/L in a YF + 70g/L xylose medium. - Fig.5 is a graph illustrating the production of ethanol (Y-axis, in g per kg of medium) as a function of the fermentation time at 32°C (X-axis, in hours) for two yeast strains according to the invention EG8 and EG10. The tested clones were inoculated at 0.25 g yeast dry matter per kg of YF medium containing 70 g/L xylose as the sole source of carbon.

DETAILED DESCRIPTION OF EMBODIMENTS

Thus, the first object of the present description is a method for preparing a Saccharomyces cerevisiae yeast strain capable of producing ethanol from a medium including at least one pentose (notably xylose) and which comprises the following steps: (i) selecting (or providing) a Saccharomyces cerevisiae yeast strain (ii) integrating the following expression cassettes into the genome of the yeast of step (i), a. the association of the open reading frame (ORF) type of a gene encoding an enzyme capable of transforming any carbohydrate, notably xylose, into xylulose under the dependency of a promoter and of a Saccharomyces cerevisiae terminator, said cassette being flanked upstream and downstream with recombinogenic regions allowing its targeted integration into the genome, b. the association of the open reading frame (ORF) type of a gene encoding an enzyme capable of transforming in a single step any pentol, notably xylitol, into xylulose under the dependency of a Saccharomyces cerevisiae promoter and terminator, said cassette being flanked upstream and downstream with recombinogenic regions allowing its targeted integration into the genome, (iii) inducing the expression of at least one gene of each step of the non-oxidative portion of the phosphate pentose route as well as of at least one gene encoding xylulokinase (XKS1) by placing them under the dependency of a promoter of a gene, notably a glycolysis gene, neither repressed by anaerobiosis nor by catabolic repression and strongly expressed during alcoholic fermentation, and (iv) deleting at least one copy or preferably at least two copies of the open reading frame (ORF) of the Saccharomyces cerevisiae GRE3 gene encoding an aldose reductase. Preferably, all copies of the open reading frame (ORF) of the Saccharomyces cerevisiae gene GRE3 gene are deleted.

The XKS1 gene is preferably the gene reported in GenBank under number 853108.

The GRE3 gene is preferably the gene reported in GenBank under number 856504.

Preferentially, the gene of step (ii)a is a gene XI encoding the xylose isomerase enzyme selected from those present in the genomes of the Clostridium, Pyromyces, Bacteroides, Streptomyces, Haemophilus, Burkholderia, Enterococcus,

Thermotoga, Fusobacterium, Geobacillus, Arthrobacter, Ciona, Physcomitrella, Cellvibrio, Chitinophaga, Saccharopolyspora, Salinibacter genera.

The XI gene is preferably selected from a gene of Clostridium phytofermentans, Saccharopolyspora erythraea, Salinibacter ruber or Piromyces sp. E2.

According to a preferred embodiment, the sequence of the XI gene is the SEQ ID NO:l nucleotide sequence (which corresponds to the sequence of the XI gene from Clostridium phytofermentans, described in document DE 102008031350). Alternatively, the XI gene has a sequence which has at least 70% identity, preferably at least 75% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least 98% identity, or at least 99% identity, with SEQ ID NO:l, and it encodes a functional xylose isomerase enzyme.

According to another embodiment, the XI gene has a sequence encoding a polypeptide having the amino acid sequence SEQ ID NO:2 (which corresponds to the sequence of the XI protein from Clostridium phytofermentans described in document DE 102008031350). Alternatively, said polypeptide has a sequence having at least 70% identity, preferably at least 75% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least 98% identity, or at least 99% identity, with SEQ ID NO:2 and it has a xylose isomerase activity.

According to the present invention by «transforming any pentol into xylulose in a single step » is meant direct oxidation of xylitol into xylulose and this by the same and single enzyme (xylitol dehydrogenase).

Preferentially, the gene of step (ii)b, is a Pichia stipitis gene encoding the xylitol dehydrogenase enzyme XDH. Preferably, it is the XYL2 gene, the sequence of which is the sequence reported in GenBank under number 4852013, or a sequence at least 70 % identical, preferably at least 75 % identical, or at least 80 % identical, or at least 85 % identical, or at least 90 % identical, or at least 95 % identical, or at least 98 % oidentical, or at least 99 % identical, to said sequence reported in GenBank under number 4852013, and encoding a functional xylitol dehydrogenase enzyme.

Preferentially, the yeast strain of step (i) has an endogenous xylitol dehydrogenase XDH activity of less than 150 mKat/g of proteins. The xylitol dehydrogenase activity can be measured in the conditions set forth in the article by Xu et al. entitled Characterization of Ethanol Production from Xylose and Xylitol by a Cell-Free Pachysolen tannophilus System, in Appl. Environ. Microbiol. 59:231-235 (1993).

It is known that during the processing of the biomass intended for alcoholic fermentation, certain fermentation inhibitors appear. Among them, mention may be made of phenolic products, of furfural or further acetic acid. It is also known that these inhibitors are detrimental to the performances or even the survival of the yeast.

In order to solve this additional problem, the Applicant suggests selecting the strain of step i, from industrial strains having resistance to phenolic derivatives.

Another advantage of the XI route, is the possibility of « grafting the bacterial arabinose route in parallel » as described for example in EP 1 499 708 or yet in WO 2008/041840, this combination then allowing an increase in the final degree of alcohol in the case of the presence of arabinose in the media to be fermented.

The method for preparing the yeast of the present invention takes into account both the constraints of the yeast producer and those of a final user in its applications notably in terms of industrial production of ethanol with low cost and high yield.

The method according to the invention has, in particular, the following advantages:

For the yeast producer, it allows: - construction of a prototrophic aneu/polyploid, Saccharomyces cerevisiae yeast strain in order to allow production of biomass on simple sources of carbon, nitrogen, phosphorous in inexpensive media such as the byproducts of the sugar industry like, for example, molasses, - availability of a Saccharomyces cerevisiae yeast strain having a maximum growth rate (μ max) comprised between 0.37 h1 and 0.5 h1, - availability of a Saccharomyces cerevisiae yeast strain which, when it is produced according to a method as described in the reference book «Yeast Technology» (2nd edition, 1991, G. Reed and T.W. Nagodawithana, published by Van Nostrand Reinhold , ISBN 0-442-31892-8), offers the possibility of obtaining a biomass production yield of at least 45g of yeast dry materials for lOOg of saccharose equivalent applied, - availability of a Saccharomyces cerevisiae yeast strain, resistant to the drying process as described in patent documents EP 511108 and US 5,741,695, the loss of fermentative activity after drying should not exceed 30%, - production under industrial conditions (in particular, inexpensive medium, good biomass yield, dry ready-to-use yeast) of a fresh or dry yeast from a genetically stable, Saccharomyces cerevisiae yeast strain notably robust because it is tolerant to high concentrations of ethanol and capable of producing, for example from hemi-cellulose biomasses, at least 40g/L of ethanol and this at a high temperature of the order of 30 to 40°C. A prototrophic yeast strain is a strain capable of growing on a minimal medium. In particular, a prototrophic yeast strain according to the invention is capable of synthetizing all amino acids and bases that are necessary for its growth. A minimal medium is a medium comprising a source of carbon, a source of nitrogen, a source of potassium, a source of phosphorus, a source of sulfur, a source of magnesium, a source of calcium, a source of iron, a source of trace elements and water.

An example of minimal medium is the YNB medium (Yeast Nitrogen Base). The YNB medium comprises, per liter: 2 pg biotin, 400 pg calcium pantothenate, 2 pg folic acid, 2000 pg inositol, 400 pg niacin, 200 pg p-aminobenzoic acid, 400 pg pyridoxine hydrochloride, 200 pg riboflavin, 400 pg thiamin hydrochloride, 500 pg boric acid, 40 pg copper sulfate, 100 pg potassium iodide, 200 pg ferric chloride, 400 pg manganese sulfate, 200 pg sodium molybdate, 400 pg zinc sulfate, 1 g monobasic potassium phosphate, 500 mg magnesium sulfate, 100 mg sodium chloride, 100 mg calcium chloride, 5 g ammonium sulfate, final pH 5.4.

According to another preferred alternative of the method according to the description, when in step (ii) the expression cassette consists in the association of the open reading frame (ORF) type of the gene XI encoding the xylose isomerase enzyme of Clostridium phytofermentans / promoter and terminator of Saccharomyces cerevisiae, said cassette being flanked upstream and downstream with recombinogeneic regions allowing its targeted integration into the genome, said method then further includes a step of saccharification and simultaneous fermentation (SSF) in the presence of polymers of hexoses, predominantly consisting of glucose, and of at least one enzyme capable of hydrolyzing them.

Moreover, for the ethanol producer, the advantage of the method according to the invention is also to have an active (fresh - liquid or compressed, pressed together or dry) yeast, obtained according to a production method as described in the textbook « Yeast Technology », from a Saccharomyces cerevisiae yeast strain as defined in the preceding paragraph which is: - capable, under the SSF conditions described in patent document WO 2004/046333, of fermenting at 32°C a hydrolyzate of cereals up to a minimum ethanol concentration of 16% (w/w), - capable, under the SSF conditions described in patent document WO 2004/046333, of fermenting at 35°C a hydrolyzate of cereals up to a minimum ethanol concentration of 14.5% (w/w).

The results of the method according to the invention are all the more remarkable when they are obtained from a prototrophic aneu/polyploid so-called industrial strain and in fact having a clearly more complex genetic material than that of a so-called laboratory strain, at the very least making the consequences of modifications of said industrial strain unpredictable. This complex genetic background, specific to industrial strains, makes it all the more difficult to obtain, in the end, genetically modified strains free of markers of resistance to antibiotics, in particular when many genetic targets have to be modified. Strains free of markers of resistance to antibiotics are quite obviously preferable for health and environment reasons.

The prototrophic strains according to the invention have the advantage of growing on simple sources of carbon, nitrogen and phosphorus.

But this feature causes the transformation vectors available in the scientific community (vectors using auxotrophy markers) to be inoperative.

It is therefore necessary to have available tools/vectors using markers of resistance to antibiotics, these so-called tools/markers being advantageously constructed in order to allow in fine excision of these markers. By way of example, use can be made of the Cre-lox technology. In brief, loxP sequences are provided on each side of each selection marker. Excision of the selection markers is performed by transforming the yeast strain by the lithium acetate method (Schiestl et Gietz, 1989, Current Genetics, vol 16, p.339-346), using a plasmid comprising the Cre recombinase gene and a selection marker different from the selection marker(s) to be excised. The expression of the Cre recombinase in the yeast strain makes it possible to excise the selection marker, leaving only a loxP sequence, possibly together with its flanking sequences. It is then possible to induce the loss of the plasmid comprising the Cre gene by culturing in non-selective conditions, i.e. in an enriched medium in the absence of antibiotics. For example, the construction of yeasts compliant with the invention required the use of 4 different positive markers giving resistance to 5 different antibiotics (geneticin, phleomycin, hygromycin, blasticidin and nourseothricin).

The strains of the invention are preferably aneuploids or polyploids: this is a feature generally encountered in industrial yeasts which stem from the natural medium. The phylogenetic past of these strains is at the origin of this particularity.

But this is an additional difficulty encountered when disrupting/inactivating all the copies of a given gene is desired. However, this aneu/polyploidy feature is generally at the origin of many interesting properties of industrial yeasts (growth rate, resistance to different stresses, phenotype stability).

Further, the Applicant after long research work surprisingly noticed that with the method according to the invention, applied from the selected strain: - the introduction of expression and deletion cassettes by no means made fragile the modified yeast, which experiences improvement in its genetic heritage.

In particular, the inventors have shown that with said strain, it is possible to achieve: - the deletion of at least two copies of the gene GRE3 of S. cerevisiae (the Gre3P enzyme being an aldose reductase which consumes NADPH,H+ which is produced for a major part via the oxidative portion of the pentose route) in said industrial strain according to the invention allowed reduction in the consumption of NADPH,H+ by said enzyme, by that much.

As a preferred alternative, said at least one gene of each step of the non-oxidative portion of the phosphate pentose route of step (iii) is selected from the group formed by the genes encoding the D-ribulose-5-phosphate 3-epimerase, ribose-5-phosphate ketol-isomerase, transketolase and transaldolase enzymes, and notably from the group of the RPE1, RKI1, TKL1 and TALI genes. Preferably, said promoter of a strongly expressed glycolysis gene during alcoholic fermentation is the TDH3 promoter for RPE1, RKI1 and TKL1, and PGK1 for TALI.

The TALI gene is preferably the gene reported in GenBank under number 851068.

The TKL1 gene is preferably the gene reported in GenBank under number 856188.

The RKI1 gene is preferably the gene reported in GenBank under number 854262.

The RPE1 gene is preferably the gene reported in GenBank under number 853322.

According to complementary or alternative features in the method for preparing a Saccharomyces cerevisiae yeast strain according to the invention: - the Saccharomyces cerevisiae promoter of steps (ii)(a) and (ii)(b)is selected from the group comprising the promoters of genes encoding glycolysis enzymes and those encoding the alcohol consisting of ADH1, ADH2, PGK1, TDH3, PDC2 and GAL1/10, preferably ADH1. The terminator of Saccharomyces cerevisiae is formed by CYC1 or by the specific terminator of the gene of the non-oxidative pentose phosphate pathway. A subsequent directed evolution step is preferably contemplated, which includes the following successive steps consisting of subjecting the obtained yeast to (i) mutagenesis, (ii) growth in cyclic cultures under limited O2 in a medium including said at least one pentose, and (iii) selection by aerobic growth on a solid medium containing glycerol as a single source of carbon, so as to provide respiratory non-deficient mutants of said yeast which exhibit growth in anaerobiosis in the presence of a medium including said at least one pentose (notably xylose).

Preferably in this alternative, the mutagenesis of step (i) is performed under « mild » conditions, i.e. moderate mutagenesis with 100 to 500J/cm2 and still preferably 300J/cm2 of ultraviolet radiation at 254 nm. These conditions only cause mortality of 7% to 16% of the population subject to UVs.

The inventors have thereby surprisingly shown that with such a so low controlled mortality, it is possible to reduce by a factor 10 the duration of the directed evolution step with cyclic cultures required for obtaining mutants capable of fermenting said at least one pentose (notably xylose). The survival rate is determined by spreading out on medium dishes, the carbon source of which is glucose, an identical volume of cell suspension before and after mutagenesis. The number of colonies is determined after 48h of growth.

Preferably, the O2 limitation of step (ii) of this alternative is achieved by partial overpressure in the equipments used (for example vials or fermenters) due to overpressure consecutive to production of produced CO2.

The cyclic cultures according to this alternative, under fermentation conditions, offer the possibility of enriching the population in mutants capable of fermenting said pentose (notably xylose) and this within a period from 2 to 6 weeks, and preferably from 3 to 4 weeks, which is relatively short and highly interesting as compared with what would be obtained by chemostat, as described by Kuyper et al. (2004), FEMS Yeast Res. 4, 655-664.

Although the « petite » respiratory deficient phenotype may coincide with the fermentation criteria of said at least one pentose, in this alternative, the present inventors carried out a step for removing « petite » yeasts since this phenotype is incompatible with the methods for producing industrial yeasts in the sense of the invention.

The researchers noted that the directed evolution step as explained above made it possible to significantly increase the xylose isomerase activity, which is characterized by an increase in the xylose consumption rate.

Without wishing to be bound by theory, this unexpected effect seems to be attributable to an increase in the number of XI copies in the modified strain.

The object of the present invention is further the EG6 Saccharomyces cerevisiae industrial yeast strain directly obtained using the method according to the invention after the step of directed evolution and which consists in the yeast strain deposited on November 23rd 2010 at the C.N.C.M (Collection Nationale de Cultures de Microorganismes of the Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris, France) under No.1-4399 under the terms of the Budapest treaty.

The object of the present invention is also the EG7 Saccharomyces cerevisiae industrial yeast strain directly obtained using the method according to the invention after the directed evolution step, deposited on November 23rd 2010 at the C.N.C.M (Collection Nationale de Cultures de Microorganismes de l'Institut Pasteur) under No. 1-4400 under the terms of the Budapest treaty.

The object of the present invention is also the EG8 Saccharomyces cerevisiae industrial yeast strain directly obtained by the method according to the invention after the directed evolution step, deposited on December 14 2010 at the C.N.C.M (Collection Nationale de Cultures de Microorganismes de l'Institut Pasteur) under No. 1-4417 under the terms of the Budapest treaty.

The object of the present invention is also the EG10 Saccharomyces cerevisiae industrial yeast strain directly obtained by the method according to the invention after the directed evolution step, deposited on October 5 2011 at the C.N.C.M (Collection Nationale de Cultures de Microorganismes de l'Institut Pasteur) under No. 1-4538 under the terms of the Budapest treaty.

Other strains according to the invention are strains derived from one or more strains according to the invention, for example from one or several strains obtained using the above method, and notably from one or more of the strains deposited at the CNCM under No. 1-4399 on November 23, 2010, under No. I-4400 on November 23, 2010, under No. 1-4417 on December 14, 2010 and under No. 1-4538 on October 5, 2011.

By the expression "derived strain" is meant in particular strains derived by one or more cross-breedings and/or by mutation and/or by genetic transformation.

The strains derived by cross-breeding can be obtained by cross-breeding a strain according to the invention with the same strain, or with another strain according to the invention, or with any other strain.

The strains derived by mutation can be strains which have undergone at least one spontaneous mutation in their genome or at least one mutation induced by mutagenesis. The mutation(s) of the derived strains can be silent or not.

By "mutagenesis" is meant both random mutagenesis obtained by applying radiation (e.g. UV) or by mutagenic chemicals, and insertional or directed mutagenesis, by transposition or by integration of an exogenous DNA fragment.

The derived strains which are within the framework of the description are those which comprise at least one exogenous XI gene and one exogenous XDH gene and which are capable of fermenting xylose to produce ethanol, and notably with an average yield of ethanol produced by consumed xylose greater than or equal to 0.2 g, preferably 0.3 g, 0.35 g or even 0.38 g ethanol per g of consumed xylose.

These derived strains also preferably exhibit a deletion of the GRE3 gene and/or a control of the XKS1 and/or RPE1 and/or RKI1 and/or TKL1 and/or TALI genes by a promoter of a gene which is not repressed by anaerobiosis or by catabolic repression induced by any source of carbon, and strongly expressed during alcoholic fermentation, such as a promoter of a gene encoding a glycolysis enzyme or encoding an alcohol dehydrogenase enzyme, preferably the ADH1, PGK1, TDH3, PDC2 or GAL1/10 promoter.

Still preferably, - the obtained Saccharomyces cerevisiae yeast strain is practically or totally free of markers, notably markers of resistance to antibiotics.

Preferably, the Saccharomyces cerevisiae yeast strains prepared according to the present invention and to the criteria defined above retain, after introduction of the genetic modifications and other mutations generated during the directed evolution step, their genotype and phenotype characteristics after a complete industrial production process. In particular, the yeasts produced have kinetics for producing alcohol, kinetics for consuming xylose and/or arabinose and a maximum produced amount of alcohol, strictly identical with those of the yeast strain before applying a complete industrial process.

Moreover, the industrial characteristics of the selected strain before manipulation, as described earlier (growth rate, production yield, drying capacity) remain unchanged.

The object of the present description is also a method for producing ethanol from a medium including at least one pentose, by fermentation with yeast according to the invention, mentioned above, or such as obtained with a method according to the invention as it has just been described.

Preferably, the method for producing ethanol has the following alternative and/or complementary characteristics: - Said at least one pentose is xylose or a mixture of xylose and arabinose. - Said medium is selected from the group consisting of lignin, cellulose, hemi-cellulose dextrin and starch hydrolyzates. - In the case of an SSF, the average rates for releasing the hexose, in majority glucose, are of the order of 2.8 to 5.6g/L/h with zero extracellular concentration of hexose, predominantly glucose. - The average yield of ethanol produced by consumed xylose is greater than or equal to 0.38 g ethanol per g of consumed xylose, for example it can be approximately 0.40 g ethanol per g of consumed xylose.

The concentrations of sugars which can be applied (for example 70g/kg of xylose or 150g/kg of xylose) to the knowledge of the Applicant are the maximum concentrations which may be encountered in practice. All the published tests referring to fermentation of xylose were conducted with much lower concentrations of total sugars.

Other features and advantages of the invention will become still better apparent upon reading the exemplary embodiments which are given purely as an illustration and not as a limitation and, for the understanding of which reference will be made to the appended drawings. EXAMPLES Example 1

The selection of the strain is as described in the description above.

All the DNA sequences which were used for the different transformations aiming at the overexpression of a gene were obtained from a known vector type (pUC type) in which are provided: - the integration targets; - the promoters/terminators selected per gene of interest and - the resistance markers which will be removed subsequently (see below).

An exemplary vector used for the overexpression of the XDH of Pichia stipitis is illustrated in Fig. 1.

An exemplary vector used for the overexpression of the XI of Clostridium phytofermentans is illustrated in Fig. 2.

For disrupting the copies of the GRE3 gene of the selected industrial strain, the inventors used PCR amplicons from a plasmid of the pUG6 type (Gtildener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. Nucleic Acids Res. 1996 Jul 1; 24( 13) :2519-24).

The transforming step of the yeast was performed 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, EG6, EG7, EG8 and EG10 respectively, were deposited at the CNCM and No.1-4399, No.1-4400, No.1-4417 and No.1-4358 were respectively assigned to them.

The strains according to the invention: - have the following genotype:

Ethanol Red™, Delta CRE3, BUD5::pADHl-XKSl-tCYCl, TALI::pPGKl-TALI-tCYCl, TKL1: :pTDH3-TKLl-tCYCl,RPEl: :pTDH3-RPEl-tCYCl, RKI1::pTDH3-RKIl-tCYCl, HO::PsXYL2-HYGRO, BUD5::CpXI-BLAST - are free of any residual marker (by the action of ere recombinase).

Example 2

Mutagenesis of these strains obtained in the previous example was performed in a moderate fashion i.e. from 100 to 500J/cm2 and preferably 300J/cm2 of UVs at 254nm.

After a week of culture at 32°C in a YE type medium (0.5% Yeast Extract) containing 7% of xylose, with stirring, without ventilation - the O2 limitation being achieved by means of partial overpressure in the vials due to CO2 produced during fermentation - one ml of the culture is used for re-seeding the same medium. This operation is repeated 6 times. The cells are finally spread out on a gelose YE 20g/L glucose medium. Isolated colonies are sampled and then successively cultivated on: - YE 20g/L glycerol and in aerobiosis for removing the « petite » i.e. respiratory deficient mutants; - YE glucose for checking their growth rate; - YE xylose for identifying the most interesting clones.

Example 3

After obtaining the EG6 strain, the copy of the XDH gene which was added to Example 1, was substituted with the gene of resistance to kanamycin. The new obtained strain is called EG6 - XDH. The xylose fermenting capacity of the relevant strain was compared with that of the EG6 strain. The result of this comparison is shown in Fig. 4.

Claims

1. Gærstamme, kendetegnet ved at den omfatter exogene gener fra xylosemetaboliseringsvejen, hvor de exogene gener xylosemetaboliseringsvejen, der er stede i gærstammen, består af mindst en kopi af et exogent gen, der koder for en xylose-isomerase, og en kopi af et exogent gen, der koder for en xylitol-dehydrogenase, og ved at den har gennemgået et efterfølgende trin med kontrolleret evolution omfattende følgende successive trin bestående i, at den opnåede gær underkastes: (i) en mutagenese, (ii) en vækst i cykliske kulturer, under begrænset O2 i et medie omfattende mindst en pentose, især xylose, og (iii) en selektion for aerob vækst i et fast medie omfattende glycerol som den eneste carbonkilde.

2. Gærstamme ifølge krav 1, hvor det exogene gen, der koder for en xylose-isomerase, er et gen stammende fra Clostridium, Piromyces, Bacteroides, Streptomyces, Haemophilus, Burkhoideria, Enterococcus, Thermotoga, Fusobacterium, Geobaciiius, Arthrobacter, Ciona, Physcomitrella, Cellvibrio, Chitinophaga, Saccharopolyspora eller Salinibacter, og er fortrinsvis et gen stammende fra Clostridium phytofermentans eller Piromyces sp. E2.

3. Gærstamme ifølge krav krav 1 eller krav 2, hvor det exogene gen, der koder for en xylitol-dehydrogenase, stammer fra Pichia stipitis.

4. Gærstamme ifølge et hvilket som helst af kravene 1 til 3, som er valgt blandt Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kiuyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp. og Debaromyces spp., og fortrinsvis er en stamme af Saccharomyces cerevisiae.

5. Gærstamme ifølge et hvilket som helst af kravene 1 til 4, hvor mindst en kopi, fortrinsvis mindst to kopier, af et gen, der koder for en aldose-reduktase, er deleteret.

6. Gærstamme ifølge krav 5, hvor det deleterede gen er CRE3.

7. Gærstamme ifølge et hvilket som helst af kravene 1 til 6, hvori et endogent gen der koder for xylulokinase, fortrinsvis genet XKS1 , er under kontrol af en promotor for et gen, der ikke undertrykkes af anaerobe tilstande eller ved katabolisk repression induceret af en hvilken som helst carbonkilde, og som udtrykkes stærkt under alkoholfermentering.

8. Gærstamme ifølge et hvilket som helst af kravene 1 til 7, hvor mindst et endogent gen fra den ikke-oxidative del af pentose-phosphatvejen, fortrinsvis valgt blandt generne RPE1, RKI1, TKL1 og TALI, og alle disse gener særligt foretrukket er under kontrol af en promotor for et gen der ikke undertrykkes af anaerobe tilstande eller ved katabolisk repression induceret af en hvilken som helst carbonkilde, og som udtrykkes stærkt under alkoholfermentering.

9. Gærstamme ifølge et hvilket som helst af kravene 1 til 8, hvor promotoren er promotoren for et gen, der koder for et glykolyseenzym, eller der koder for et alkohol-dehydrogenase-enzym, fortrinsvis promotoren fra ADH1, PGK1, TDH3, PDC2 eller GAL1/10.

10. Gærstamme ifølge et hvilket som helst af kravene 1 til 9, kendetegnet ved at den er en aneuploid eller polyploid stamme og/eller en pro tot rof stamme.

11. Gærstamme ifølge et hvilket som helst af kravene 1 til 10, kendetegnet ved at den omfatter mindst to kopier af det exogene gen, der koder for en xylose-isomerase, fortrinsvis mindst tre kopier eller mindst fire kopier af det exogene gen, der koder for en xylose-isomerase.

12. Gærstamme ifølge et hvilket som helst af kravene 1 til 11, kendetegnet ved at den er en industriel stamme som er resistent over for fermenteringsinhibitorer, der stammer fra hydrolyse af biomasse, såsom fenoliske produkter, furfural eller eddikesyre.

13. Gærstamme ifølge et hvilket som helst af kravene 1 til 12, kendetegnet ved at den producerer en ethanolkoncentration på mindst 16%, fortrinsvis på mindst 17% v/v, i et kornhydrolysat, under betingelser for simultan saccharifisering og fermentering ved 32°C.

14. Gærstamme ifølge et hvilket som helst af kravene 1 til 13, kendetegnet ved at den er valgt fra gruppen bestående af Saccharomyces cerevisiae stammen deponeret hos CNCM [National Collection of Microorganism Cultures] den 23. november 2010 under nummer 1-4399, Saccharomyces cerevisiae stammen deponeret hos CNCM den 23. november 2010 under nummer 1-4400, Saccharomyces cerevisiae stammen deponeret hos CNCM den 14. december 2010 under nummer 1-4417, og Saccharomyces cerevisiae stammen deponeret hos CNCM den 5. oktober 2011 under nummer 1-4538.

15. Gærstamme ifølge et hvilket som helst af kravene 1 til 13, kendetegnet ved at den er en stamme afledt af en eller flere af stammerne defineret i krav 14.

16. Fremgangsmåde til fremstilling afen gærstamme, omfattende indføring, i en udgangsgærstamme, af exogene gener fra xylosemetaboliseringsvejen, hvor de exogene gener xylosemetaboliseringsvejen indført i udgangsgærstammen består af mindst en kopi af et exogent gen, der koder for en xylose-isomerase, og mindst en kopi af et exogent gen, der koder for en xylitol-dehydrogenase, og også omfattende et efterfølgende trin til kontrolleret evolution omfattende følgende successive trin bestående i at den opnåede gær underkastes: (i) en mutagenese, (ii) en vækst i cykliske kulturer, under begrænset O2 i et medie omfattende mindst en pentose, især xylose, og (iii) en selektion for aerob vækst i et fast medie omfattende glycerol som den eneste carbonkilde, til opnåelse af ikke-deficiente respiratoriske mutanter af gæren, som udviser vækst under anaerobe tilstande i nærvær af et medie omfattende den mindst ene pentose.

17. Fremgangsmåde ifølge krav 16, deom endvidere omfatter deletering af mindst en kopi, fortrinsvis mindst to kopier, af et gen der koder for en aldose-reduktase, fortrinsvis genet GRE3, i udgangsstammen.

18. Fremgangsmåde ifølge krav 16 eller krav 17 til fremstilling afen gærstamme ifølge et hvilket som helst af kravene 1 til 15.

19. Fremgangsmåde ifølge et hvilket som helst af kravene 16 til 18 til fremstillinng af en Saccharomyces cerevisiae gærstamme, der er i stand til at producere ethanol fra et medie omfattende xylose, og som omfatter følgende trin bestående af: (i) at vælge en stamme fra Saccharomyces cerevisiae, (ii) at integrere følgende ekspressionskassetter i genomet hos gæren fra trin (i), a. kombination af typen åben læseramme (ORF) af et gen, der koder for en xylose-isomerase under kontrol af en Saccharomyces cerevisiae promotor og terminator, idet kassetten er flankeret opstrøms og nedstrøms af rekombinogene områder der muliggør målrettet integration deraf i genomet, b. kombination af typen åben læseramme (ORF) af et gen, der koder for en xylitol-dehydrogenase under kontrol af en Saccharomyces cerevisiae promotor og terminator, idet kassetten er flankeret opstrøms og nedstrøms af rekombinogene områder, der muliggør målrettet integration deraf i genomet, (iii) at inducere ekspression af mindst et gen for hvert trin af den ikke-oxidative del pentose-phosphatvejen, og også mindst et gen, der koder for xylulokinase (XKS1), ved at placere dem under kontrol af en promotor for et gen der ikke undertrykkes, hverken af anaerobe tilstande, eller ved katabolisk repression induceret afen hvilken som helst carbonkilde, og som udtrykkes stærkt under alkoholfermentering, og (iv) at deletere mindst to kopier af den åbne læseramme (ORF) for Saccharomyces cerevisiae-geuet GRE3, der koder for en aldose-reduktase.

20. Fremgangsmåde ifølge krav 19, kendetegnet ved at genet, der koder for en xylose-isomerase, er et XI-gen, der koder for enzymet xylose-isomerase, valgt blandt dem der er til stede i genomerne hos Clostridium, Piromyces, Bacteroides, Streptomyces, Haemophilus, Burkholderia, Enterococcus, Thermotoga, Fusobacterium, Ceobacillus, Arthrobacter, Ciorta, Plyscomitrella, Cellvibrio, Chitirtophaga, Saccharopolyspora eller Salinibacter.

21. Fremgangsmåde ifølge krav 19 eller krav 20, kendetegnet ved at XI-genet er valgt blandt et gen fra Clostridium phytofermentans eller Piromyces sp. E2.

22. Fremgangsmåde ifølge krav 19 eller krav 20, kendetegnet ved at genet, der koder for en xylitol-dehydrogenase, er et gen fra Pichia stipitis, der koder for enzymet xylitol-dehydrogenase.

23. Fremgangsmåde ifølge et hvilket som helst af kravene 19 til 21, kendetegnet ved at gærstammen fra trin (i) har en endogen xylitol-dehydrogenase-XDH-aktivitet på mindre end 150 mKat/g proteiner.

24. Fremgangsmåde ifølge et hvilket som helst af kravene 19 til 23, kendetegnet ved at gærstammen fra trin (i) er valgt blandt industrielle stammer med en resistens over for fermenteringsinhibitorer, der stammer fra hydrolyse af biomasse, såsom fenoliske produkter, furfural eller eddikesyre.

25. Fremgangsmåde ifølge et hvilket som helst af kravene 19 til 24, kendetegnet ved at Saccharomyces cerevisiae-promotoren fra trin (iii) er valgt fra gruppen omfattende promotorerne for gener, der koder for glykolyseenzymer, og dem, der koder for alkoholdehydrogenaseenzymer.

26. Fremgangsmåde ifølge et hvilket som helst af kravene 19 til 25, kendetegnet ved at gruppen består af ADH1, PGK1, TDH3, PDC2 og GAL1/10, fortrinsvis ADH1, og ved at terminatoren fra Saccharomyces cerevisiae består af CYC1 eller den egentlige terminator tilhørende genet af den ikke-oxidative pentose-phosphatvej.

27. Fremgangsmåde ifølge et hvilket som helst af kravene 19 til 26, kendetegnet ved at stammen fra trin (i) er en industriel stamme valgt blandt stammer, der er i stand til at producere høje ethanolkoncentrationer på mindst 17% v/v, på et kornhydrolysat, under betingelser for simultan saccharificering og fermentering(SSF) og ved 32°C.

28. Fremgangsmåde ifølge et hvilket som helst af kravene 19 til 27, kendetegnet ved at den også omfatter et eller flere trin til indføring af antibiotikaresistensmarkører, et eller flere trin til udvælgelse af stammer i overensstemmelse med kriteriet for deres antibiotikaresistens, og et eller flere trin til fjernelse af antibiotikaresistensmarkørerne.

29. Fremgangsmåde til fremstilling af ethanol fra et medie omfattende xylose, ved at fermentere en gær ifølge et hvilket som helst af kravene 1 til 15 eller en gær opnået med en fremgangsmåde ifølge et hvilket som helst af kravene 16 til 28.

30. Fremgangsmåde ifølge krav 29, kendetegnet ved at mediet omfatter en blanding af xylose og arabinose.

31. Fremgangsmåde ifølge krav 29 eller krav 30, kendetegnet ved at mediet er valgt fra gruppen bestående af hydrolysater af lignin, af cellulose, af hemicellulose og af stivelse.