HUE034441T2 - Xilóz metabolizálására képes és inhibitoroknak ellenálló élesztõ törzsek, eljárás elõállításukra és alkalmazásuk - Google Patents

Description

YEAS!' STRAINS CAPABLE! OF METABOLIZING XYLOSE AND RESISTANT TO INHIBITORS. METHOD FOR OBTAINING SAME AND USE THEREOF

YIOMUAL FIELD

The present invention relaies» lo Ihe field of yeast strains capable of metabolizing xylose and also exhibiting resistance to at least onit.%meö^tí®p..khibkor,: ip, the lor ohtnrnrng same, and to the use of iiese yeast strains lor producing a fermentation product in a medium comprising xylose, opionstly in the presence of at least orte ter.mentat.iou inhibitor.

Document PCI' I P201 i o"lolOdescubes \casts for producingalcohol in.medib: at least one pentose. via the expression of the XI X Dl l metabolic pathway. Said document deseriiesfor fie Írat lime: ite au ovetexpression of xylose isomerase (XI)

activity with an overexpresston of xyUtol dehydrogenase (XDB) activity and shows that the overexpression of xyhtol dehydrogenase activity makes it possible to prevent the Íiilulsitloa, by xylitoh of xylose tsomeraso aetnity, Document POT ΓΡΆΙΙ I 0716.1b describes, for example the yeast strum deposited at the CNCM (National Collection of Microorganism Cultures) on Uctdher 5( 2Öf I, verier munher L45M

Tie liidhstrial prodbdtoh of alcohol bfsP&Cttof Iptoceilulosle hydsolyxsies requires not only ihe genetic machinery lor metaholixing pentoses,/hut u1.so:'|^$efiieS·oÉÂtSfenotrm- it«: fermentation inhibitors preset# in these ligtKicelhriosie hydrolyxares.

The fermentation inhibitors ; are produced during the pmUeslment and the hydrolysis of the IlgaoeeiMosid biomass. Among the feri»<ÄtÄS:'lhbibite are thralriehydM |teiïml, BMP), phenolic compounds aval organic acids (acetic add, ievuhmc acid, formic acid).

Various means have been .described ibr eountering the effect of fermentation inhibitors, among which are detoxification of the ienneniation thedium, improvement of the fermentation process, or Improvement of the resistance o f yeasts to fermentation irdrihi tors.

The resistance of yeasts to fermentation inhibitors has, tor example, been improved genetically, by directed evolution or by aeelimatioation (Almeida and Hahn-Hägerdal, International Sugar Journal, 2009, Yoi 111, No. 1323),

Toyoma mat fBloresonr, 1Vchnoi,ÿ 2011, 12: 7917-7024} describe yeast strains that ferment xylose and that are resistant, to acid,.-oi?if^îr^î:«î thesó yéásilTdrig obtained:by íhere^ip|;:iÍfó''^an^iÍ0l»(TAI.) andformatedei|dirogepase (EDH.) activities. lib\v«v«r, it appears to be difficult to provide an industrial yeast strain winch gives yeasts which fife both ef&amp;ethe for alcohol production in a fennentation medium eonmrising at least one |^nte^äafresisÄi:tO:älioast one fermentation inhibitor.

There is thus a reef need to provide novel yeast strains which are capahfeof nmtabohxmg at least one pentose, tor example xylose, even in the presence of at least one fermentation inhibitor, stieb as aéèticttslà* -sali y««tóM.|pMng yeasts which^atX5 elfeeive tor pfodueing alcohol, even in the presence of at least one fermentation inhibitor. AAbbject of the inventipngpiates to the yeast strain deposited oohla.y 2% 2012 ai îlnCNÇM [National Collection of Mieroorganism Cultures] under number 1--1027,

Another object of the invention relates to .a method for obtaining a yeast strain capable of metabolizing xylose and resistant to acetic acid, eompttaingthe; steps oil -- crossing the yeast strain deposited on October 3, 2011 at the €NCM [National Collection -of Mismerganisrn Cultures] under number MS38 with the yeast strain deported on May 24, 2012 at the CMC ’M undet number 1-4n2"·. so as tv·· ebnen at least one hybrid, - selecting at least one hybrid capable ot metabok/mg xylose and teswunt to acetic acid. Ibcfreschtidoeernent also describes a yeÄstPhP: capable ofmetabolizing xylose and resistant to acetic add, which can be obtained by means of the method as defined above.

Also described is a yeast strain derived from a yeast strain as defined above, characterized in Utal said derived yeast strain is capable of metabolizing xylose arai resistant to acetic add. Also described is a yeast obtained by culturing a yeast strain as defined above or by culturing a derived yeast strain, as defined above.

Another object of the Invention relates to a method ibr producing at least one fermentation product, comprising a step of fermentation, under ahaerhbie eottdilions, uamg ä yeast as defined above,

Another object of the invention relates to the use of a yeast as defined above, for producing at least one lerjÉmfetfon ^dipbjrpfMbly in a liMéntstlon medinmmompfsmg xyfose^^M one fopnicptailpp: iabibitop

Other objects of the invention relate to a culture comprising a yeast siralmM debped above,uud a block yeast comprising a yeast strain as defined above. mi.ILM.SCaiPliON..QF.lttB..! .Kilim

Usure 1: Ethanol production (in g/kg of fermentation: medh») over time (in hours) in. the fePGX-Ac fenrsematson .medium, at 32;'C, by the I-4S3I yeast stram (deunood), the III ye&amp;si Simiti (square), the EDI yeast strain (triangle), the EDIyûmîiStiÄiptxde) Md the EOS ÿmâï strain: (eroes).

Figure 2: Ethanolfreduction #mg/fcg of lennenlation medium) overtime in imtws) in medium. .¾ at 32CC, by the M§|| ym$ eimin (diamond), the EDI yeast strain (Mangle) and the ED2: yeast strain icircle). lihure 3: Ethanol production (in g/kg of fermentation medium) over time (in hoorsfm medinm A, at 35*C, by the jM53d yeast strain, (diamond),. the EDI yeast strain (triangle), the ED2 yeast strain (circle) end the Bi.33 yeast; strain (cross).

Figured: Ethanol production (in gfegof fermentation mediutn|opr time(inhours) Inmedlunt 8, at 35°€, by the 1-4538 yeast stram (diamond!, the EDI yeast strain (triangle), the EM yeast strain (circle) and the ED3 yeast strain (cross),

DEFINITIONS

The expression “yeast slmltf: .rmkiiv%: ImÄpneo® pofulttiaa of past eells, jk yeast strain Is obtained from the isolation of a clone, a clone being a cell population obtained pore; a single yeast cell.

The term “exogenous” gene is intended to mean a gene winch is net naturally present in a past Strain; of the species under consideratlou.

The; term gene is intended to mean a gene: which Is naturally present in a. past strain of the species under confederation, A xylose isomcrase or Nil denotes herein an enzyme capable of conves tmg, in a single step, I)-xylose to D-.xyiulosc and which corresponds to the dass I C => T 1.5> A .xyliiol dehydrogenase or XDH denotes hpelp ap; enzyme capable of converting, in a single step, xyliiol to D-xylulose and wind; corresponds to the class EC 1.1,1.0.

Xylose reductase Or XR denotes herein an enzyme capable of converting, lb a single step,: ©~ aylo.se to .xyliiol and which corresponds to the class EC 1.1.1.307, A D~xyhdoki.uase or XKS denotes herein art enzyme capable of converting, in a single step, D~ xylulose to D-xyiulose~5'pho:Sphafe and which corresponds to the class ED 2,7,1.17,

The GR.E3 gene encodes an aldose reductase pgyme; which: eonespunds to the class EC 1.1.1.21.

The RPR 1 gene-encodes a t>ribulose-5»phosphaw 3*eptmcraae enzyme whkh corresponds to-the class EC 5 I <3.1.

The RK.il pac encodes a ribosecS<fhosghafe Ebtoi^lsombraso Miapie which corresponds to the class EC 53.1.6.

The 1 K.Li gene encodes a transketoiase enzyme which corresponds to the class EC 2.2.151. The TALI gene encodes a tnmealdolase enzyme which corresponds to the class HO 2.2.1.2. A prototrophic yeast Amin Is a yeast strain capable of :pawmg on a minimum medium. In gariicutmy strain is capable of synthesizing all the ammo acids end bases required tor its growth.. A nttemumimedium is a medium comprising;« carbon so:nrce :(ÇsHvQ^.mterg««àl::nihoim. source, a potassium sourse, a phosphorus : source, « suffer source, a magnesium «nerce, a Mlciitm source, anTronsoofee, -An example of ®Ιμ® medium I«:ihe%® mediums sggioe lTtjr exaxnple a-supr) and an inorganic: mtrogen source (for examipe apimonitmt suilhiel are added.

The medium ebmpriscs, per liter; 2 gg biotin, 400 |tg cbleldm ;panIollrenate, 2 pg Iplo awd 2iM)n> mw ok Ut1 m n arm telne p-antmbu mi v. d 40o}.g p\ doxine ibydroehloHde,: 200 ggrihotevin, -400 pg thiairäi^: :|ty|pßcMoi:d^ 5§0 g.g botfc acid, 40 gg copper -sulfate,. líMl pg; potassium Iodide, liMhgg: feric chloride, 400 ug manganese snli|tei: 20b:|tg::Sbdmm ntolyhdehate, 40(1 pgAmc sulfate, ! g mobOfeasicsporassium phosphate, S0Ù mg magimsium Sidfiito-. 100 mg Mikim chlorides IIÄtng; calcium: ehlotide, 3 §/l ammonium sulfate, hnai pH 5.4.

The expression “derived yeast strain" denotes a jeast shnin derived by one ;or more crosses: and/or by mutation and/or by genetic transformation. A yeast: strain derived by crossing can he obtained by crossing: a;yeast swain-according: m inyenttoh with the same yeast stein, wiir Another y to the irsvention, or with any other yeast strain. A yeast strain derived by mutation can be a yeast strain having undergone at: least one spontaneous mutation in its genome or at least one mulatton induced by mutagenesis. Fite mutaüon(s) of a derived strain may or may no; be silent.

The expression "mutagenesis" denotes both random mutagenesis obtained by applying: .radiation (for example UV radiation) or using mutagenic chemical agents, and mserliouior xbkn directed mutagenesis, by transposition or by integration of an exogenous DMA fragment, A yeast sfcmk derived by genetic framlbnnabon is a yeast strain into. a ON A sequent o whuh is préfet ahh provided b\ .» plasmid oi integrated dtreetlv suie the genome.

He goal of the pesent mventien is to provide a yeast strain capable of metabolkmg at least one pentose, in particular xylose, even in the presence of at least one fermentation inhibitor, such as acetic acid, for the purposes of the invention, a yeast strain capable of metabolizing xylose is a yeast strain eepabie of psoducing etlanol in a medium comprising xylose, under anaerobic céödMdm In particular, a yeast strain capable of metabol ixing xylose is a yeast strain capable of converting xylose to

He conversion of xylose to ethanol results from the direct or indirect isomerization of xylose to xylulose, followed by the use of the resulting xylulnae in the nonoxidaiive pain of thn pentose phosphate pathway.

For the purpose offne invention, a yeast strain capable of metabolizing xylose is a yeast strain which:: converts at least ?Q%. preferably at least fd%, mom .preferentially at least §01«: of: the xylose to ethanol in 60 hours in a fermentation medium comprising 55 g of glucose and 45 g of xylose per kg of fermentation medium, under atfebtobic conditions.

The inoculation with the yeast strain used to of ft* ethanol is preferably 0.25 g of dry maffePhgof fermepfdlbU medium,

Tie period of 60 hours is calculated starting.ftohSIfebiifcPPulatkm of the yeast strain1 into the le rrn en t at ton medium. 1 he fermentation medium used to measure the percentage of xylose converted to ethanol: is: preferably a synthetic medium, A svnttKt^ medium, w a medium id nhkh me exact chemical compositum is known. A synthetic tnedium usrôMift ;;#ht|lis€S.:;i::iosfhi)n mwm* a nitrogen sources a phosphorus source, and also the vitamins and minerals essential for the growth of a yeast strain.

The fermentation medium used to measure the percentage of xylose converted to ethanol is preferably thé YFGX synthetic medium comprising 55 g/kg of glucose, 45 g kg of xyloses tO g%g of yMsi mtraci, 10 g/feg of pofoné mà 2000 ppm ofoaeehO Mid, 0¾ pH: being âdpstsi ίο 5 with KOH. % the YFCDC medium the acetic: acid present at a concentration of 2000 ppm at:pH S las no inhibitory effect. 'rbofotmootatloaIs preferably carried out ai 32&amp;C. with moderate stirring, for example 90 rpm. TlMsbuing is moderate so as not to be oxygenating.

The pH of the medium is preferably controlled^ par example by the buffering eapaeity of an acid-base pair, (or example the acetic acid acetate buffering capuedx m the VK'*K medium The anfonnt of ethanol present in the fermentation medium is measured: by any appropriate means known to those skilled in the art.

It may involve a direct measurement of the ethanol produced or art indirect:measurement χ ta a parameter which correlates with ethanol production, such as the loss of mass.

For example, the alcohol production can be measured by chromatography^; in particular by HFLO i High iforfomannee m enxymatic kit (for example the ethanol assaying kit from Hoeiiflnpfk öt*ß assay using potassium diehrptpaln, lie .amount of xylose present in the fermentation nfodium is nfoaaured by any appropriaie means known to those skilled in the art, preferably by Chromatography, in particular by HFLC. The use of a fonneniatfon medium comprising both glucose and xylose makes It possible to evaluate the conversion of xylose to ethanol using a quantify of biomass which ;lt eonrparable for the various yeast strains evaluated. Indeed, tint yeast strains fust forment the glucose of the glucose and xylose mixture, and then the glucose and the xylose;

Th#- tb: .foetuhaîteé; xylose in the presence of at least one formentatiou inhibitor is described as a resistance to said fermentation inhibitor. in the coMMt of the present: invention, the fermentation inhibitor is preferably acetic acid.

It is the nontouized form of acetic ahlíj Siarflug from: a certain eoneeniraiioa, termed toxic concentration, which is responsible for its toxiciH <nul therefore for its tnlnbuorx e fleck i'o$ example a eoncemmnon of 4000 ppm of acetw foul at pH 4.4 ts a toxic euncentmdÄfof: strains not resistant to acetic acid* such: as thei lfo-SSS yeast strain.

During fennen ration: under ánaefobie Conditions, the inhibitory effect of..ae§tib-4PM.^sulb:|p:. particular in a delay of the initiation of the conversion of t he sugars to biomass ami to ethanol, lie resistance to acetic acid is measured herein relative to the delay Of Initiation of the conversion of the sugars to ethanoL be. to the delay of initiation of äfeohöie fomentation.

The alcoholic fermentation curve representing: the aurout of alcohol produced as a fotfoiou of taue generally comprises three phases: - a latency phase, during which there is no sthano! production., ~ un alcoholpoducttdh phase, and - ύ plateau phase, which corresponds to the end of the lerMeptaiony

The delay of the initiation of alcoholic ftatea&amp;Uon corresponds herein to the Pattis at the origin of the line representing the maximum derivative of the alcohol production nft More sunplx. the delay ol the ininaoou ot aieohohe fenuertfatton uonesponds hercat m the x-axis at the origin of the Uric corresponding to the slope of the alcohol products onphasc. A yeast strain resistaret to acetic acid Is defined heroin as a yeast strain having, a delay of initiation of alcoholic fermentation of less than 30 hours, preferably less than W hours, more prêtèrentiaiiy less than .18 hours, in a fomentation medium eoroprisirtg 4000 ppm of aeotio acid at pH 4.4.

The fermentation medium used for evaluating the resistance to acetic acid is preferably a spthebc rnediutnf moro profoehihlly the YFAe medium.

The Cent position of the YfAe ntediuin. is the following: ISÖg/kg ;:f g/fcg-'PÍf csat extract, 4..7 g/k g of DAP idlammonium phosphate), Π.5 gkg of citric acid, 4gdrg of acetic acid, 13.5 g/kg of sodium mtfafo lrnl/kg of Tween 8(f 2 ml/kg of ZoSC)* (or iffri gdY 3.3 nii/kgoCMgSO.4,7H2O:'Clât::4i0'''id|j I Ml/kg of thianrine tMf§,2d g/l), 1 Ml/kg of pyridoxine fat 5.28 g/l}, 1 rnl/kg of biotin (at 1.70 g/1), 1 ml/kg of pantothenate (p Ytgi), 3-5 i1é| of nicotinic acid (at 8 g/l). i ml/kg of mesolnosu.nl (at 50 g/l), 1 nn/kg of rihoflavm (at 1 gd), I ml/kg of para~aminohenzoate (at 1.2 g/l). pH adjusted to 4,4 with KÖM.

The inoeulalun with the yeast strath used tor evaluating the resistance to aeetlu acid is preferably 0.25 g of dry- mutter kg of fermentation merilum.

The tune t 0 ot the Pen rohe fomentation curve corresponds to the time at strain is inoculated into the fermentation medium.

Tie; iieohoiic fermentation is carried out under anaerobic conditions, preferably at $20Q and with tuoderaie stirring, lor example 90 rpm.

The stirring immoderate so as not to bo oxylohaini.

The pH of the Medium is preferably controlled, tor examplè-'ly^h^MÂÎ^g capacity of on yeid/haxe pair, for example the acetic aeid/aceiate buffering capacity in the YFAc medium*

In the Y F Ac fermentation medium, a yesM strain not résistant tu acetic acid can have a delay úMtMmim of alcoholic fett8@mat>on of at legst 40 h.

Ute inventors have sought to obtain a yepi; strain capable of.mutabPl»g xylose, even in the presence of acetic acid,: starting Irons a yeast strain which meieetiveln temB: oíYöitewsionaitf xylose to ethanol.

This yeast strain which is ; effective in terms of conversion of xylose to ethanol, chosen as starting a view to acetic acid, is the 1-4538 yeast 5;v20í 1, with the CNCM (National Collection: ofMieroorganikm f'uitmeS},:;I&amp;4UCdp OocteptfSôdN» 75 ";24 Paris eedex 15, France.

The 1-4538 yeast strain converts §t least 00% of xylose to ethanol in 60 hours in the YFGX fermentation medium, comprising 55 got glucose and 45 g of xylose per kg Of medium, However, the M55S ^east attain ts particularly sensitive to acetic acid; it has a delay of initiation of alcoholic fermentation of at least 40 hours in a YFAc fermentation medium comprising 4000 ppm of acetic add at pH 4,4.

The 1-4538 yeast strath is a Saedt^r^ces &amp;mmmm mmnjMßimä^ êmemé evolution from a:y«s^-®lrhm-p^ti^yiíK>dííí«á by: ~ the Itaseriion of at least one copy oh an, exogenous genereneoding <* xylose isomerase {MJ of ClbMmämm phytqßrmmims:f under the eohiftil Of the pADH 1 promoter and of the CYC! terminator, « (he insertion of at least one copy of an exogenous gene encoding ia xylitol dehydrogenase (XPHfpf Picki# sti0is, under the control of the pADH i promoter and of (he CYC1 iemiinator, - ai least one copy of the TA !.. 1 endogenous gene placed under the control of die pPGJO promoter, al least one copy of the T;$£Ll endogenous gene placed under the control of the pTPI-13 promoted - at least one copy of fne RPF 1 endogepous gene plicedifelder tfe control of the pTDH3 promoter, - at least one copy of the R.KÍ1 endogenous gene placed under the control of I he pTDH3 promoter. ». the inso don of at least one copy of an endogenous gene cncodiog a xylulokinase tXKRi) undo! the control of the pADHH promoter and of the CYC1 terminator, am! - the delellfifs of $t least ppeipopy of the open trading frame of the 011.113 endogenous gene encoding an aldose reductase.

The 1-4538 yeast strain Is devoid of any residual selectable marker.

The 1-4538 yeast strain does not comprise a gene encoding an XRpf exogenous origin, nor an uraA, araE or AmD gmW:·

The exogenous gene; encoding a xylose Isornerase (XI) of Cbsiridium phyiqfermenimis h the IIPS^f|^|?tttózed..sgfnMce SHQ ID NO: 3 dispáheá in. ààmmm&amp;$t$lô2QQ&amp;0%13S|.

The exogenous gene encoding a x y Idol dehydrogenase (XDIi) of Pichia sUpdri is the gene of reference sequence X.5S3U2.I on Genbank.

The pADHl, pPGKl and pTDi-B promoters are promoters of ddathuronmrex eemihne.

The CYC1 tennlnatt b a promoter of Mmækmmnvees mmmme*

The 1-4538 yeast Strain is an Industrial yeast strain which is aneuplokt The 1-^338 yeast átram is prototrophic.

In order to conter a properly of reststanocito: aeetle atiá on the 1-4538 psast strain, hot without substantially modifying its ability to metabolise xylose, the inventors have chose« to use the strain crossing teeíhuque·:

To the knowledge of the imemors, it is the first nme-tbitil#· etOkiiöilTS'ete^öd 'M used to improve a genetically modified aueuploid yeast strain.

Indeed, those skilled in,the art would not ât first giâ»eë.4ÉivMô&amp; «siögfiiaf jüô^smg.têéhmqu.é,, when the starting yeaM strain: - comprises no inker than eight geuutic ntodifications wltiChmus! u priori ho iranstemxf to the selected segreganis, - is an ancuploid yeast strain and Éero are .segregation problems in the absence of strict diploidy*: nod - has nndorpne a step of mutagenesis in the context of directed evohUiefiy winch maybe : responsible for difficulties tor the crosses, in particular in the: ease Of chromosomal irauslocatidn responsible for germination problems.

Grosses were thus carried out between the 1-4538 yeast strain and yeast strains which arc resistant to acetic acid and not genetically modified, i.c, do not possess the genetic machinery required for xylose fermentation.

The past strahis resistant to acetic seid,: osed: for the: crosses, have a demy or initiation ot

IhandA h m the \ ΙΆο fermentation otOiOt.ua,: TN resalta ol'lhO: orossos were not conclusive:; while the chStrietoftslie öt resistance to aoctic aad was indeed irmistetred to the %brids, hte latter wem: much loss effective than the starfeig I-4538 yeast strain for converting xylose to ethanol.

At best, the hvhnds obtained converted 4t)% ot tbc xylose to ethanol ot bO ! tours ityfhbfe bviX léîtmovdatlotr ateditini comprising 55 g of glucose and 45 g of xylose per Kg of medium, fhelhxmutors Med to- imptpvf the xylose fertnenfatlottoof those hybrids: by directed evolution, but without farther success:,

The immtm have themfote demonstratedUthstAin: order to transfer the characteristic of resistance to acetic add by crossing, without losing the ability to efficiently metabolize xylose, tt is necessary to cross the 1-4538 yeast strain with a yeast strain resistant to acetic add which has a similar genetic background, i.e. wlvidt also has the g^etiß-'maefeayÄrmetahötöng:. xylose, but without necessarily being effective in terms ofmetabolixitig xylose.

The yeast strain used, which both is resistant to acetic acid and also has the genetic machinery for metabolizing. xylose, is the Smehärom^ms mfmMm sdabrae^ under the treaty o| Pouapest on May 34, : .2d 12, with the CiMCiM: (ÂfefflÉ Collection ot rde do Docteur lonx, 25224 Paris Cedex 15¾ Francéi Thus, by crossing the 1-4538 yeast strain wrth the 1-462? yeast strain, the inventors have ^Msm^l:':y©ast';'sfraîixy .which are both effective itr metabolizing xylose and resiatantho acede acid.

An object of the present invention is thus the yeast strain deposited with the CNCM under number lMb:2 7,

The 1-162? strain constitué one of the two starting nfraiM Ihr obtaining tilg yeast: strains according to tin.' invention.

The 1--4627 yeast strain: is: a novel yeast strain, obtained by means: of,m original method of crossing resistam id áeétio acid and; not genetically modiibd, with a: genetically modified anenploid yeast strain.

The·1-462? yeast strain has thus been nhtálöeá:iS:lhMpllbwing''Wdyr· >okctm< ot a toast \fu n \m\ , t to xetts aenl, 11* Ά \ t mo a defe\ ot matador ot alcoholic fermentation of less tlum 1 Tbdu Jte YFAc ionnentauon medium, - selection of a yeast strain genetically modified by: £5 iïé Insertion of si: least one copy of au exogenous gene encoding a xylose bo.auira.se ( X1} of Clostridium phyiofermema?^ under the control of the p.A DH 1 promoter and of the C YC i terminator, o- the hberhon of at least: one copy of an exegeuonS: gene encoding a ®y|to! deiydrogehase: (ICiCi of Fichm mipWky Wider the control of the p APMf promoter and of the CYC 1 terminator, o at least one copy of the TÄ1, endogehoos gene placed tender : the MPirol of the pPGK I promoter, o at lea s t one espy of the Tilt 1 endogenous gene placed under the control of the pTDR3 promoter, o at: least one copy of the llPB 1 endogenous gene placed under the control of the pTDH 3 promoter, o atleast one copy of the RID endogenous gene placed under the control of the p i DH3 promoter, o the Insetlloo nfat least onecopy of no endogenods pne encoding a xylolok inasé pi s 1 ) under the control of the pADMI promoter and of the CYC I terminator, and o the: deletion, of at least one copy of the open reading frame of the CRI3 enddgenoos pne eneodin| au aldose redoebbe, , crossing of said yeaststrsin resistant i&amp;- seelieaeid. withbald genedeaily modthed, yeaaf strain., so as to obtain a hybrid, - directed evolution of said hybrid, so as to obtain the 1-4627 yeast strain, the 1462? yeast:strain composes at least one copy of an exogenous gene encoding a, xylose bomerase (XI} of Clos iridium phyioßmtäntam* undor th eeomro!: of the: pADHl promoter and of the CYC I terminator, at least one copy of an exogenous gone encoding a xyhto! <. eKchugco.ue (YD ID <n bM<> ^ nun, unde? the , onhul of t to v\DH1 promoter and o, We CYC1: terminator,, at: least one copy of the TAM endogenous gene placed rrndes the eotrhwloi the pPCK ! promoter, at least one copy of the TKLI endogenous: gene placed ander the control of the gCDl-B protnoter, at: least one1 copy of the HVK1 endogenous gene placed under the control of the pIllRS promötefyat least one copy of the IIMII endogenous germ placed under the : control of die pTOlid promoter, at least one copy of an endogenoos gene encoding a xyhdokimse (ΥΚΥΙι under the control of the pAOHl promoter and of the CYC1 terminator. and at least one copy of the open reading Iramé of the GRID endogenous gene encoding Si-aldose reductase which ruse beer; deleted.

As for the 1-4338 yeast strain, the exogenous genes encoding a xylose köntöspS'h ot < n-n; 'fa w ' vm is .be go m of ojr.mned sequence v ! Q 10 NO * dos nbed k document DE 1020080313 SO and the exogenous gene encoding a xylitol dehydrogenase (XDH) of Pi(Mm siïpüU is The: gene of ieiMOhee sequopeo XSSI9¾. i on Gertbank. flte Me3" was! sttatn ia dcu'ulcfoiw re'-'du.il sdectaHe tnarkcr.

The 1-46:21 yeast strain does not comprise a gene encoding an XR of exogenous origin, nor an: ara A, araB or AraD gene.

The I-4627 yeast strain is an industrial yeast strata and it is aoedplold.

The 6-062/ yeast strain is prototrophic.

The resistance to aeeife add of the )4637 yeast strain is less than that of the starting acetic add-rskskofyeasi siraih -usetl lor the crosGng, bhi Its fesktäike ttkiêtidiÂ! is widely acceptable tor an industrial application.

Thus, the 1-4627 yeast strain has a delay of initiation of alcoholic fermentation, of less than 2C) hours in the VF Ac fermentation medium.

As regards the effectiveness on xylose, the l4^if:^si«Ä^ÄVieÄ^aj>pfe}ximateiy 60% of xylose to ethanol, under anaeroMc eoildilions, in 60 hoars, in the YFGX fermentation sued.·am comprising 35 gof§theose and 45 g of xylose per :kg of modi om.

The Ii-4627 yeast strain is thus an original yeast strain which possesses resistance to acetic «cto and: which pôSâ^MÂ;ïfMtie:machinetÿfot mctahohkng xylose, hot which is nof capable of metabolizing xylose tor the purposes of the Invention, i.e. which is incompatible with an industrial use with a view to ethanol production.

The inventors have therefore crossed the 14538 yeast, strain with the 1-4627 strain, m order to obtain a yeast strain capable of metabolising xylose and resistant to acetic add,

An object of the present invention is kerekre a method for obtaining a yeast strain capable of métákdiXing xylose and resistant to acetic acid, comprising the steps of: - erossini; the yeast strain deposited at die CNCM [.Rational Collect ion of Miemorganistn Cultures] under number 1-4538 with, the yeast strain deposited at the CNCM under the number 1-4627, so as to obtain at least one hybrid, « selecting at deast ©ne ^#^fef«íéaisö| x#km and pistant tehette add, so as to obiam a yeaststrahf xylose and resistant to acetic add.

As dellned above, the yeast strain e##fo of and resistant to acetic acid according to the Itwadion hm t he following properties: - it converts at least 70%, peferably at least SC1%, mote preierentlally at least 90% of xylose to ethanol in 60 hours in the YFGX fermentation mediant comprising 55 g of glucose and 45 g of xylose per kg of fermentation medium, under anaerobic conditions, and - it has a delay of initiation of alcoholic fermentation of less than 30 hours, preferably less than 20 hours, more preferentially less than 2b hours., in the YFAe fermeatatton medium comprising 4000 ppm of acetic acid at pFl 4.4.

The crossing step is carried out aecoiding ht the conventional tecbni|ncs, such as those taught in chapter 7 “Sporulation and Hybridisation of Yeast” by R.R. Fowdl, of the reference hook 'The Yeasts'5, volume 1, edited by Adi. Rose and J. S. Harrison, i960 - Aeadetnic Press. in order to improve the efficiency of the method lor obtaining a yeast strain according to the invention, it is padlcdlarly advantagedus: to perform a Aiôi:ô» the segregants derived ttom the l:~4S3b yeast strain andior on the segregants derived

The segregants of foe 1-4538 yeast.strain which are used for the;crossing are preferahly selected on the basis of their ability fo metabolize xylose. t he segregants of the 1-4627 yeast strain that are used for the crossing are selected ön the hasis of their resistance to acetic acid.

The method as seined above may he; comprises: a step of sporulation of the yeast strain deposited at theCNCY I National Col lection of Microorganism Ctiltureaf tmder nambër^MSii^so as to obtain M least önéxe an optional step of evslhation of the conversion of xylose to ethanol by at least orte segreganFK, · a step of sporulation of the yeast strain deposited at the CNCM under numfeer 1-4627, so as to obtain at least one segregant Y, - m option a i step o f evaluation of the resistance to acetic aeld of at ! east one sepegant Y> s· a step of kybillÄQ« of ut foatbfnc segregant X with ;u least one segregant Y\ said1: segregam X being capable of convening xylose to ethanol mtd/or said segregant Y having resistance to acetic acid, so as to obtain at least one hybrid.

The stops of sporulation, of evaluation of the conversion of xylose to ethanol and of evaluation of thprosistaneo io acetic aeil ean bo carded put in airy desired order, as long as the prfá« of a given yeast strain is performed before the evaluation of its sepregants, lor example, a method, as defined above, may id ebaraeferihed in that said crossing step comprises: * a step of sporulation of foe yeast strain deposited at the C'NCM [National Collection of Microorganism Cultures] under number 1-4538, so as to obtain at least one segregant X - ap; optional ' Step of measmemeet of foe percentage of xylose converted to etimnoi by at least one se|piphfX> under anaerobic conditions, in 60 hours in a fermentation medium dOfoprÄg 55 f:0f glucose and 45 g of xylose per kg of said medium, « a step of sporulation of foe yeast strain deposited at foe CÎNCM under number 1-4627, so as to obtain at least one segregant Y, an optional step of measurement of the delay of initiation of aleobol ie formentaiion of at least otie segregant Y in a formen ration medium conrprkmg 4ÖÖÖ ppm of acetic acid at pH 4.4, a step of hybridisation of at least; one segregant X with at least one segregant Y, the segregant X converting M least 60% of xylose to ethanol in 60 hours and/or the segregant Y having a döí^ipf 30 hours, so as to obtain at least one hybrid.

The steps of tporfoarion:, of measurement of the percentage of xylose converted to ethanol and of measnretnent of foe delay ©f initiation of alcoholic formentation can he earned out in any desired order, as long as the sporulation of a given yeast strain is performed before the measurement of the selection parameter applied to the segregants of fois strain .

The inoculation used for; measuring the percentage of xylose emiverted to efoanhl by at least one segregant of the 1-4538 yeast strain is preferably 0.2.5 g of dry mader/kg of fermentation medium.

The Jlrmentation nrndfom: used for measupPiifoe pereetdage of xylose converted to efoanol by at least one segregant of the f-4538 yeast strain ixpreferably the Y FGX fermentation medium.

The segregants of rise 1*4538 yeast strain are naturally less effective titan the starting aneuploid grain in terms of metabolizing xylose. Consepeniiy, the seleetion criterion applied regarding tie conversion of xylose to ethanol is less demanding than the ability of die starring 1-4538 yeast strain.

Thus, a seg.rega.nt of the 1-4.538 yeast strain may be selected if under anaerobic conditions, it eouyeris at least 60% of xylose to ethanol Irt it) hours m a feîtmWÂMsàîtÂ. -dm^áng 55 g of glucose and 45 g of xylose per leg of said medi um, pyfirahly at least 65% of xylose, evee more peferentially at least 708¾ of xylose.

In order to optimize the duration of the segregat'd selection step, it is possible to measure the conversion of xylose to efcnel fey the segregants of the 1-4538: yeast: strain oyer: the course of a shorter period, tor example over the course of 48 hours.

Thus, a segregant of the 1-4538 yeast strain may be selected il under anaerobic conditions,, it converts at least 60% of xylose to ethanol in 48 hours in a fermentation medium comprising 55 g of glucose and 45 g of xylose per feg: of said medium, preferably at least 65%, of xylose, even more prefer gats ally : at: least 70% of x ylose. À segfegant of the 1-4:633 yeast Tfeaia may be seleeted i f if has a delay of initiation: of alcoholic lermenlütion of less than 30 hours. pretbnhiy1:gi§'.Ifesa. If rcsfeeiiially: ÏIS# Ä88. 28 hours, in aiemtenlatsoa medimn comprising 4000 ppm of acetic acid at pH 4.4.

However, surprisingly, the majority of the SegrepPts of the 1-4627 yeast strain are more resistant to acetic acid than the starting 1-4627 yeast atraim fee, their delay of initiation of alcoholie fermentation is less than that of the starting 1-4627 yeast sham.

The selection criterion applied regarding the resistance te aeetie acid is therefore preferably more demanding.

Thus, a aegregant Of the 1-4627 yeast strain may be selected if it has a delay of initiation of alcohol ic term en tattoo of less th an 25 hours, prefer ably : legs than 20 hours.

The fermen táti oh medium used tor measafihg: the delay ofibitiation of alcoholic fermentation #f FA c fermentation medium.

The .inoculation^ used Éfe measurihg the delay of ifeitiatiofe-ef fifeehofec lennentation of at least ope segregam of the 1-4637 yeast strain is preferably fe2 5: g of #y matterilgof femeutafiou; medium:.

The segreganis of the 1-4627 yeast strain can also be seleeted on their ability to couvert xylose to ethanol. H0wevervs«?|>nstaglyvth« best hybrids have been obtained using segregants of fee 1-462? serai« selected only on the basis of their resistance to acetic acid.

Preferably, the hybridfeulion step is earned out with. ~ at least ©s@ s^regant II which converts at least 60% of xylose to ethanol, preferably at least 65% of xylose., eben m&amp;mát least 70% of xylose in 66 hours, preferably in 48 honrs, and -- at least one nepeggnt Y which has a delay oflnltiation of alcoholic fermentai ion of less than 10 hours, preferably less than 20 hours, more preferentially fess more preferentially less; than 25 heurs, even more ^preferentially less than 2(f hours.

The hybrids thus obtained are then selected on die basis of thé: two criteria;: their ability to mot Jvh © mlose ami then wfestanee m acetic acid, Λ method, as defined above, may be eharaeferfefefM that said; step of selecting at least one hybrid cap J do of metabolizing vriose and roMStan to acetic add comprises the stops of. - measuring the: pereddtage of xylose converted to ethanol by at least one: hybrid under anaerobic conditions in 60 boms in a fermentation medium comprising 55 g of glucose and 45 g of xylose per kg of fermentation medium, - measuring the delay to nutrition of alcoholic fermentation of at least one hybrid m a febnent&amp;hon medium oötii0Äg,#il#'.ippö'tof âdufcâeit at pH 4,4, - selecting at least one hyhÄÄÄiöÄi^ at least 70%, preferably at least 80%, mór© preferentially at least 90% of xylose to ethanol in 60 hours and of which ihe; belay of initiation of alcoholic fermentation is less than 30 hours, preferably less than 29 hours, mow peferemihliy less than 28 hours, so as to obtain a yeast straib eapabfe of rnetiboMing xylose and resistant to acetic acid.

The: order of the steps ôf to- ipihMisge- of converted to ethanol and of measuring the delay of inifiation: of alcoholic :it ef no he espied out one after feeother, In one direction or the other, or at the same time, 'Bio eondfeon® for measormg the percentage of eonyersion of xylose to ethanol and; Sie delay of initiation of alcoholic fermentation are as defined above.

Is described boréin a method fer Obtaining ayeast strain capable of metaboiiahig xylose and resistant to acetic acid, comprising the steps of: « crossing of ihe yeast straw deposited at the CNCM. [National Collection of Microorganism Cultures] under Hate 1-4538 With the yeast strain deposited at the CNCM under number 1-4627, said otossihg slop comprising the steps of: o sporulation: of they east : swindef ositod at. the CNCM under number 1-4538, so as to obtain at least one segreganl X, O measurement of tire percentage of xyloseroMve&amp;^rtp. ethanol; by at least one segregant X in 60 bouts, preferably in 48 bouts, in a femehiatiou medium comprising 55 g of glucose and 45 g of xylose per; kg; of said medium. o selection of at least one segreganl X which converts at least 60% of xylose to ethanol in 60 hours, preferably in 48 hours, ο sporulation ot ihe yeast strain deposited at the CNCM undermiufeer 1-4627, so as to obtain at least one segregant V, o measurement of the delay of initiation of alcoholic fermenlation by at least one segregant Y in a fermentation medium comprising 4000 ppm of acetic acid at pH 4.4, o selection of at least one segregant Y which has a delay of initiation of alcoholic fermentation of less than 50 hours, hybridization of at least one segregant which, converts at least 604¾ of xylose to ethanol in 60 hours preferably in 48 hours, with at least one segregant Y which has a delay of laibatiou ot alcoholic ferruuntatfen of loss than Id hours, so as to obtain at least om hybrid, - selection ot at least one hybrid capable of membol king xylose and resistant to acetic acid, suid selection step comprising foe steps of: o measuring the pereeutage of xylose converted to ethanol by at least one hybrid, tmder anaerobic conditions, in 60 hours, tn a fermenMion medium uomprising 55 g of glucose and 45 g of xyiose ner kg of fermentation medium, o measuring the delay of initiation of alcoholic: fermentation of said at least one hybrid in a fermentation medium comprising 40011 ppm of acetic add at pH 4.4, o selecting at least one hybrid which converts at least 7(1%, preferably at least 805«. more preferentially affeasi#£)¾¾ of xylose to efeauul In: 60 hours and of which the delay of initiation oi alcoholic fermentation is less than 30 hours, preferably less than 20 hours, mom preferentially less Érán ;2ihours, so as to obtain a yeast strain capable of metabolfeing xyiose and resistant to acetic acid. M indicated above, of the steps of the cmssmg step Is of 00 importance, fust s$ the order of the steps of the step of selecting at least one hybrid is of no impoîtanes.

Preferably, the hybridization step is earned out between: - at ÍMsr one segppnt 1:: which at least 60% of .xylose to: ethanol, preferably at. least 65% of xylose, ebek mord: preforentlslly at least 70% of xylose:, in #0 hours,, preferably to 48 hours, and: ^ at least one segregant Y which has a delay of ini hátion of alcoholic fermentation of less :¾¾¾¾. :íi©Sii^:jí*¥da^ ^ihijiars* mom prefommf ally lesaÉtm 2B hours, even :tporc.;fPïÎbt^y|yl.e^#^::.25 hours, even more preforentMIy less than 20 hoars.

The method can optionally comprise a directed evolution step.

The directed evolution step may be carried out on a hybrid not selected during the: selection step of the method tor obtaining a yeast ptralpyapÄe of xnotabtihalpg,xylose: apd psi4s.nt to acetic acid ns defined. above, with a view m rendering said hybrid capable of metahoiizmg, xylose to uÉMaoliÄ#Äf«iii.dering it resistant to acetic aeid.

Is described: herein a: method m defined shove tor obtaining; a yeast strain eaphie of metabo lizing xylose and: resistant to acetic acid, comprising the steps ok - crossing of the yeast strain deposited at the CINGM patlonal Golleetion of MIctoorganisM Cultures] under number 14538 with the yeast strain deposited at the Od£M under number 14627, so as: to obtain at least one hybrid, - directed evolution of at least one hybrid, « seieotldn of at least one hybrid capable of nwlaheiiaing xylose: and resistant to acetic acid, so as to obtain a past strata capable of metabolizing xylose and resistant to acetic acid.

The directed evolution step comprises the following snhsteps: - mutagenesis, so as to obtain mutants, and - multiplication of the mutants, m cycle cultures, under hypoxic conditions,in a culture: tuudlum comprising xylose and/or a fermentation inhibitor.

To simplify, the mutants obtained at the end of the directed evolution step are still described as hybrids.

The expression “under hypoxic conditions” signifies under conditions of oxygen deficiency.

Ai) oxygen deficiency is defined herein by an oxygen percentage ; A less than 20% of fee air. The tuufagenestkstep is pfëthrdbiy a. hmolefate” mutagenesis, i.e. obinhtedTfy exposure of fee yeast cells to U V radiation ai 254 itthaaF bstb'Ohh: I höiand 500 J/esvfe for example 300 j/cmf These con<i nions cause a ixmMÊW ratepfymiy 7% to 16% of the cell ponulahon subjected to the UV radiation.

The mortality rate Is determined by plating out, oh dishes of medium ofwhich the eMhon: source is glucose, an Identical volume or the cell suspension before and after mutagenesis; and by eornpnrtngThe nUbiberefcokmf^ hours of : growth.

The mtdliplication of the mutants in cyclic eulturesy phier tryptic conditions,: it 0 cuter# medium comprising xylose anchor an inhibitor comprises*fe example: a) the culture of the cel! pogubuon subjected Te îp ifehtpenesis. in. a culture:medtom, with stirrlöi* under bypoxle hondidciuSs preferably M .12*01,: bi Jo smtp * u ^ , - oiume W ·, >,*. Us. re o u t ted m -top a) to? -, cas ?p -, out· n I as . inoculating it into a culture medium having the same composition as m step 0¾ stems at and b) berna ropeatvO at kast '< ..mes. p? eie ably ut leuM 4 unes, tor oxaripk o times, 7 times or 8 times.

When a fermentation inhibitor is present in the culture rcedmm during the multiplication of the metán??, u is preferably acetic acid.

The multiplication of the mutants in cyclic cultures rmdet hypoxie conditions nitty he carried, out in' a culture medium. comprising xylose and in the absence ofaceuc acid.

The culture medium tor the multiplication of the mutants comprises, lor example, 7% of xylose. A?i example of a culture medium ihr the multiplication of The mutants Is the YFX medium having the following composition; 70 g/kg of xylose, 5 g/kg of yeast extract, 47 g/kg of OAF {dkmrhOnium: phosphate), 1:1.5 g-kg of citric acid:. 13.5 g/kg of sodium citrate, I ml/kg of Tween S0, 2 mhkg of XnSCh (atfiOJ#1!), 2.5 ml/kg of MgSO.?.7lbO (at 400 g/lfe I. ml/kgof tip albino (at 18.24 g/I), T ml/kg of py?4doxhte :fat S..28 gd).. 4 ml/kg of biotin (at 1.76 g/i), I uf kg ot paniothcnavO tat 7fg » fiaiké of meotmn aesu (atlgli. I mhkg of mesoinositoi (at 50 g. If I ml/kg of riboflavin (at 1 gd), 1 ml·kg of pataminirKfeencoate tat .1.2 g/I), pH adjusted to 4,4 with K.OB,

The hypoxic psltai«, for example, obtained by virtue of a partial «pessure m the equipmmt jmä {ßvmm^s Hasis or icrmcnters) duefo an overpressure subsequent m tie production of 0¾ pmdueéd during the termemutnm, fedélé the absence oi aeration.

Preferably, the duration of foe culture a) Is such fo# foi of the xylose of the medium is consumed. tire dpralon of a culture is, for example from 1 week to 4# peurs, lienumber of cultures carried out is variable, forexampfo from 5 to 20, mutants. A noa^pirit^dtiilMf mmmis.&amp;mmu captfoio of multiplying «oder conditions of oxidative metabolism.

In. pattieulam a nptnmspirato^foeftdent: mutant Is capable: of multiplying, under aerobp conditions, at 30°C, in a medium which contains gymmi m sole carbon source, for example a medium i^^pisi^-.äö.g/kg^f'llydkili 5 g/feg of ammonium Mámmá i.7 g/kg of YNB (Yeast Nitrogen Base) meditált, Ér example the YN® medium tum Pilco,

When the culture medium docs not comprise a fermentation inhibitor, a substep of emUipiieatkm in a culture medium comprising acetic acid can be edmed nut after certain or each substep(s) of multiplication in cycle cnlturen In order to verify that the medium still contains at least one mutant resistant to acetic acid.

The éiijt&amp;ûm :ôf thi-miltiplidÄü $£&amp; culture medium composing: néetie acid is preforahly greater than 20 b and less than 75 h.

For example, the duration of the subsiep of multiplication in a eultum medium comprising acetic acid is included Item 70 ft to "5 ft.

It is also possible to carry out a step of selection of the mutants which have an appropriate growth rate, by culture in a medium: comprising glucose, "îêt example a medluin omhprislng 20 gd of glucose,: and selection of the rftutants which haue the appropriate growtlt rate, A directed evolution step can also be earned ρηΐ·'ΡηΙη^:,ΐ^Ρ·^ϊίΙΙί.1 selected at the end of die selection step-of the process for obtaining a yeast .strain capable cd' metabolizing xylose and resistant to acetic acid as defined above, for example itt order to lufthet· improve its abdity to oietabolixe xylose and er its resistance to acetic acid. fsbosedbed béréin a yeastshnfn chF^Me of-^isisiairiilii -tç> acetic acid, which can he obtained by means;ibfXhfi iltethoitäs·

Tbc Ti 1 yeast strain was thus obtained by means of the|$ijl&amp;ö$ yeast strain äa defined above (c£ example 1),

The H1: yÄt&amp;IÄT -it.Isast #0¾ of xglese to ethanol under anaerobic conditions in 00 hours MaYOfinitiniiou of alcoholic Ikmerhation of less than 22'fito'tbe YFAc fermentation mediunn

Is also described herein a yeast strain capable of metabolizing xylose and: resistant to acetic acid, obtained by niehns of the inethod as defined above, which tä chosen from the yeast strain deposited at the (iM^M [National CoU^fc»fMl!^oor^Ät.Äk«3^1 under nurnber MiSd, the y,\vO strain deposited at the < '\t ’hi under numbei i-4o25 and the xcast strain deposited at the CNCM. under number 1-1626.

The 1-4624,1-4625 and I-4626 strains are Saccharomyces cûwvMâà sitfáí»s4e|tóí*ed under the treaty of Budapest on May 24, 2012, with the CNCM (National"Collection of-Microorganism Cultures:}, 25. me du Docteur Roux, 75724 Paris codex 15, France,

The yeast htraht number 1-4624 converts at least 90% of xylose to ethanol under nMerdble conditions in 60 hours in the YFGX medium and: has n delay of initiation of alcoboHe letinentation of less than 23 hours in die YFAc tementalion mcditixn.

The yeast strain number 1-4625 convents at least &amp;D% of: xylose to etlianbi ::under anaerobic conditions in 60 hours: in the YFGX tnedttns and has a delay of initiation of alcoholic fementation of less tbán 35 hours in the Y FAc iicrmentation medium.

The yeast strain nuntber 1-4626 converts at least 90% of xylose to etlxanol under anaemble conditions in 60 hours in the YFGX medium and has a delay of urns,dm s m akoho-iv lermentation of less than 22 hours in fire: YFAc iertnentadon medium.

Is also described herein a yeast strain derived from a yeast strain according to the invention which shares the same properties.

Thus, a yeast strain hetived fiom a yeast strain according to the Invention, is characterized in that said derived yeast strain is eapabieof metahoixipll xylose and is resistant to aeetie acid.

A dehved yeas? strain is ay defined above in the deiniteBW lx also described bereis a yeast obtained by culturing; a past strain as defined above or by eulturinga detiyed past strain as átelfcm.

Thepastaâré obtalnedby eeliuring á::^sbáMÍ!0l:*C#Mii»g.to the invention or a derived yeast; strain. In parfieularas described in the reference hook * Yeast Technology” 2ns} edition, 199b 0. Reed and T.W. NugodfyÄän% published by Van Ästrand Reinhokl ISBN (M42-31 $92- b<

The industrial-weala r^ ofyossts poor al iyeowjplses at least the first two steps pf the set of iieifeii owing stepa; > multiplication of a past strain p several Paps, irst mám semi-aoMrobic eondltlobs and then under aerobic conditions, .* separation by « entnfugatiott of the resulting yeast from its culture medium, so as to obtain a liquid cream past eontabring approximately between 12% and M% of dp •natter, or even atiighér amount of dry matter if the cream yeast is mixed Pth osmolyte products, ». filtration of the resulting liquid cream yeast, generally on a rotary filter under vacuum, so as to obtain a dehydrated fresh yeä&amp;t .eontaini»g":&amp;m:2fi||: ofdry matter, ~ mixing of said dehydrated fresh yeast, so as to obtain a homogeneous mass, < ex trusion of the resulting yeast, so as to obtain;: o a pressed yeast in the form of fresh yeast cakes or of crumbled fresh yeast, containing approximately 30"« ofdry matter, or o a yea«i to the ibnn of part teles, generally granules, ifiheyeasl is intended to be dried, - optiouuily, drying in a sparing manner, ip a stream Of hot dir, Ihr example by fiuuivutjon. of the \east patricien obtained hy tonustom so as to ohtam dry yeast The drying step is preferably a sparing rapid drying in the presonoe of an cnndsiller.

Among the emulsifiers which can be used during the drying step, Serbian snonpsiearaim lor example at a edrmentfation of approximately 1jI% (by Weight relati ve to the weight of dry yeas%: maybe chosen.

The yeasts according to the invention can be used in any possible form, for example, a yeast as defined above, may be characterized in that it is in the forth of cream: yeast, of pressed yeast, of dried yeast or of deep-frozen yeast

The tbf smne propediés gs-Ho yeast strain from which they atp obtained by culturing,, namely::: * =s®^íl: - the vea.sts described herein are resistant tu acetic add,. the pasts described herd» are particularly advantageous fht thibpo^Udlm bf ItttetÄl. alcohol, för example intended for biofuels or for chemical industries.

Is: also described: hefoftr a mefoodifor producing a|lhast:.öt^ídsMMíáÍónpróáuet, comprising a stegof formehtatiott^undernhaerobie conditions, by a yeast asdefmefoaboye in s fermentation medium.

The fermentation product is in particular chosen from efoanok a mofahphfo obtained tient elhat toi or a seeoudindbu'eiahoiile; A preferred fermentation product is ethanol.

The ethanol production h obt.ai.ocd hy means of alcoholic fermentation.

Those skilled in. the art know bow to determine the appropriate conditions for an alcoholic iermenlation. %tw^y'ölmám#H:'t^r^d0::mayiW:mÁ:td the aieoholie fermentation conditions described1 in the reference book: ^Teast Technology*·, 2nti edition, 1191, (1 Meed and T.W. 'Mapdawifoaha, published by Van Nostrand Reinhokl, ISBN 0-442-31892-8.

The fermentation medium: comprises the Mlowmg elements: at least one fermentable oafoon source, at least one oilmgen source, at least one sulfur source, at least one phosphorna source, at least one source of vitamins and/or at least one source of m inerals.

The carbon source is, for example, provided in die fonn of a sugar which can be immediately asshmlafed hy foe pash a p(mtose such as xylose, gîvccrcfo ethanol and or a mixture thereof The carbon source ss preferably provided by a sugar which can be immediately assimilated by the yeast, and by xylose. A sugar which can he immediately assimilated by the yeast is, for efouPgie, a simple sugar of glucose, fructose nr galactose type, a disaeeharkle of sucrose type and/or a mixture of these sugars.

The carbon source can ip provided in the form ©f a glucose syrup, a fructose syrup, molasses, low-grade run-off 2 (low-grade run-off resulting from the 2nd crystallisation of sugarl, a ii\riroU/>ue of ail or pan of a pi am material, and/m a mixture thereof :Xb# lU&amp;ogæs ih % example, provided ssiÉte aí^a^oisiuní hydroxide, <ii ammonium phosphate* amxîioai^ urtm aml'or a combination thereof,

The Ml&amp;f mmm 1% Ä example, provided in the form of mmmÈm mÊÊk^ ruagnesium sulfide. .sulfuric goid. audte a^

Ile phosphorus source is, fe examplp provided In the form of plio^lonc mé< potassium phosphate, diamrnonium phosphate, monoammonium phosphate, amfor a combination thereof, liw: source of vltanhps Is, for example, provided in the hem of molasses, yeastlhydrolyzafei a? solotion: of for© vitamin or of amlxtare of pure vitamins, and Or a eotuhlpation thereof Ilié source of vitamins provides the yeast with all of the vitamins In hmohids at least :e|blvidest: to those recommended in hie reference handbooks. Several sources; of vitamins: cad he combined.

The source of minerals is, tor example, provided in the form of molasses, a mixture of mineral salts, andrnr a combination thereof.

Thé source of minerals pm vîdesdie yeast:wtth allof the macroelements and traue Yemenis in amounts at least equis. abut to those recommended In the refmence handbooks, Several sources of minoralk eapibeeombtned.

The same substance can. provide several different elements.

Is also described herein a method us defined above, for producing at least one fermentation product, preferably ethanol, comprising a step of fomentation, under anaerobic conditions, by a: yeast no dehned above: ip: as fenoepafion medium comprising;: xylose and/or at least one ten,nentation inhibitor.

The fomentation inhibitor is, for example, chosen from art organtc acid, furfural, BMF fbydroxytneth y! mrrural), one: or mom:ibpÄmi«pdWi>%:Ot>d the osmotic: pessure,

The orgastic acid is, for example, chosen from acetic acid, lache add, forme acid and kvulmic acid.

Sold at least one fèrmprtaîton Inhibitor is preferably acetic acid.

The osmotic pressure exists, for example, in a hydro! vzate of ail or part of a plant material. The osmotic pessure then originates irr particular from tire saline load provided during the method for obtaining said hydroiyxaie.

Thétferrnesnatkm medium is as defined above. fhe Iboneutaiton medium preferably comprises at least 0.2% of xylose, the percentage being expressed by svcightAveight of the fermentation medium, Λ prÄire4t^^^t«tiöa:im#di!öiii'döbi^ds#i':2%'To Ä of xylose.

Thv feMgfttàifltîgtAffl tmy also comprise a sugar Mich can he immediately assimilated by the yeasf toe example glucose.

The feroientaticm medium eoinprihesytofm horn 214; to 7% of :xy1osuóod:Sem üjlfe ίο 151-'« of glucose.

The fermentation medium may comprise xylose, glucose and aecüc acid.

Is also described herein a method as defined above, for producing: at least: o® iiuaeutahon pofodét, piafeabiy eihsnog characterized in fiat said fermentation inedium comprises at: least ame fayitoiyxaie of all or pari of a plan t material:. A hydrolyxate of all: or part of a plant material can he iihtainP by means of a step of pretfeMnfent of tic pi ant: material, tor exantple: at high tempemfore and in tic presence of orpnio acids or solvents, opfonally followed: by a partial or iota! hydrolysis of the sugar pblpwp etmyrnatically and/or chemicalfe and/or thennally.

Tic bydtolyxnfo of ab or part: of a plant material: therein®: comprises a. .mixture: of sugars: originating from thé hydrolysis of sugar plymets, sued: as: cellulose, hemleelluld:sc:apdrstarch > A tefincntation: nsedium may also contprise a hydrolysate of rill or part of a plant material and sucrose.

Is also described herein the: use of a pást: as defined above, for producing at 1 exist one iermestafion product, preferably In a fenneumdon medinm comprising xylose and/or at least one fermentation inhibitor. 3^.feHneÄfion;,prod«ctÄm defined abos®.

The^ fomicntation product Is preferably ethanol.

The fermentation medium is as defined above.

Tbc fermentation inhibitor if -as-defined abem tIther#«ra^ifte:Sind advantages of tic invention will emerge even more Cleath· ou reading: the follovylng exemplary enthodlmcnts wlrieh illustrate the munition otthont homing 0 and for the imderstanding of cdtieh: reference will be made to thép|ipcnd.ed, dippings,, tWMPl; D‘< Λ!\ NO M t \οΓΜΚ \1\ t AP Ml F Pi Ml MO 17ÏM» V I OM AND RESISTAN! TO ACETIC ACID .Materials and .methods ïfeè: 'prôpsgiiipi: medium used: fer II® propagation of a yeast: attain : ©omprfsoy $@'g#£g of yoast exit ach lO η kg ot peptone and HiO g he of glucose.

The Y FOX culture medium described below is used for measuring dm nommolon of xylose to ethanol. lire TIf4o eiiltura medium described below is used for measuring the resistance to acetic acid.. The YFOXxàe culture medium describes! below is used for evaluating the yeast strains m a onltura nrod!om \vhich contains at the same tune xylose, glucose and a fermentation inhibitor, acetic acid-

in lb© ΎΙΙΟΧ medium^ &amp;c :aceilè-:ipil: duiséeatràtioâ of 200Û ppm at phi 5 has-no ihyMmry effect.

On the other hand, in the YFOX-Ac nmdiom, the-a!bgt!o:a^d'^n^^aÄlk:öi'#.i^.Äf|?mat;pM f: has an inhibit;o > effect.

(«) XmtiMmsümmiMm 1 he yeast strain propagation is carried out in two Steps: u prceulture step: Allowed, by .s>o«|fee step.

The preeultureis carried out by inoculating a loop of the yeast strum into 5 ml of the propagation medium as deSned -s^pm.

After 24 b of culture at 30°C in a medium aerated by stirring, 2 tsiof ids precolhne arc removed and inoculated inna 50 ml of propagation medium:,

This culture is carried out for 24 h at 30°C in a medium aerated by stirring. ethanol and the résistance to acetic acid sremeastuedon the cells of yeast resulting from this culture. inxmier to determine the 0.23 g of dry mattbr of the yeaststrak propagated as indicated above are inoculated per kgof YFGX medium. rpe fermentation is carried out at 32/3.1, under anaerobic conditions, wsth moderaie voning. 1 he ethanol and.also xylose and glucose concentrations arc measured by UPt O.

The following formula is then used to obtain the degree of conversion of xylose tó: etbasoi:

in which [xylose]:«»«.»! eurrct*po»ds to die xylose concentra«»»» m the medium at the time of thy %i&amp;the yea&amp;t stpduand i xylose] ».««i so*'expends m the xUose e-rncenn uhonm the eoltttro Hleáíöih: at Φ hoofs iltmting ifom the inocPlatiool {*) M ea set oniony (YthejesiM«Ç2e^LâiMiiLa£i«i

The resistance to acetic acid of a yeast strain is determined hy the delay of initiation of alcoholic fermentation. 0.25 g of dry spader of the yeast strain propagate ás indicated: áte^d'i^dihdedÍMéd';|ii^ kg of VP Ac medium.

Tiré fÄt^Wönfs.-hiöiti^d out under anaerobic conditions, at dl^Cynyithitnodoráíe stíping,; 1 he ethanol prodnchontsioo'oan'd md'ucth hy moavnmp « te loss o fro ass Y the ten net ton son flask, this loss of mass correlating directly w-th the alcohol production.

The alcoholic lennen ration curve indicating; (he lop: of mass : as: a: Junction of lime is produced, f hep oint of intefseetioniheiween the n-axidhhd. the :*af'ïbe' loss of mass is determined: u corresponds to the delay of initiation «CteMÄ«,

It should he noted that the maximum derivative of the loss of mass gives the same result as the maximum dens alive ot dm aLohoi production rate, if the alcoholic fennentation euren indicates the alcohol production as a function of time instead of the loss of mass as a function of time. The «tore tire dufátionohíameáshorter the delay of initiation of fermentation and the more resistant the yeast strain is to acetic acid. tv) Crossing

The starting strains used for the crossing are the 1-4538: yeast strain and the 1-462? yeast strain, Step 1: Yeast strain growth A loop of the starting strain (conserved at -80°€) is inoculated at the sort ace of the agar of a Petri dish of medium A.

The composition of medium Λ is fete following: 10 g of yeast extract. 10 g of peptone, 20 g of glucose, 20 g: of agar, pH é,2 VÂ Oil, water qs 1 L The Petri dish is then incubated ior 24 hours at 3CPC,

Step 2 : Sporulation on medium 2 A loop of the previous cdldlreis inoculated at the surface oflhc agár of a Petri dish of medium B.

The composition of medium B is the following: 6.5 g of sodium acetate, 15 g of agar, pH 6 .5-7, water 6s 1 h

He Petri dishis foeultofod for '96 ho urs at 2;5%Τ The biomass oMamed at the surface of the dish Is thee harvested id » pi of sterile waten B μϊ of aymolyase ere added to .I;fO μΐ of ihm C&amp;P sttspoMlon is incubated tor 30 ndnuiesat 301¾. ÈeÊmà iei^ plated opt onto an: agar dish pfimtÉ« '1, He tetrad dissection is prefetokly earned out M totales later. Stmt: Tetrad dissection

The: tetrads are dissected with a Singer (hno fbo Petri disk is irtouhated tor 48 hours at 30' <1

Step 4: Segtegani storage

The segregants are thon stored at toCtoC is medium 1 comprising 20% of glycerol.

Step .§: Selection of the 1-4538 yeast strato sepepfos

Ike: 1-4538 yeast: mmx segregants are selected bn:fhett ability to convert xylose to ethanol The xylose conversion ia measured as in (hi), uafog a segrepnt propagated as in #i) tor Ike yeast strain, except font the measurement is earned out at 41 hours tostead of |Cfltours>

The sepnganto sel ected: convert at least 70% of the xylose: at 41 hours .

The Mat n or Mai alpha sea type of the segroptos selected is dfoemtioedby ft'R.

Stegh: Selection of the 64627 strain sepegaeta

Tke 14627 yeast siram segregams are: selected ·am the basis of their resirianee to aeetie a.eid. Tke resistance to acetic acid is evaluated as in fiv), using: a segregant propagated as to (If) lor the yeast strain.

Th e segrogants selpetod have a delay of íMtiati on of torrnenlation of toss than 2 5 hours.

Tke Mat a or Mat alpha sex typo of the segrogants selected is determined by PCR.

Step 7: Crossing and selection of hybrids Á loop of a Mat alpha haploid segregant is inoculated at the surface of the agar of a Petri dish of medium Jo A loop of a Mat a haploid topepni is mixed with tke deposit of the Mat alpha strain.

Tke composition o f medium A is the following; 10 g of yeast ex tract, i p g of peptone, 2P g of glucose, 20 g of agar, pH 6,2 tó· (),2, water qs 1 1,

The Petri disk is then iucuhatod for 24 hours at 30-C. A loop of the mixture is inoculated at the surface of the agar of a Petri dish of medium A. This step is repeated 5 tunes, touring the final inoeulation, the cells araforeaked so as m obtain isolated colonies. Teást cella arc removed from each isolated colony and enltared, their genomic ÖNA is extracted and a PCR is carried out on this genomic DMA in order to verify that the Mat a arid Mat alpha alleles are indeed present m one and the same yeast cell.

The yeast cells which have the Mat a and Mat alpha alleles arc called hybrids.

The hybrids;are sOleeied on the basis of two criteria: their ability to metabolize xylose and which läre evaluated as indicated in {id; and (iv).

The |^ri&amp;Meet^d:;8rfÄb' MIhwfng: - the hybrids: which have a delay of initiation of alcohol fermentation of less than 3() hours, - the h^fids:W&amp;k:ié«tnv^t:: at least φ% of xylose at ööhouric («} lilriTtixLe^imoo

Directed evolutionIs theeembinatKnr of a step of UV mutagenesis followed by an eöridlhOÄ: on a fermentation medium in which xylose constitutes the only carbon setrtee.

Step l: During the VWmutagenesis, 15 tnl of a suspension of yeast cells at 5% (in g of dry matter for 1.00 g of the suspension) are placed in a 9 cm Petri dish. The Petri dish is: then placed amddr n DY radiation of 500 i,'enr at 254 nm.

Step 2: Theí írradlá ted yeast cells amount of I g of dry matter/kg of YFX medium. After 96 houp rif prtibglieaîiow^ at: 327C, 100 μΐ of the culture shedldm are: used In inoculate Id ml of n glycerol me#nm containing. 20 ifkg of glycerol, 5 gTg of ammonium sulfate and 1.7 g/kg of YMS (Yeast Mmopn Base) medium from Ditco. This second culture is carried our under aerobic conditions and at 3CFC for 48 hours. ! mi of this culture .is then removed to inoculate 170 ml of YFX medium.

This cycle is repeated 8 times.

Hcsults

Several hybrids are obtained: by crossing the D455B ateain with thef-4627 strain, among which the 11 and 112 hybrids.

The 12 hybrid, winch is net capable of metahoimn g xvlose tor the purposes of the invention hut which exhibits resistance to acetic acid, is subjected to a directed evolution step: the EDI, ED2 andlBDS hybrids are then obtained, corresponding respectively to the 1-4624,1-4626 and 1-4625 yeast strains.

Table 1 indicates the percentage of xylose converted to ethanol at 60 hours in iho YfCxY medium and the delay of initiation of alcoholic fermentation in the YPAc medium (in hours) of various yeast strains.: - tbc Ac control which is a oom-ioneticdly-modificd yeast strain rssisfontfo acetic acid, which is not therefore capahlo Of mcÄolföiog xylose, • the 1-453 S standig yeast strata which is very effective for the production, of alcohol, hut

Ernőt resistant to noetic tidd, - the 1-462? starting yead:sipin which is resistant to acetic add, but is not capable of i nemin di/mg \>hwc au the purposes of die on esd fore ~ die 111 h> ht fo which -,s ,- y er,a strain n-licit is capable o Í metabolizing xylene and n Inch is resistant, to acetic acid, - the H2 hybrid Which is not capable of metabolizing xylose for the; purposes of the invention, hut exhibits resistance to acetic acid, the EDE ED2 and ED3 hybrids which are capable of metabolfomg xylose and are resistant to acetic acid. lahicj.

:MpwvwsÄ»©Äanöhpyp^teef.ihe yeasi:strains is sydnaten infhe YPGX-Ac fermentation m^imv^mpri.^ng-atlhe::s^«rteé, glucose, xylose and acetic acid as fermentation inhibitor. The HI, EDI, BD2 and ED3 yeast strains have art alcohol production which is dearly improved compared with the 1-4528 parent yeast strain ln tbis Y FGX-Ac modi am icf figure /),

EXAMPLE 2; ALCOHOL FRODl XT ION IX AN ACTUAL APPLICATION MEDIUM USING THE YEAST STRAINS ACCORDING TO 1'Iil·' INVENTION and methods the alcohol production (ifibe EDI. 1 Ù2 and i 1>3 \cwit ^mena 1$ evaluated m .in umuat application medium.

The alcohol production oft% E4338 parent yeast strain Is alsu ovaluateil hy way of emapS^Ä The evaluation of im áíeúMl prodPeidn of the yeast straths is earned out on two difceni application media: - medium A comprising: ο a hemioellulosic stdssiaiu which eetrespohds to the liquid phase obtained alter solid/liquid separation of the biomass having undergone a pretreatment and thermoohonueal hydrolysis of a wheat straw and which; comprises predominantly xylose (48 g/kgk other sugars in small amount and also those;nation inhibitors resulting from the physicochemical pretreatmem of die li gnoeel 1 ulosic biomass, and o nutrients (sources of si trogen and phosphores}, - medium B corresponding to medium A above suppletnented ssdth giueosc, in order to mimic a hydrolyxate of lighocelhddsie biomast the hemiceiluhssle finetion of which has been hydrolped and the eellidosic Ifactldn of which would have been partially hydro! yxed.

The initial pH of media A and B is adjusted to pH 5.5.

The fermenter is Inoctdaied with 1.5 g/kg of dry matter of the yeast straindo he evaluated,

The alcoholic fermentation is carried out at 35a(L A fermentation is also carried out at 32'€ with medium,Α*. lies nits

The results dhtamed with medium .A at 32*Cfand;35c;C3 are gtvm respecfiyely in figures: 2 and 3, and the results with medium B at 35 '€ in figure 4,

It should he noted that, in figure 3, the points have stopped at 60h itrlte Strahn, since the plateau phase has already been reached.

The ED! and ED2 hybrids show a real advantage in application medium A at 32CC compared with the 1-4538 parent, yeast:strain,

The ElTl, ED2 and ED3 hybrids show a mal advantage; in appheatiou medium: A at 3S°€ compared with the :1-4538 parent yeast strain, indeed, even though: theethanol pmdnetien is better in the first 60 horns for the ! A 53S;yeasi;stAti:tg the maximum amount of ethanol produced is 6.3 g per kg of medium, whereas amounts of ethanol .greater than 10 g/kg: are· obtained with the EDI, EM and ED3 hybrids. lie ED! mid ED2 hybrids show, once agaiig a mal advantage in application medium B at 3SaC compared with the I-4.538 yeast strain, The ED3 hybrid is a little behind in this application medium.

Claims (4)

1. Szoba Éátmi 4 .4 CHÇM-nél 2012, mtáus: 24-έη 1-4627 -pâmia»· áftjétbe- he%Äl&amp; ijesztő; term,

   1, Eljárás xilôz n-etabol ízálására képes és eeetsavnak ellenálló elosztó: tfe§. élé* állítására, amely a következő lépéseket foglalja «mgábsrj: «&amp; CNCM-nél 2011, október 5-én 1-4538 számon letltfee helyezett élesaíp íifös fereezîezése: a CN CM· net 2012. május 24-ën 1 -4627 számost letétbe helyezett élesztő törzzsel, legalább egyMbrtdeM - legalább egy xlléz nmiabobzálására képes és eeetsavnak ellenálló hibrid kiválasztása,
2. 3, A X igénypont szerinti eljárás, azzal jellemezve* hegy a kefésztezesi lépés tnagában foglalja: - a CNCM-nél 2011. október Join :1-4538 számon letétbe heiyMsl^ileszto törzs apőrázMási lépését, legalább egy X szegregáns nyerésére. - a CNCM-nél 2012. május 24-én 1 -4627 számon letétbe helyezett élesztő törzs spófáziatási lépését, legalább egy Y szegregáns nyerésére, - legalább egy X szegregáns hibridizációs lépését legalább egy Y szegregánssal, legalább egy hibrid nyerésére, amely X szegmgáM képes xllózt etanoiiá alakítani ésNagy amely f szegregáns eéetsavnab ellenálio. 4. Λ 2. vagy 3, igénypont szerinti eljárás, azzal jellemezve* begy a keresztezés lépés magában foglalja: - a CNCM-nél 2011. október S-én 1-4538 számon letétbe helyezett sf&amp;a^íásl lépését, legalább egy X szegregáns nyerésére. - a CNCM-nél 2012. május 24-én 1-4627 számon letétbe helyezett .élesztő törzs Spéráziatásl lépését, legalább egy Y szegregáns nyerésére. - legalább egy X szegregáns hibridizációs lépését legalább egy Y yzegregánssal, legalább egy hibrid nyerésére, amely X szegregáns a xüó?. legalább 6§ 34-át 60 óra alatt etanoiiá alakiba és/vag}· amely Y szegregáns az áikoheios erjesztés elindítását kevesebb, mint 30 órával késlelteti, 0. A 2-4., igénypontok bármelyike szerűm eb tms azzal jellemezve, hogy a sn iózt etanoiiá alakítani képes és ecelsavnak ellenálló: legalább egy hibrid kiválasztási lépése a következő lépéseket foglalja magában: ~ á legalább egy hibrid által anaerob körülmények között 66 éra alatt a közeg kg-ra számítói 55 g glükózt Is 41 g xilézt tartalmazó fortüentáciös közegből etanoiiá alakított xflöz százalék mérése. ·· 4000 ppm mmsmrn «Énazó fermentációs közegben pH 4,4-nél legálább egy hibrid alkoholos erjesztés indítására gyakorolt késleltetésének mérése, » legalább egyMbtM kiválasztása, arndy a xilőz legalább 70 %-ál 60 óra a lan etanoüá alakiba és amely az alkoholos erjesztés elindításéi kevesebb, mint 30 érával késlelteti 4 .4 2-4 Igénypomnk bármelyike szermti spál, amely továbbá egy Iráöyilöti evolúciós lépési tartalmaz. 7*: Élesztő ifes, amelyet az alábbiak teil a O^CMmél 2012, májM 24-éii 1-4Ő24 számon letétbe helyezeti élesztő tÄS, CNCIvl-nel 2012. május 24-én 44620 számon letétbe helyezeti élesztő törzs és a CMClMméi 2012. mt|as ;24rán 1*4626 szá* mim letétbe helyezett élesztő törzs. 8. !a|átás legalább egy fermeitíáemiS termék előállítására, amely a ?, igénypont szabni élesztő törzs állal anaerob körülmények közölt egy iátmaniMős közegben •végzett fermentációs lépést, tmáalmaz, tv A. ;|, igénypont szerinti eljárás, azzal jellemezve, |ogy a rármentáelós médium gilőzl és/vagy legalább égy fermentációs inhibitort tartalmi 10. A. 4 vagy 9. Igénypont szerinti eljárás, azzal jeilensezve, hogy a fermeníáélős oráriium egy teljes növényi anyag vagy annak részének legalább egy MdrráizÉtnmát tártál-mázzá. 14 A 7. Igénypont szerinti élesztő törzs alkalmazása egy lenoeniácié# tettnél: elő-állítására, előnyösen xllózt és/vagy legalább egy íermenfáelés inhibitort tartalmazó förmaorá-elés közegben.
3. 12. Tenyészet, amely egy ?. igénypont szerinti élesztő törzsei tartalmaz. 13* Élesztős kenyér, amely egy ?, igénypont szerinti élesztő törzset tartalmaz,
4. 14, A ÍJ,: igénypont szerinti tenyészel, azzá! jeiiemrzvg, hogy száraz élesztő alakú.: