HUE031826T2

HUE031826T2 - Eljárás ipari élesztõ elõállítására, az ipari élesztõ és alkalmazása etanol elõállítására legalább egy pentózból

Info

Publication number: HUE031826T2
Application number: HUE11794083A
Authority: HU
Inventors: Thomas Desfougeres; Georges Pignede
Original assignee: Lesaffre & Cie
Priority date: 2010-12-03
Filing date: 2011-12-02
Publication date: 2017-08-28
Also published as: PL2646544T3; ES2608784T3; BR112013013696A2; AR084087A1; WO2012072793A1; CA2819323A1; US20120142067A1; CL2013001569A1; PT2646544T; BR112013013696B1; CA2819323C; EP2646544A1; AU2011334846A1; EP2646544B1; FR2968313B1; DK2646544T3; MY180908A; MX347050B; HRP20161752T1; AU2011334846B2

Description

MIT»D wm FREPARINfl AN »UST1ÄL ? EAST, INDUSTRIAL YEAST ANP OSI IN THE FRODUOf I« £)F ETHANOL FROM AT HAST ONE

PENTOSE

TECHNICAL FIELD

The present description relates To the leld 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 genera! aspect relates to a method for preparing yeasts from so-caiied industrial strains of Saccharomyces c&r&visise. to said yeasts and their application to the industrial production of ethanol from industrial media containing at ieast one pentose, notably xylose,

TICNNfCAL 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 s laboratory conditions.

Indeed, scientific literature as well as patent documents analysed 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 auxofrophy 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 procîiî0tÂ:re£|Uif®m@rm--ofi:^n industrial scale, re. to only mention tnree öt them: ettdg growth rate, drying capacity, storage stability, if so-called fermentative performances (anaerobic ethanol production capacityl are obtained in synthetic or defined media, so-calied iahoratory media, with these previous strains, they generally cannot be transposed to industrial media including, for example, complex mixtures stemming from cellulose-processing residues which contain toxic compounds which can Inhibitthe yeast's cell mechanism at different levels, notably furfural, Hilf, phenolic derivatives, acetic acid. Furthermore, the « scale up » or scale transposition capacity of these earlier ethanol production methods is seldom documented. :Oeoumeht..WO-20ÔB/1'ii^It#achÔ$'fbië production of alcohol from a yeasf strain with a « genetic background » of the type: ~~ Mutated SIP I S gene (F117S,Y1S6H, K218R). ···· Exogenous genes encoding XI / Xft / XDH or XK. “ΧΓ designates xylose isomerase, ,:XR’: designates xylose reductase, “XDH" designates xyiitol dehydrogenase, and 7XK” designates D-xyluldkinase.

Document WO 2005/113774 describes a recombinant operon comprising two nucleic acid sequences respectively encoding an XI of Ε,οοίί and an XDH of Triahoderma r&esei in the context of the production of xyiitol.

The document: PioS Genetics of Gavin Sheri ok et al, published on May 13th 2010, describes an XDH1 gene which is present in some specific Cmohammyma cerevisiaa strains, which may encode a xylitoi dehydrogenase,

The document in the name of David Brat, Eckard Boles and Beate Wiedemannn, in Appl. Environ. Microbiol., April 2009, Vol. 73, No.8, p. 2304-2311, describes the expression, in Saocharomyc&s mrmmm, ofthe xylose isomerase gene from Clostridium phytofemi&ntans. it emerges from the review of these documents of the prior art, an Weil as from the work of the inventors Of the present invention, that given the very different genetic backgrounds/heritages of the strains of Saccharomyces mmviaim yeasts used with the purpose of growing on and/or fermenting xysose, œ consequences, tor example, of an overexpressson and/or or thr deletion of native genes and/or of the introduction of one or more heterologous fines cannot ho predicted/

SUiMAil^ OF THE INVENTION

Thus, the Applicant having studied many strains of the alcohol, brewery and batey 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 nueíeis acid encoding X! is not sufficient by itself for efficiently fermenting xylose.

In general, the Applicant noticed that the introduction ©f expression cassettes of a gene encoding an enzyme capable of transforming any carbohydrate (notably xylose) into xylulose (D-xyiuiose) and of a gene encoding an enzyme capable of transforming any pentoi (notabiy xyiitoi} into xylulose in a single step, made all the strains thus modified particularly effectieni át 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 xyiitoi into xylulose In a singie step“ Is meant a xyiitoi dehydrogenase enzyme. indeed, $#Appfteant eonfirmedthat, unlike the so-called fungai 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 xyiitoi which is a metabolic Intemiedlale present «η the fungal route and which may significantly reduce the ethane! production yield.

Very recently, in certain S. œmmm strains, notably those for winemaking, a gene XDH 1 was identified as being essential for the metabolism of xylose of said strains (Rbdtl genetics 201Ö, 6, 1 -11% Also, the Applicant noticed that even by suppressini the GRE3 gene of the modified strains, there are ether patasile activities that may transform xyiose into xylioi (aldose reductase activity), which is detrimental for the Xi activity thereby lodueing the sought ethanol yield.

The work carried out by the Applicant shows that reinforcement of the x'yllto! dehydfogenasi 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 medluni Inciudihi at feast yylose, with s good kinetic yield.

In other wofdi, in the prior art, two different pathways were explored so as to make possible the fermenfatidn of xyiose by yeast: the so-cailed 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 ah improved result

The invention is defined la the appended claims.

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

By -exoiendus” 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 if 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 if 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 stipitm, - Fig. 2 illustrates an overexpression vector of XI from Clostridium phytof&rm&ntans. - Hg. <5 fs a graph iustrating the production of ethanol versus the fermentation time at 32*0 for two yeast strains according to the invention after directed évolution* and for the Ethanol red™ strain. The tested clones were inoculate! with an amount of 5g of dry mass/L in a YF 4 70 g/L xylose medium, ™ Fig. 4 is a graph illustrating the production of ethanol versus the fermentation time at 32*0 for two yeast strains* one according to the invention aier directed: evolution, the other strain resulting from the first one but after substitution of the copy of the KPÍ gene by a marker of resistance to kanamycin CKaofytX4). 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 efhanoi (Y-axis, in g per kg of medium! as a function of the fermentation time at 323C (X-axis, In hours) for two yeast strains according to the invention EG8 and EG10. The tested clones were inoculated at Ö.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 cercvisiaa yeast strain capable of producing ethane! from; a medium including at least one pentose (notably xylose) and winch comprises the following steps'. (i) selecting (or providing) a Sacchammyma yeast strain (if) Integrating the following expression cassettes into the genppe of the yeast of step (i), a. the association of the open reading frame (ORF) type of a gene encoding ah enzyme capable of transforming any carbohydrate, notably xylose, into xylulose under the dependency of a promoter and of a Sacchemmyaes ocremim terminator* said cassette being flanked Upstream and downstream with recomhlnogenic 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 íranafórmihg in a single step any Pántol, notably xylitol, into xylulose under the dependency of a Saccharomyces ommdmee promoter and terminator, said cassette being fianked upstream and downstream with recombinogenic regions allowing its targeted integration into the genome, fii| inducing the expression of at least one gene of each step of the nao-oxidsfixe portion of the phosphate pentose route as well as of at least one gene encoding xylulokinase (XKSi) 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 strongiy expressed during aicoh oi ic ferme π tats ο η, and (iv) deleting at least one copy or preferably at least two copies of the open reading frame (ORF) of the Sacohammyms cerevisiae GRE3 gene encoding an aldose reductase Preferably, all copies of the open reading frame (DRF) of the Saccharomyces cerevisiae gene ORES gene are deleted, the XKSI gene is preferably the gihi reported in GenBank under number 863108.

The GRE3 gene is preferably the gene reported in GenBank under number 858504

Preferentially, the gene of step (ii)a is a gene XI encoding the xylose isomerase enzyme selected from those prisent in the genomes of the Clostridium, Pymmyces, Bact&midm. Stmptonv/ces, Haemophilus, Burkheiderm> Enterococcus, Thermotoga, Fusobacterium, GeobacíHus, Arihföbacter, Cioaa, Pbyscomitrella, Cellvihrio, Chitinophaga, Saccbaropolyspora, Selmibacter gene ra.

The XI: gene is preferably selected tom a gene of Clostridium ppytgfadrmntam, Saccbaropolyspora myth race, Salinibacler ruber or Piromyces $p, E2,

According to a preferred embodiment, the sequence of the XI gene is the SEQ ID NO:1 nucleotide sequence (which corresponds to the sequenct of the XI gene from Ciostndium phytofemmntans, described in document DE 102008031350), Alternatively, the XI gene has a sequence which has at leas!; 70% identity preferably ai lernt 75% Identity, or at least 80% identity, or at least 85% identity, drat ieast 90% identity, nr at least 95% identity, or si least 88% identity, or at ieast 91% ideate wife SEQ ID NO:1, and it encodes a functional xylose Isemerase 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 priytofmwmam described in document DE 102008031350). Alternatively, said polypeptide has a sequence having at least 70% identity preferably at ieast 75% identity, or at feast 88% identity, or at least 85% identity, or at least 80% identity or at least 85% Identity, or at least 98% identity, or at ieast 99% identity, with SEQ ID NO:2 and it hal a xylose Isomerase activity.

According to the present invention by « transforming any pentoi into xylulose In a single step » is meant direct oxidation of xyiito! into xylulose and this by the same and single enzyme (xylltoi dehydrogenase).

Preferentially, the gene of step (ii)b,. is a Plchia siipiUs gene encoding the xyllol dehydrqpnase ehEyme XDH, Preferably, it Is the XYLÊ gene, the sequence of which Is the sequence reported in Gen Bank under number 4052013, or a sequence at least 70 % Identical, preferably at least 75 % identical, or at ieast 80 % identical, or at least 85 % identical, or at least 80 % identical, or at least 95 % Identical, or at ieast 98 % oidentical, or at least 98 % Identical, to said sequence reported in GenBank under number 4852813, and encoding a functional xylltoi dehydrogenase enzyme.

Preferentially, the yeast strain of step (I) has an endogenous xylltoi dehydrogenise XDH activity of less than 150 mKat/g of proteins. The xyíitol dehydrogenase activity can be measured in the conditions set forth in the article by Xu et ai. entitled Characterization of Ethanoi Production from Xylose and Xylitol by a Cell-Free Pachysoien iannophllus System, in Appl. Environ. Microbiol. 89:231 »235 (1993).

If is known that during the processing of the biomass intended for alcoholic fermentation, certain fermentation Inhibitors appear. Among them, mention may ho made of phenolic products, of furfural or further acetic acid. it is also known tkot these inhibitors are detrimental to the performances or even the survival of the yeast in order to solve this addition a I probiere, the Applicant suggests selfctlnf fie Strain of step I, from industrial strains haying resistance to phenolic derivatives.

Another advantage of the XI route, Is the possibility of « grafting the bacteria! arabinose route In parallel » as described for example in EP 1 499 708 oryetdWWÖ^0ÜS/O4ilMÖ, 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 past producer and those of a Inal user In its applications notably In terms of industrial production of ethanol with low cost ar# high yield.

The methodaccording to #|i..invention· has, in particular, the foilowing advantages:

For the yeast producer, it allows: ~ construction of a prototrophic aneu/polyploici. 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, rnoiasses, - availability of a Saccharomyces cerevisiae yeast strain having a maximum growth rate (μ ma#comprised between 0,37 h'1 and 0.5 h'1, avaifabily of a Mamfmmmycm cerevisiae yeast strain which, when it Is produced according; to a method as described in the retef^hci bdpN t Tlast Technology » (Ind edition, 1991, G. Reed and T.W. Nagodawithana, published by Van Nostrand Reinhold , ISSN M4M1S92#), offers the possibility of obtaining a biomass production yield of at least 45g of yeast dry materials for iOOg of saccharose equivalent applied. - availability of a Saccharomyces cerevisiae yeast strain, resistant to the drying pmeeas as described in patent documents EP 511108 and US 5,244,095, the loss of fermentative activity altar drying shouib not exceed 30%, - production undor industrial conditions fin particular, Inexpensive medium, pod biomass ylsii,ready^usa yoestl of a fresh or dry yeast from a genetically stable, Saccharomyces eer&visiae yeast strain notably robust because it is tolerant to high concentrations of ethanol and capable of producing, for example from hemi-'Ceífulose biomasses, at least 40g/l of ethanol and this at a high temperature of the order of 30 to 4CPC. 4 prototrophic yeast strain Is a strain capable of growing on a minimal medium. In particular, a prototrophic yeas? strain according to tbs invention is capable of syntbetizing all amino acids and bases that are necessary for its giowth, 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 «ter.

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~ aminohenzoic acid, 400 pg pyridoxine hydrochloride, 200 pg riboflavin, 400 pg thiamin hydrochloride, 500 pg boric acid, 40 pg copper sulfate, 1ÖÖ pg potassium iodide, 200 pg ferric chloride, 400 pg manganese sulfate, 200 pg sodium molybdate, 400 pg zinc sulfate, i 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 (li) the expression cassette consists in the association of the open reading frame (QRF) type of the gene Xi encoding the xylose Isomerase enzyme of Clostridium phytofennentans l promoter and terminator of iBacchammyces cerevisiae, said cassette being 'flanked upstream and downstream with recoffibinogeneic regions allowing its targeted integration into tie genome, said method then further includes a step of saecharfication and simultaneous fermentation (SSF) In the presence of polymers of hexoses, predominantiy 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) past, obtained according to a production method as described In the textbook « Yeast Technology », from a Saccharomyces ceœvkiaa yeast strain as defined in the preceding paragraph which is: - capable, under the SSF conditions described In patent document WO 2ÖÖ4/G46333, of fermenting at 32X a hydrolyzate of cereals up to a minimum ethanol concentration of 18% (w/w). - capable, under the SSF conditions described In patent document WO 2004/046333, of fermenting at 35X 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/pelyploid so-called Industrial strain and in fact having a clearly more complex genetic material than that of a so-calecl laboratory strain, at the very least making the consequences of modifications of said Industrial strain unpredictable. This complex genetic background, spéciié 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. i is therefore necessary to have available toois/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 Credox technology, in briefs loxP sequences ore provided on each side of each selection marker. Excision of the selection markers is performed by transforming the yeast airain by the ilhibfh acetate method (Schiestl et Gietz, 1989» Current Genetics, voi 16, p.339-346), using a plasmid comprising the Cre recombinase gene and a selection marker dlfforshf from the selection marker(s) to be excised. The expression of the Cre recombinase in the yeast strain makes it possible to excise the seiection marker, leaving only a loxP sequence, possibly together with its fianking sequences. It is then possible to induce the loss of the plasmid comprising the Ore gene by culturing In non-selective conditions, /,e. In an enriched medium intbe absence of antibiotics. For example, the construction of yeasts compliant with the invention required the use of 4 difforenf positive markers giving resistance to 5 different antibiotics (geneticin, phieomycin. hygromycin, biasticidin and nourseothricin).

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

But this Is an additional difficulty encountered when dlsruping/iiiacivating all the copies of a given gene is desired. However, this aneu/poiypioidy feature is generally at the origin of many interesting poperies of industrial yeasts (growth rate, resistance to different stresses, phenotype stability').

Further, the Applicant after iong 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 exper sences improvement in its genetic heritage.

In particular, the investors have shown that with said strain, it Is possible to achieve: - the deletion of at least two copies of thé gene GRE3 of S. a&r&visiae (the Gre3P en^me being; an aldose reductase which consumes ΝΑΡΡΒ,Η* 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-f by said enzyme, by that much.

As a preferred aiamatiym said at least one gene of each step of the non-oxidative portion of the phosphate pentose route of step (if; is selected from the group formed by the genes encoding the O-ribuiose-5--phosphate 3~ epimerase, ribose-S-phosphate ketoMsornerase, transketolase and fransaiioiase enzymes, and notably from the group of the RPE1, RKH, TKL1 and TALI genes, Preferably, said promoter of a strongly expressed glycolysis gene during aicohoiic fermentation is the TDH3 promoter for RPEL RKil and TRkf, and WGKi for 1)411,

The TAL 1 gene is preferably the gene reported in Gen Bank under number 8S1Ö65,

The TKL1 gene Is preferably the gene reported In GenBank under-number 856188.

The RK11 gene is preferably the gene reported in GenBank under number 854262.

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

Aoeording to compiambntatT or alternative features in the method for preparing a Saccharomyces cerevisiae yeast strain according to the invention: - the Saccharomyces cemvisiae promoter of steps (ii)(a) and (ii)(b)is selected from the group comprising the promoters of genes eheodthg glycolysis enzymes and those encoding the alcohol consisting of ADH1, ADH2< Μ3Κ% TDH3, RDC2 and GAL1/1Q, preferably ADHt The terminator of Samhwvmyces cerevisiae Is formed |y CYC1 or by the specie terminator of the gene of the noo-PkidStive 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 O? in a medium including said St least one pentose, and

Cili) selection by aerobic growth on a solid medium containing glycerol as i single source of carbon, so as to provide respiratory nondeficient mutariS of said yeast which exhibit growth In anaerobiosis in the presence of a medium including said at least one pentose inotabl^acyloseb

Preferably in this alternatives the mutagenesis of step (0 is performed under « mid » conditions, i.e. moderate mutagenesis with 180 to SööJ/crn2 and still preferably 300J/cma of ultraviolet radiation at 264 nm. These conditions only cause mortally of 7% to 18% of the population subject to UVs.

The inventors have tlareby surprislhgfy shown that with such a so low controlled mortality, it is possible to reduce by a factor TO the duration of the directed evolution step with cyclic cultures required for obtaining mutants capable of fermenting said at leas! one pentose (notably xylose). The survival fileJsr 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 Q2 limitation of step fill of this alternative Is achieved by partial overpressure in the equipments used (for example vials or fermenters} due to overpressure consecufive to production of produced CDs.

The eyoilo cultures according to this alternative, under fermentation conditions, offer the possibility of enriching the population In mutants capable of fermenting said pentose (notabiy xylose) and this within a period from 2 to 8 weeks, and preferably from 3 to 4 weeks, which is relatively short and highly interesting as compared with what would be obtained by ohemostaf, as described by KuyperφM (2004), FEMßYmsi Res. 4, 865-664,

Although the « petite » respiratory deficient phenotype may coincide with the fermentation criteria of oaii it least one pentose, in this alternative, the present inventors carried oaf a stepferram^ petite » yeasts since this phenotype is incompatible with the methods for producing industrial yeasts in the sense of the invention.

The resea?chers noted that the directed evolution step as explained above made it possible to significantly increase the xylose isomerase activity, which Is characterized by an increasi 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 effect of the present invention is further the Εββ Saccharomyces c&revisiae 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 201Ö at the C.N.C.M (Collection Nationale de Cultures de Microorganismes of the Pasteur institute, 25 rue du Docteur Roux, 75724 Paris, Franc# under Νο,Ι-4399 under the terms of the Budapest treaty.

The object of the present invention is aiso the EG? ÊmëMmmymâ cerevisiae industrial yeast strain direotiy 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 Cuitures de Microorganismes de l’Institut Pasteur) under No, 1-4400 under the terms of the Budapest treaty.

Ihe object of the present invention is also the EGS Samhammyms oer&visiae Industrial yeast strain directly obtained by the method according to the invention after the directed evolution step, deposited on December 14 2010 it the C.N.C.M (Coiiection Nationale ele Cultures de Miercorganisrnes de í’ínstitut Pasteur) under No. 1-4417 under the terms of the Budapest

The object of the present invention is also the EG 10 Saccharomyces œrevisiae industrial yeast strain directly obtained by the method according: to the invention after the directed evolution step, deposited on October 5 2011 m the C.N.C.y (Collection Nationale de Cultures de Microorganismes de íirístM Fasteurl under No. 1-4538 under the terms of the Budapestireaty. Öther strains according to the Invention are stralns derived from one or more strains accordtnf to the invention, for example tom 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, Í-440Ö on November 23, 2010, under No, I-4417 on December 14, 2010 and under No, t-4538 on October 5: 2011.

Dy 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 crossbreeding 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 strilns which have undergone at least one spontaneous mutation in their genome or at least one mutation induced by mutagenesis, The mutaiion(s) of the derived strains can be silent or not % “mutagenesis” is meant both random mutagenesis obtained by applying radiation (e.g. UV) or by mutagenic chemicals, and insertionai or directed mutagenesis, by transposition or by intolralon of an exogenous DN& fragment.

The derived strains which are within the framework of the description aire 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 ÖJ g, preferably 0,3 g, 0.35 g or even 0,30 g ethanoi 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 RK!1 and/or TKL1 and/or TALI genes by a promoter of a gene which is not repressed by anaerobidsis or by oatabbllo repression induced by any source of carbon, arid strongly expressed during alcoholic fermentation, such as a promoter of a gene encoding a glycolysis enzyme or encoding an alcohoi dehydrogenase enzyme, preferably the ADH1, PGK1, TDH3t PDC2 or G Ail/10 promoter.

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

Preferably, the Saccharomyc&s carevisiae yeast strains prepared according to the present invention and to the criteria defined above retain, after intrdduotion of the and other mutations generated during the directed evolution step, their genotype and phenotype charaetenslics after a complete industrial production process. In particular, the yeasts producert have kinetics for producing alcohol, kinetics for consuming: xylose and/or arahinose and a maximum produced amount of alcohol, strictly identical with those of the yeast strain before applying a complete industrial process.

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

The object of fie present description is also a method for producing ethanol from a medium including at: least one pontosé, by fermentation with yeast according to the invention, rhohtionert abbvdi Of such as obtained with a method according to the invention as if has just been described.

Frefeiably, the method for producing ethanol has the following alternative and/or comple me nia ry c h a rscieristics : ······ 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, hemkcellulose dextrin and starch hydroiyzates.

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 extricollular concentration of hexose, predominantly glucose, - The average yield of ethanol produced by consumed xylose is greater than or equai to 0;|8 f 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 1 SOg/kg of xylose) to the knowledge of the Applicant are the maximum concentrations which may be encountered in practice. AH tie published tests referring to fermentation of xylose were conducted with much tower concentrations of total sugars.

Other features and advantages of the invention will become still better ippareh! upon reading the exemplary embodiments which are given purely is an lustration 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 strafe Is as described the description above.

All the ONA sequences which were used for the different transfomiatiohl 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 overdxpressidn of fee XDH of Pichfa stipifis Is illustrated In Fig, 1,

An exempiary vector used for the overexpression of the Xi of CbMndmm phytofenvenîansm illustrated in Fig. 2.

For disrupting the copies of the GRES gene of the seiecteci industrial strain, the inventors used PGR ampltoens from a plasmid of the pUG8 type (Güldener U, Heck S; Fielder Ts Beinhauer J, Hegemann JH. Nucleic Acids Res. 1996 Jul 1:24(13):251^24).

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 yeas! strains according to the; invention, EG6, EG7. EG8 and EG 10 respectively, were deposited at the CNCfvl and No, 1-4399, No. 1-4400, NoJ*441? end NoJ-4358 were respectively assigned to them.

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

Eihsnoi Delta GRE3< 8 U05::pA DH 1 ~XKS 140 Y Cl, tku::ptdh3-tkl 1-RKI1::p TOH3~RKI1~ tCYCi, HO:mXYL^H¥QRO, 8υ05;:€ρΧί-ΒίΑ8Τ - are free of any residual marker (by the action of ore recombinase).

Example 2 fdutagenesis of these strains obtained in the previous example Wio performed in a moderate fashion l.e. from 109 to iOOj/ont2 and preferably OOOJ/onl- of UVs at 254nm.

Mar a; week of culture at 32°C in a YE type medium (9.5% Yeast Extract) containing 7% of Xylose:, with stirring, without ventilation - the O% limitation being achieved by means of parfiai ivet|)ressure in the vials due to CÖ2 produced during fermentation - one mi of the culture is Used for reseeding the same medium. This operation is repeated 8 times. The pgfis are finally spread out on a gelose YE 20g/L glucose medium, isolated colonies are sampled and then successively culivafed 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. ixameM 3

After obtaining the EG6 strain, the copy of the XfDH gene which was added to Example 1, was substituted with the gene of resistance to ksnamycin. The new obtained strain is called EG6 - XDH. The xylose fermenting capacity of the relevant striih was compared with that of the EG6 strain. The result of this comparison is shown in Fig. 4. SEQUENCE LISTING <110> Lesaffre et Compagnie <120> A method for preparing an industrial yeast:, itttetii'âl yeast and application to the production of ethanol from at (east one pentose <n0> 3873*02101 <]60> 2 <170> Patent! h version 3.5 <21 (.» ! <211> i 317

<212> DNA <213> Clostridium phytofenSènlam <40C)> 1

akgaáaa&bt. aet:.tfccoaa& tgktecagsa gtaaasfcacg aaggccca»» fctô&sœg&afc SO ccatfcfcgcstvfc ti'-Saat&tt& tg&cîgc.aaaÆ KS.agti.gtxg cgggtaaa&c sxfcgsx&gsg X20 exctgbegtt fctgaátfc&fcc kiggfeggaxfe aæfcctttgbg csggfcggfcgc- fcgafcccxttc 100 ggfcgbs&oss ct&*££&«*£ ascsfesggga astateapag *%G«sm%W*· aefcfeçetaag 240 qmm&gtt% aegct-ggitt actasattag ga&fcfe<j»&fcc gfctetgttee 1SÔ ©afcgaogcag tóakfegeköe .xgaxggtgat actbtagaag sgicxa&gxs gsatcvit-kb 360 gaa&cegfetg afct&e&tcaa agagaagstg gatesgacr,«! gtatoaagtfc abtatggggfc 420 aetgefc&ata aetteagtea te.casgattfc ätgea&ggfcg cr.tcscacsfcc feeggaaegcsa 4S0 gacgaatfcsg eabasgetgc fcgcta&gakt &&gs.akg«sat, bsgatga&ác a&etaaatka $40 ggcggtaaag g-kt.atgt.acfc ctggggtggt cgtgaaggv.fc atgaaacact tcffcaataca SÖ0 gatbtaggac fckgagcttg& iaatstggct sgacttatga aga&ggsfegt is.gxgtxkggo 660 e.gfcg«<aaafcg gtfcttgatgg cgacfctssfeafe aetgagcc-aa agccaa&gga acc&aaaaag 72» öafcäSafeafcg akkCfegsfese' agsaaçéçta cttgctfctoc ttegca&afea feggtótfeagga 7§ç aasgafebfeg« &gatg&a<siík catgcfcacfcc tfcgcággfces fcagotbtgaa 040 c.&tg»aofctg e&afcggcfc&g· agr-taar.ggt gc.attfcggfct ctgfcag&fcgc asscesgggt- $00 gsfcsçj&a&gg bfegg&fcggg& t&cggaeeaa keesesacbf afcgfcfcc&fcsg kgcaaefeefct 060 gça&fcgekcg aagfca©fc.fca& ggcfcggfcggs fcgg&efc*««g gegg&áfckaa ©fcfcfcg&fcgga X020 àaggt-aâgaç gkggkfcecfefc «gaattagaa g&fc&fcfcgcafc «BfgfefcafcSfe fcge&ggaatg :tÖS0 gafcaotkttg easfcfcggfcfcfe &&fcfcg»ggçfc gctgagstta teg&eg&fcgg taga&tcg©« 1140 «»atbfegfc&g atgaCegtfca tggs&ggfcäfc assacaggaa ttggtaaagc: aafckgtggst 1200 ggaaetag&fc «fccfcfcgasgs afctsgageag tatgttttaa öaoatagbga acos.gfc&sfcg 126ö eagagt-ggé« g-fccaggaagfc 't'őfetgaaaea áfcögfcáa&fcs atákkttatfe tagafcaa 131?

<2 K» 2 <215> 438 <212> PRT <2!3> Clostridium phvm fermentant <400> 2 írni Aya Äan Ayr PA« Ktt-o äs« Val Pro Glu Val Ays ’üyx Glu Gly Vxo X ' 5 JO 15 Äs« Ser Tb.r Äs« äso PS« Ala Äh« Ays Gyr Tyr &sp Ai« As« Aya Val 20 25 30

Val Ala Gly Sys Ähr Hst Ays Gitt His Cys Arg Äh« Ala fee« S«r l’rp 35 40 45

Tvp Gis Thr X«u Cys Ala Gly GXy AI a Aá$ Sro Ohe öly :ShÄ Ihs 50 55 SO «et Asp Arg ihr Tyr Gly As« 11« Uhr Asp író »£ Glu A®« Als Ays m 70 7S 80

Als Ays Val Asp Aia Gl y Oh« Gl« lau Siet ïhr Aya A©u Gly XI« Gitt SS 30 35 Äh« *hé :6ye Äh« Hia Äap Ala Asp 7 le Ais Pro Gl« Gly Aap SA« Äh« 3.Ö0 3 05 110

Glu Gitt S«r Aya Aya As« !<©« Ob.« Glu T.l« Val üsp Tyr lia Aya Glw 115 120 ISS %S » % ati: S?hr Gly u* Aya Aa« A«tt ï.rp Gly Thr Al«. Aa« As« ISO 135 140 Äh« Ser Kis y.va Ay§ ptt« Mat iiis Gly Ala Ser ihr 8 er Oys As« Al » 143: ISO ISS 160

Tht 11« Ays Sa« Gly Gly Aya Gly Tyr Val Ph« Trp Gly Gly Arg Gl« ISO 185 1.90

Gly S'yr Gitt ihr Aaa Saw &«« Ghr Asp A«tt Gly A«ü: Glu Α«ώ Asp Asa :1 SS 200 ...... 2 OS mt Ala Arg A<Stt *&Sh Aya Met Al» Vei Gitt %r Gly Arg- Ala Assv GXy 210 21S 220

Pha Asp GXÿ: ¾Ph® Ty* XX« Slu Pr« Lys p.t® Lys Siî » Th* Lys 225 230 235 246

Hiss <3X ή Ty* Asp Ph® Asp Th* A3.« 'ïhr Vai À«« Ai a Ph« Lsu Arg Lys 245 250 255

Tyr GXy hass Giss Lys Asp Aïs.« Lys Ptefc Asn XX®: GX« AX® A«G HXa ATS: 2«0 265 270

Thr Asm Ai a CUy SU s ï'hr Ph® Glu His Ma La« Ala Mat Ai» Arg Val 2A5 288 263 Äs« SXÿ AXa Ah« Gîy See VaX Asp AXa. As® GX® ÔXy Asp Pro As« Lau 286 2SS 300 SXy Trp Asp: Thr Asp SX« Ph® Pro Thr Asp VaX Hia S«* Aia Th* La® 305 Si8 3X5 320

Als H»« ïssp ®Xw val t>m Lys AJ.® SXy Siy Sfcj*. '*(*# *»» SX y SXy Leu 325 330 335

Asn Ph® Asp AXa Lys Val Asrp Arg GXy Sur Ph« Siu Phs &so Asp XX® 340 345 3a0

Ai» Tyr CU y Tyr XX« AXa GXy Mat Asp Th«.' Ah®: AXa Ls« OU y X<®« U« 355 300 365

Lys AXa AXa Si» XX« XX« Asp Asp «Xÿ A*§ XX® AI* Lys PA® V&î Asp 370 3X5 380 hsp: Ah®: Py:p AXa H®* tpr X<ys Thr GXy XX« Sly Lys Als XX® V&X Asp 385 ' 3$ô 305 400 GXy Thr Thr Sar Lau SX« Gl« L®« Si« SXh VaX Ls« Th r Hi s S«r 405 410 4X5 81« Pro VaX Mat Plu Ser GXy Arç GX« SX« Vai Leu Glu th«· XX® Val 42Q 425 430

As a As« Xi« I<®« S?h.a Arg 433

Claims

$z8bsdais*it Igénypontok

1, Élesztő törzs, äz&&! jeilé»?ezve, hegy a xilóz rneíabólilats útvonal exogén génié-Irtarlaimazza, ahol az élesztő törzsben jelenlévő xilóz metabólíkus útvonal exogén génje! egy xílóz»Í2omerázt kódoló exogen gén |i^Âsip'.:i%li|àbôl, és egy xllit-dnbídrógenázt kodolő exogén gén kópiájából állnak, és mmi bogy tmután egv irányított evolúciós lépésen ment á| amely magában foglapi a lővefcm egymás diám lépéseket, amelyeknek m élesztő törzset alávetjük: (i) mutagenézis, (ii) növesztés ciklusos tenyészetekben korlátozott ()2 alatt legalább egy pentóxf, nevezetesen xilózt tartalmazó közegben, és Cili) glicerint egyedüli széníotessklnt tartalmazó szilárd közegen aerob növekedés szerinti lekció. S, ÀJ. igénypont szerint! élesztő törzs, ahol a xilóz-izomerázt kódoló exogén gén egy alábbiakból származó gém CïmmÊMm Piromyces, Baeteroides. Strepíotnycm, Haem&pMIm, :BmkhoMmai Bßterococcus, Thmmmiigm Fímmacíenim, GmbaciBm: ártémkacfer, ftl FhysmmdmUa, Ceíhíhrw, ChitimpMűga^ Saccharúpöiymam vagy SaHmbacter, és előnyösen egy alábbiakból származó :g#b, €ksirf$jum·· vagy Bímmyces sp. E2.
3, Az k vagy 2:, tgénypöhi szerinti élesztő törzs, ahol a áilŐzAzomerázt kódoló exogén gért Píchia snphis-böl származik,
4. Az 1-3. igénypontok bármelyike szerinti élesztő törzs, amelyet a Saccharomyces spp,, Schizosaccharomyce? spp., Pichia spp., Faffio spp,, Kluyveramyces:;§pjpk»,CÄÄÖ s|p,s Talaromyces spp,, Brettmmiycei spp.. Pachysokn spp. és Debaromyccs spp. közül vlja^íopk, és előnyösemé Eaccharomvces cerevisiae törzs,
5. Az 1-4. igénypontok bármelyike szerinti élesztő törzs, ahol egy aldoz-rédttkiází kódoló gén legalább egy kópiáját, előnyösen legalább két kópiáját eitávolitottuk.
6, Az 5. igénypont szerint! élesztő törzs, ahol a GRE3 gént távolítottak el,
7, Az 1-6. igénypontok bármelyike szerinti élesztő törzs, ahol egy xilulokmázt kódoló endogén gént, előnyösen a XKSÏ gént, az anaerob körülmények áltál vágy bármely szem forrás kiváltotta katabolikus represszió által nem represszálí gén promoíerénefe nienőrzése alá helyezzük, és erősen expresszáljuk alkoholos termontáció során.
8. Az 1-7. igénypontok bármelyike szerinti élesztő törzs, ahol a pentóz-íoszíat útvonal nem-oxidaisv részének legalább egy endogén génjét, amelyet előnyösen a RPEÎ, RKIL fiSi és TALI gének közül választunk, és különösen előnyösen ezen gének mindegyikét, az anaerob körülmények által vagy bármely szén forrás kiváltotta kaiaboltkas testesítő aha run représszált gén promoterének ellenőrzése alá helyezzük, és erősen expresszáljuk alkoholos fermentáció során.
9. Aï 1-8, igénypontok bármelyike szerinti élesztő törzs, áhoi a promoter egy gUkolÍKts enzimet vagy egy alkohol-dehidrogenáz enzimet kódoló gén promoter«, előnyösen az ADNI, PGK1, TDH3. PDC2 vagy GAL1/I0 promoter« '10. Az 1-9. igénypontok bármelyike szerinti élesztő törzs, ftg&sd jellemezvén Ipgy az egy aneuploid vagy poliplnid törzs és-'Vagy egy protoiróf törzs.
11. Az -WC), igénypontok bármelyike szerinti élesztő törzs, jnleinezves hogy a xilózózomerázt kódoló exogén gén legalább két kópiáját, előnyösen legalább barom kópiáját vágy a xilóz-izomerázt kódoló exogén gén legalább négy kópiáját tartalmazza,

11, Az 1-11, igénypontok bármelyike szerinti élesztő törzs, az&sil hogy az egy Ipári törzs, amely eílenál! á btöiöassza hidrolízisből 'eíed&ifemeittáőti' Mfbteroknak, mint a^ feolos termékeid vagy eceísavnak.
13, Az 1-1:3, igénypontok bármelyike szerinti élesztő törzs, azzal ptteÄezve» hogy legalább lő %, előnyösen legalább 1? % etanol %ppfontráeÍH: ierpei* gabona blőrolizátttmon, szimultán cukrösitisl is fermentációs körülmények 32ô€*onu
14, Az 1-13. igénypomok bármelyike szerinti élesztő törzs, azzal jelkiaezve* hogy az alábbi törzsek alkotta csoportból válaszok; Saceharomyc&s cmmmme törzs letétbe helyezve a CNCM-néi [National Collection e£ Microorganism Cultures} [Âfe^yg^àzrmtsiBéRyésZéték Nemzeti Cjyyjternébfej 2010, november 23-án 1-4399 számon, Smiéim&mye&s c&rmmm törzs törzs letétbe helyezve a ÇNCM-nél 2010. november 23-án N44ÖÖ; számon, Smeharúrnyces eërevïsiae törzs törzs: letétié helyezve;& CNCM-néi 2010. december 14-én 1-4417 számon, és a $m$km&wt%wMMtëVîsim-ièwê,i^^htéthe. helyezve a CNCM-néi 2011, Oktober 5~énN4a38 ·** ..... :«* számon.
15, Az 1-13, igénypontéi;h.teői$tk&ifeéiinii élesztő törzsi aizaí jellemem7«, hogy a 14, igénypont szerinti egy vagy több törzsből ered,
16, Eljárás élesztő törzs előállítására, amely magában foglalja a xiiöz metabóiikus útvonal exogén génjének beviteléi a kiindulási élesztő törzsbe, ahol a kiindulási élesztő törzsbe bevitt xilőz metabóiikus útvonal exogén gén a xilóz-izomerázt kódoló exogén gén legalább egy kópiájából., és a xüit-dehidrogenázt kódoló exogén gén legalább egy kópiájából áll, és tartalmazza az irányított evolúció következő lépését is, amely a kapott élesztőn végzett alábbi egymást követő lépésekből áll: (i) mutagenézis, (II) növesztés ciklusos tenyészetekben korlátozott O; alatt legalább egy péntózt, nevezetesen xíiózt tartalmazó közegbehj Iá ( h) glicerint egyedüli szénforráskéni tauahnazó szilárd közegen áerofe uivekedés szermti szelekció, oly módon, hogy az adött élesztőből nem-légzéshtányóá mutánsokat kigylij^ük, amelyek anaerob körülmények közöli nőnek 4 legalább egy pentőzt tarfalmaző közeg jelenlétében. ïf, át M igénypont szerinti eljárás, amely ezen felül mRalmazza az áldoz redukíázt kodoló gén, előnyösen a GRE3 gén, legalább egy kópiájának* előnyösen legalább kit kópiájának: eltávolítását a kiindulási törzsből. 18. 4 Iá vagy l:frii^ypont ólját# az 1-15. igénypontok bármelyike sze rinti élesztő törzs előállítására,
19. À lő-18. igénypontok bármelyike szerinti eljárás xilóz tázíáimáMzegMi etanol előillMsám képes Smokatm§mm eererísíae élesztő törzs élöálíításÉra, amely a kővetkezőkből lié lépéseket «almazzá; (i) egy Sacçkmomyms eermMmWm szelektálása, (ii) az (i) lépésben kapott élesztő genümjába a következő ekpressztős kazetta beépítése, á, égy S&mkammyces cmë&ae promoter és terminator irártyítása alatt egy xilóz-izomerází kódoló gén nyitott feelvasisí keret (ÖRF)-típnsú kombinációja, amely kazetta lefelé és felfelé irányban rekombinogén régiókkal szegélpzett,: amelyek lebetivé teszik annale célzott beépülését a genomba, b. egy Saccharomyces cerevmm promoter és terminator irányítása blatt egy xilít--débidrogenázt kódoló gén nyílott leolvasási keret (ORFKípnsó kembmáéíója, amely kazetta lefelé és felfelé irányban rekombinogén régiókkal szegélyezett, amelyek lehetővé teszik annak célzott beépüléséi a genomba, fül) a pentóz-foszíat útvonal nem-oxidaiív részének minden egyes lépésére legalább egy gén, valamint a xilulokmázt (XKS1) kódoló legalább egy gén expresszlójártak előidézése oly módon, hogy ezeket sem az anaerob körülmények által, sem bármely széníbrris kiváltóin katabolíkus represszió által nein represszált gén prontoterénelí ellenőrzésé: álálelyezzük, és erősen expresszijük afcholos fermentáció során, és (ív) egy aldóz-reduktázt kódoló o)mchatomsin icnunac GRES gen nysvn leoKesé-j ke*el (ÜRF) legalább két kópiájának eltávolítása. 20. A 19. igénypont szerinti eljárás, azzal jellemezve, légy a xilóz-izomerázt kódoló gén égy xilóz-ixomeráz enzimet kódoló XI gén, amelyet a Closiridnm, Piromyces. Bactemíáed, Streptomyces, Haemophilus. Bitrkholderia, Enterococcus. Thermotoga, Fuxdbmtmim CkabaciUuS', Arthrobacter. Ciorta, PlyscomitreUa. CeUvibrio. Chidrtophaga, SacchtwapMyspma vagy Satunbaaer genomjában jelenlevők közül választunk. il, A 19. vagy 20. igénypont szerinti eljárás, azzal jellemezve, hogy a XI gént a Clostridium phytofermemans vagy Piromyces sp. E2 génjei közül választjuk. 22 A 19, vagy 20, igénypont szerinti eljárás, azzal jellemezve, hogy a xiít-- dehidrogenázt kódoló gén a PícMa síipitis xHil-dehídrogenáx enzimet kódoló génje. 23, A 19-21, igénypontok bármelyike szerinti eljárás, azzal jellemezve, begy az (í) lépés szerinti éleszti törzs endogén xilít-dehidrogenáz XDH aktivitása kisebb, mint I SO mkat/g fehérje. 24. A 19-23. igénypontokszerinti eljárás, azzaí Jellemezve, hegy az (i) lépés szerinti élesztő törzsei olyan ipari törzsek közöl választjuk, amelyek a biomassza hidrolízisből eredő fermentáció inhibitorokkal szemben, mini íenolos termékek, furfural vagyeeetsáy, ellenállók. 25. A 19-24. igénypontok bármelyike szerinti eljárás, azzal jelléMezve, hogy az (Hí) lépés szerinti Saccharomyces ver évit iae promoter! választjuk a giikolízis enzimeket kódoló gének promotereit tattalma/ó csoportból és az alkohol-dehidrogenáz enzimeket kódolók közül. 26. A 19-2$. igénypontok bármelyike szerinti eljárás, aszal jellemezve, hogy az. adóit csoport ADMl, FGKi, TDH3, PDC2 és GALHiO-bői, előnyösen ÁDH1~\M áll, és azzal, hogy a Saccharomyces cerensim terminátor CYCJ-h&í áll vagy a oem-oxidalív pentóz-foszfát útvonal gén megfelelő terminators. 27. A 19-26. igénypontok bármelyike szerinti eljárás, azzal jellemezve, hogy áz 0) lépés szerinti törzs egy ipari törzs, amelyet nagy eíanol koncentrációkat:, gabona alapon legalább 17 térfogat/íérfogat%-ot, szaccbarifikácios és egyidejű férmentáéII§rpSf)LÍ^lmények közöd. 33^00 termelni képes törzsek közül választunk. 28. A 19-27. igénypontok bármelyike szerinti eljárás, azzal jellemezve, hogy továbbá tartalmaz egy vagy több lépést antibiotikum rezisztencia ntarkerek beépiíésére,: egy v®gy több lépést a törzsek antibiotikum rezisztencia kritériumok szerinti kiválasztására, ék egy vagy több lépést az antibiotikum rezisztencia .markerek eltávolítására. 29i. Eljárás xiiózt tartalmazó közegből eíanol előállítására az 1-Íf. igénypontök bármelyike szerinti elevm, vagy az lo 28 igénypontok bármelyike szerint előállítón élesztő íennentálásával.
30. A Ä igénypont szerinti eljárás, azzal jellemezve, hogy a közeg xilóz és arabin őz keveréket tartalmaz. 31, A 19, Vágy 30. igénypont szerinti eljárás, azzal jellemezve, hogy a közeget a iignm-j eeildloky hemiééjMé^Âëi^nyitô-hidrôîMtumbôl álló csoportból választjuk.