Bioethanol strains of Saccharomyces cerevisiae characterised by microsatellite and stress resistance

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Biotechnology

and

Industrial

Microbiology

Bioethanol

strains

of

Saccharomyces

cerevisiae

characterised

by

microsatellite

and

stress

resistance

Vanda

Renata

Reis

_,

_Ana

_Teresa

_Burlamaqui

_Faraco

_Antonangelo

_,

Ana

Paula

Guarnieri

Bassi

_,

_Débora

_Colombi

_,

_Sandra

_Regina

_{Ceccato-Antonini}

a_{UniversidadeFederaldeSãoCarlos(CentrodeCiênciasAgrarias),DepartamentoTecnologiaAgroindustrialeSocio-EconomiaRural,} LaboratóriodeMicrobiologiaAgrícolaeMolecular,Araras,SP,Brazil

b_{UniversidadeEstadualPaulista,InstitutodeBiocienciasdeBotucatu,DepartamentoParasitologia,Botucatu,SP,Brazil} c_{GenotypingLaboratóriodeBiotecnologiaLtda,Botucatu,SP,Brazil}

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Articlehistory:

Received3January2016 Accepted19September2016 Availableonline22December2016 AssociateEditor:GiseleMonteirode Souza Keywords: Fermentation Stresses Yeasts Microsatellite Contaminants

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StrainsofSaccharomycescerevisiaemaydisplaycharacteristicsthataretypicalofrough-type colonies,madeupofcellsclusteredinpseudohyphalstructuresandcomprisedofdaughter budsthatdonotseparatefromthemothercellpost-mitosis.Thesestrainsareknownto occurfrequentlyinfermentationtankswithsignificantlowerethanolyieldwhencompared tofermentationscarriedoutbysmoothstrainsofS.cerevisiaethatarecomposedof dis-persedcells.Inanattempttodelineategeneticandphenotypicdifferencesunderlyingthe twophenotypes,thisstudyanalysed10microsatellitelociof22S.cerevisiaestrainsaswellas stressresistancetowardshighconcentrationsofethanolandglucose,lowpHandcell sed-imentationrates.TheresultsobtainedfromthephenotypictestsbyPrincipal-Component Analysisrevealedthatunlikethesmoothcolonies,theroughcoloniesofS.cerevisiaeexhibit anenhancedresistancetostressfulconditionsresultingfromthepresenceofexcessive glucoseandethanolandhighsedimentationrate.Themicrosatelliteanalysiswasnot suc-cessfultodistinguishbetweenthecolonyphenotypesasphenotypicassays.Therelevant industrialstrainPE-2wasobservedinclosegeneticproximitytorough-colonyalthoughit doesnotdisplaythiscolonymorphology.Auniquegeneticpatternspecifictoaparticular phenotyperemainselusive.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:antonini@cca.ufscar.br(S.R.Ceccato-Antonini).

http://dx.doi.org/10.1016/j.bjm.2016.09.017

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

TheBrazilianindustrialfermentationprocess forfuel alco-holproductionhascertainatypicalcharacteristicsthatallow fortheentryandgrowthofwildyeaststrains.Theconditions aresoconducivetowild-yeastgrowththatoccasionallytheir developmentisfoundtocompetewiththatoftheselected starteryeaststrain.Oneofthemajorreasonsforthisisthatthe methodologyfollowedbytheBrazilianethanolicfermentation industry does notrigorously implement sterile conditions; otherprominentreasonsincludeyeastrecyclingalongwith sugarcaneharvesting.1 _{Asaresultofthe abovementioned}

reasons,contaminationbywildstrainsofSaccharomyceshas a very frequent occurrence in the bioethanol industry. At timesithasbeenobservedthatthe growthpatterns ofthe indigenousstrainsaresorobustthattheydominatethe fer-mentationprocesstotheextentofreplacingthestarteryeast strain. The status or influence of the contaminant strains inthefermentationprocess isdependentupon characteris-ticssuchasthefermentativeperformance,cellsedimentation rate,filamentationaswellasbiofilmdevelopment.2

Indige-nousstrains withrough colony morphologyare frequently observedduringtheethanolicfermentationprocessandare associatedwithpseudohyphalgrowth andhigh sedimenta-tionrate;thesestrainsresultinproblemsthataresimilarto thoseobservedforflocculentstrains.3,4_{Asawordofcaution,}

itistobenotedthatthecellaggregationcausedasaresultof pseudohyphaeshouldnotbeconfusedwithflocculation.

Chainformationinyeastisobservedwhentheyounger budfailstoseparatefrom themothercell5_;undersuch

cir-cumstancesthe newer cell remainsattached tothe parent post-mitosisleadingtotheformationof‘snowflakeyeasts’.6

AstudyconductedbyReisetal.,4_{comparingrough-colony}

strainswiththeirsmooth-colonycounterparts,demonstrated that the rough-colony strains havesignificantly lower and slowerfermentativekineticswhenmonitoredinabatch sys-tem over a48-h period underconditions wheresugarcane juicewasusedasthesubstrate.Highresidualsugar concen-trationhasbeen documented tobea factorthat isclosely associatedwiththepresenceofwildS.cerevisiaestrainsinthe fermentationprocess.3,4

Environmental conditions are known to be key factors capableofinfluencingandaffectingdifferencesincolonyand cellmorphology.7,8 _{In addition,signalling cascadessuch as}

theMAPK,TORC,SNF1andRIM101pathways,arealsoknown tobeinvolvedininfluencingmorphologicalchanges.8

How-ever,inthelattercase,theresultantmorphologicalchanges areusuallyofatransitorynature.9,10

Curiously, in spite of the presence of clear demonstra-bledifferencesincolonymorphologyand cellarrangement betweensmooth-colonyandrough-colonystrains,the restric-tionanalysisofmitochondrialDNAandPGFE(chromosome karyotyping)bothfailed touncover anyunderlying genetic differences.Thedifferencesinmorphologywere concluded tobeaconsequenceofenvironmentalconditionsthat influ-enceandcausedifferentialgeneexpression.11_Kuthanetal.12

reportedthatTy-codinggenesandsubtelomericgenesthatare inducedbystressconditionsinterferewiththecolony mor-phologyofyeasts.AreportbyCavalierietal.13_thatanalysed

metabolicpatternsindicatedthatthereweresignificant dif-ferencesinthegeneexpressionprofilesofthecolonyvariants (filigreed,roughandsmooth)especiallywithrespectto ammo-niaandaminoacidtransporters.

Inthatdirection,astudybyRatcliffetal.6_thatcompared

a unicellularstrain ofS.cerevisiaeand anevolvedstrainof snowflakeyeastshowedthat1035genesweresignificantly dif-ferentiallyexpressedbetweenthetwo.Theauthorsnotedthat sevenofthetenmostdownregulatedgeneswereregulatedby thetranscriptionfactorACE2inconditionswhereinbothACE2 alleleswereidenticalinthediploidstateoftheyeast.Astudy byRodrigues14onspontaneousderivativesofJAY270/PE-2 pre-sentinganalteredcolonymorphology(roughenedsurfaces, irregularedges,cellsedimentationresemblingflocculationin liquidmedia)revealedthatlossofheterozygosityofthegene ACE2(asaresultofframeshiftmutation)wasresponsiblefor thedevelopmentoftherough-colonyphenotype.PE-2isoneof themostimportantindustrialyeaststrainsusedinthe Brazil-iandistilleries.3 _{ACE2heterozygosityshouldbeinvestigated}

intheyeaststrainsdisplayingrough-colonymorphology fre-quentlyisolatedfromtheethanolicfermentationtoassessthe realoriginofthisphenotype.

In spite ofclear differences in colony morphology and cellarrangement,indepthanalysisintogeneticdifferences between smooth and rough-colony strains have failed to reveal the presenceofany underlying variationsata DNA level so far. The PCRmicrosatellite methodology has been extensivelyusedforS.cerevisiaestrainidentificationespecially whenassessingthewinefermentationpopulations15–17_;more

recentlythistechnologyhasbeenusedforassessingthe bio-diversityofnativebioethanolyeaststrains.18_{Thistechnique}

hasbeenrevealedtobesensitiveandrobustenoughtodetect theextensivegeneticdiversityoftheindigenousstrainsofS. cerevisiaeinBrazilianethanol-producingunits.18

MicrosatellitesorSSRs(SimpleSequenceRepeats)areshort segmentsofDNAthatarerepeatedintandemandareknown tobeco-dominantlyinheritedanddispersedthroughoutthe genome.19_{ThesixteenchromosomesofS.cerevisiaegenome}

are known to be very rich in the presence of microsatel-litesaswellasnumerouspolymorphicalleles.20_Perezetal.19

evaluatedthegeneticvariabilityof51isolatesofS.cerevisiae using the microsatellite methodology. With the use of six microsatellitestheyuncoveredatotalof57allelesand gen-erated44genotypes.

Despite the result of loss of heterozygosity of ACE2 to be the probable origin of rough-colony morphology in S.

cerevisiae,6,14 _{previousstudiesherereportedweremore}

con-clusiveregardingtothedifferencesingeneexpressionthan tothegeneticdifferentiationatDNAlevelbetweendifferentS.

cerevisiaephenotypes.Inviewoftheremarkablyhigh

discrim-inatorypowerofthemicrosatellitemarker-basedassessment, thistechniquewasappliedinourstudyinanattemptto eval-uate the genetic variability amongst strains ofS. cerevisiae isolated from industrial ethanol units. The ultimate objec-tiveofthestudywastodiscoverageneticpatternthatcould beusedtodifferentiatebetweenthetwocolonyphenotypes (roughandsmooth).Additionally,thephenotypic character-istics such as resistance to stress and cell sedimentation werealsosurveyed.Itwashypothesisedthattheassociation betweenmoleculartraitsandphenotypicfeaturescouldhelp

indifferentiatingbetweencolonyphenotypeswhichinturn woulddiscriminatethemintoseparategroupstobetreated differentlyinthefermentationunit.

Materials

and

methods

Yeaststrains,identificationandcultureconditions

Yeastculturesofbothsmoothandroughcolonieswere col-lected fromdifferent ethanol-producingunits(Table1)and subjectedtoPCRandITSregionsequencing(primersITS-1and ITS-4)21_{toconfirmidentityasS.cerevisiae.ThePCRconditions}

employedforITSamplificationwereasdescribedbefore.18

Fol-lowingidentityconfirmation,twenty-twostrains(elevenfrom eachcolonyphenotype)wereselectedformicrosatelliteand stressresistanceanalysis.Thesestrainsweremaintainedon YPD(w/v:1%yeastextract,2%glucose,2%peptone,2%agar; forbrothpreparation,agarwasomitted)slantsat4◦Cwith regulartransferstonewmedium.

Microsatelliteanalysis

A total of ten microsatellite loci (Table 2) were analysed bycapillaryelectrophoresisand thepatternsthus obtained wereusedforcreatingaphenetictree.Population1.2.30 soft-warewithalgorithmMinimumGeneticDistance–DM22 _for

distancemethod and UnweightedPair Group Method with ArithmeticMean(UPGMA)astheclusteringmethodwereused forthis purpose.Thereaction conditions formicrosatellite amplifications, capillary electrophoresis and diversity pop-ulationanalysis were described previouslyby Antonangelo etal.18

Phenotypiccharacterisation

Phenotypic characterisation was performed based on the resistancetothestressandcell sedimentationrate.To ver-ifyresistancetolowpH,yeastcellsweretransferredfromthe YPDdishestoasalinesolutionandthecellconcentrationwas adjustedsoastoyieldafinalcountofca.108_{cells/mL.A10␮L} aliquotwasplatedontoaPetridishcontainingYPDwiththe followingpHvalues:6.0,5.0,4.0,3.0,2.0,1.75,1.5,1.25,1.0. Theinoculateddisheswereincubatedat30◦Cfor4days.Yeast growthwasscoredas(1)andabsenceofgrowthas(0).Inorder toavoidliquefactionoftheculturemediumduetothelow pH,agarwassterilisedinalowamountofwaterseparately fromthesolutioninwhichtheothernutrientsofthemedium were dissolved. Thenutritivesolution was adjustedto the requiredpHvaluepriortosterilisation.Followingautoclaving, bothsolutions(agarand nutritivesolutions)weremixed,at 55◦C,andimmediatelypouredintosterilePetridishes.

Thesame procedure was utilisedfor thepreparationof PetridishescontainingYPD(pH6.0)withethanolinfinal con-centrations of 0,3, 6, 9, 12, 12.5,13, 13.5, 14,14.5, 15,18, 21% (v/v).Subsequent toinoculation, thePetridishes were wrappedinplasticfilminordertoavoidethanolevaporation. Growth wasevaluatedintheinoculatedPetridishesafter4 daysofincubationat30◦Candscoredasdescribedbelow.

YPD(pH6.0)withglucoseinthefinalconcentrationsof100, 150,200,250,300,400and500g/Lwasalsoutilisedtoverify resistanceoftheyeaststrainstohighsugarconcentrations. Theproceduresfollowedforyeastinoculation,incubationand growthscorewerethesameasdescribedabove.

The cell sedimentation assay was performed according to the protocol described by Wang et al.23 _{and modified}

by Reis et al.4 _{After growth in the multiplication medium}

(sugar cane juice with approximately 4%reducing sugars),

Table1–Saccharomycescerevisiaestrainsanalysedbymicrosatellitemarkers,forstressresistanceandcellsedimentation rate.

Strain Ethanol-producingunit Codeofisolation Colonymorphology

2 UsinaSantaAdélia VF4 Smooth

3 UsinaSantaAdélia VF5 Smooth

4 UsinaSantaAdélia VF6 Rough

6 UsinaSantaAdélia VF8 Rough

7 UsinaSantaAdélia VF9 Rough

8 UsinaSantaAdélia VF10 Rough

9 UsinaSantaLúcia 45 Rough

10 UsinaSantaLúcia 47 Rough

12 UsinaSantaLúcia 51 Smooth

15 UsinaSantaLúcia 2 Smooth

16 UsinaDiamante 385 Rough

PE2 UsinadaPedra PE-2 Smooth

18 UsinaSantaLúcia FM Smooth

19 Unknown URC Rough

33 UsinaSantaAdélia A1 Smooth

35 UsinaSantaAdélia M1 Rough

36 UsinaSantaAdélia M2 Smooth

CAT1 UsinaVOCatanduva CAT-1 Smooth

BG1 UsinaBarraGrande BG-1 Smooth

SA1 UsinaSantaAdélia SA-1 Smooth

47 UsinaSantaAdélia VF-1 Smooth

Table2–MicrosatellitelocionSaccharomycescerevisiaechromosomes,OpenReadingFrames(ORF)andprimersequences (forward/fw;reverse/rv).Thenumbersinsuperscript(inLociname)arerelatedtothereferenceswhereintheprimer designswereobtained.

Lociname Chromosome–coordinates ORF Primersequence

G115,30 _{XVI–536832-536675 SC8132X Fw:5}_{CTGCTCAACTTGTGATGGGTTTTGG3}

Rv:5CCTCGTTACTATCGTCTTCATCTTGC3

G219,27,31 _{XIII–87140-86862 YMLO91C Fw:5}_{AAAAGCGTAAGCAATGGTGTAGAT3}

Rv:5AGCATGACCTTTACAATTTGATAT3

G315,16,27,30,31 _{XV–823021-822769 YOR267C Fw:5}_{TACTAACGTCAACACTGCTGCCAA3}

Rv:5GGATCTACTTGCAGTATACGGG3

G415,16,27,30,31 _{XVI–536841-536424 YPL009C Fw:5}_{AACCCATTGACCTCGTTACTATCGT3}

Rv:5TTCGATGGCTCTGATAACTCCATTC3

G516,19,27,31 _{IV–778622-779023 YDR160W Fw:5}_{TGGGAGGAGGGAAATGGACAG3}

Rv:5TTCAGTTACCCGCACAATCTA3

G616,27 _{VI–210262-210403 YFR028C Fw:5}_{GTGTCTTGACACAATAGCAATGGCCTTCA3}

Rv:5_{GCAAGCGACTAGAACAACAATCACA3}

G716,19,27,31 _{VII–245604-245701 YGL139W Fw:5}_{CTTTTTATTTACGAGCGGGCCAT3}

Rv:5_{AAATCTCATGCCTGTGAGGGGTAT3}

G816,19,27,31 _{VII–467642-467744 YGL014W Fw:5}_{CAGGTCGTTCTAACGTTGGTAAAATG3}

Rv:5GCTGTTGCTGTTGGTAGCATTACTGT3

G1215,16,19,31 _{XIII–86902-87140 SCAAT1 Fw:5}_{AAAGCGTAAGCAATGGTGTAGATACTT3}

Rv:5CAAGCCTCTTCAAGCATGACCTTT3

G1316,19,31 _{IV–776163-778721 YDR160W Fw:5}_{TGGGAGGAGGGAAATGGACAG3}

Rv:5TTCAGTTACCCGCACAATCTA3

thecellswerecollectedbycentrifugation,washedtwicewith sodiumcitratebuffer(50mM;pH3.0)containing5mMEDTA and then washed againwith waterat4◦C. Thecells were resuspended in chilled distilled water and diluted or con-centrateduntil anOD (600nm)of2.0was reached(Thermo

Biomate® spectrophotometer). After vigorousshaking,

sam-plesfromtheupperportionofthetubeweretakenat0minand 10minintervalsandtheOD(600nm)wasobtained.The sed-imentationrate(%)wascalculatedasfollows:sedimentation (%)=[(OD0min−OD10min)×100]/OD0min.

ThePrincipal-ComponentAnalysis(PCA) was appliedto theresultsofphenotypiccharacteristics,consideringabinary matrixofdatainwhichthelinescontainedtheyeaststrains and the columns represented absence of growth (level 0) or growth (level 1) for each characteristic analysed (resis-tance to low pH, high sugar concentration, high ethanol concentration).Forcellsedimentationassay,thelevels0and 1 were applied to results below 10% or higher than 10%,

respectively.Thefunction‘prcomp’ofthestatisticalsoftware RwereutilisedforthePCA.24

Results

Themolecular-typingwork wasperformedwithagroupof

22S.cerevisiaestrainsisolatedfromalcohol-producingunits

which was separated by the characteristics of colony and cellmorphologyintwogroups:‘smooth’and‘rough’colony phenotypes. Theaspects ofcolonyand cell morphologyof bothphenotypesareillustratedinFig.1.Geneticanalysisby microsatellite assay byusing 10 microsatellite loci didnot revealtheclusteringofyeastswithinthegroups‘smooth’and ‘rough’colonyphenotypes.Thegreatestgeneticsimilaritywas observedindeedbetweenyeaststrainsfromdivergentcolony types,asstrains47and35,or33and52(lowerleftbranchofthe tree).Interestingly,theindustriallyrelevantstrainPE-23_was

Colony

Cells

Colony

Cells

Fig.1–Colony(growinginYPDmedium)andcells(magnificationof400×atopticalmicroscopy)ofasmoothcolony(left) andaroughcolony(right)ofS.cerevisiaestrains(ReprintedfromReisetal.4_).

12

Fig.2–Phenetictreecreatedwiththemicrosatellitelociby usingthePopulation1.2.31softwareandUPGMAclustering method.Thelegendinsidetheboxisreferenttothestrain code(asinTable1).Rough-colonystrainsaredenotedin circles;smooth-colonystrainsinsquares.

foundclosetorough-colonystrainsintheupperleftbranchof thetree(Fig.2).

Phenotypiccharacteristicsbasedontheresistancetothe stress and cell sedimentation rate were then analysed in an attempt to cluster the strains. Upon applying the PCA methodtothesetoffourphenotypiccharacteristicsandtheir sublevels, a biplot was obtainedand shown in Fig. 3. The PCA-1explained73.62%ofthedatatotalvariability.Themost pronouncedeffectobservedinFig.3isthe strongopposite contributionsmadetoPC1bytheresistancetolowpH(1.25 and1.5)displayedbyindustrialyeaststrainsCAT-1andPE-2in therightupperquadrant,ontheonehand,andrough-colony yeasts(eightoutofelevenstrains)totheresistanceofboth highethanolandsugarconcentrationsbesidessedimentation rateintheleftatthemiddle,ontheotherhand.Other rough-colonystrainswerefoundintheupperquadrant(strain36),a littledistantfromtheindustrialyeaststrainsbecauseofthe highsedimentationrateaswell;thestrain06duetothe exclu-sivelyresistancetohighethanolconcentrationinthemiddle ofthePCAbiplot;andthestrain52duetothesensitivityto lowpHbutresistancetohighsugarconcentrationandhigh sedimentationrate,closertotherough-colonystrainsthanto thesmooth-colonystrains.

Theisolationofthemajorityofsmooth-colonystrainsin therightinferiorquadrant(6outof11)inFig.3revealedits sensitivitytolowpH,highsugarandethanolconcentrations andsedimentationratebelow10%.Onesmooth-colonystrain wasputtogether toPE-2and othertwo(strains12 and15) wereplacedinthemiddleofthebiplotduetotheresistance toethanol.Otherparametersanalysedinthisstudy(<300g/L glucose;<13%and>14.5%ethanol;pH<1.25and>1.5)didnot

representsignificantvariationforthisgroupofstrains.Forthis reasontheycrowdedatpointzeroofthebiplot(Fig.3).

Insummary,therough-colonystrainsarefoundintheleft sideofthebiplot1(exceptforstrain06whichisquitecloseto thelimit)inwhichtheparameters:sedimentationrate,high sugarandethanolconcentrationstogetherwithlowpHwere key determinantsin the distributionofthe strains; on the otherhand,allthesmooth-colonystrainswerefoundonthe rightsideofthebiplot,togetherwithresistancetoethanoland lowpH.

Discussion

An analysis of results obtained from the phenotypic tests byPCArevealedthatunlikethesmoothcolonies,therough coloniesofS.cerevisiaeexhibitedanenhancedresistanceto stressfulconditionsresultingfromthepresenceofexcessive glucoseandethanol.ResultsobtainedbyVoordeckersetal.8

verifiedthatwrinkly(rough)coloniesofS.cerevisiaeweremore resistanttootherstressorssuchasheatanddesiccation.

Della-Bianca etal.25 reportedthatPE-2 cells exhibiteda superiorsurvivalrateundernon-proliferativeconditions,such aspH1.5,ascomparedtoregularlaboratoryorcommercial yeaststrains.AstudyconductedbyBassietal.26_demonstrated

thatPE-2wasonlyminimallyaffectedbycelltreatmentsbased onlowpHandorethanoladdition.

The microsatellite analysis was not successful to dis-tinguish between the colony phenotypes as phenotypic assays. Analysisby RFLP-mtDNAand electrophoretic kary-otyping(PFGE)werealsoineffectivetodistinguishthesmooth strain from the rough strain.11 _{Jubanyet al.}27 _{verified the}

polymorphisms at microsatelliteloci and singlenucleotide polymorphisms (SNPs)for molecular genotyping ofS.

cere-visiaestrainsandnocorrelationcouldbeestablishedbetween

pseudohyphal growth,flocculation and SNPs inFLO8 gene, which is requiredforflocculation and filamentous growth. Antonangelo28 hasnotalsofoundSNPs forthegenesFLO1,

FLO8andMSS11whensmoothandrough-colonystrainswere

analysed.Itmaysuggestthatdifferencesbetweenthecolony typesofS.cerevisiaemaybeattributedtodifferencesingene expressionratherthanDNAsequencesasobservedbefore.11

Thegeneticapproachalsofailedtorevealthepresenceof spe-cificmolecular markersthat couldbeused todiscriminate betweentheroughandsmoothcoloniesanddefinegenetic characteristicsassociatedwithcolonyphenotype.

ThepresenceofPE-2,astraindisplayingsmoothcolonies and dispersed cells, in close genetic proximity to rough-colony strains is intriguing. PE-2 and CAT-1 are the two most popular and relevant strains of S. cerevisiae used by the Brazilian distilleries.3 _{PE-2 is an extremely persistent}

and dominant yeast strain in the fermentation tanks sce-nario. This importantindustrial strain isnon-flocculant in pureculture.29_{AccordingtoBassoetal.,}3_{roughcolonieswere}

observedinselectedindustrialstrains(0–10%ofthecolonies foreachstrain),includinginPE-2,duringtheirimplantation intoethanol-producingunits.

There was no association between the microsatellite marker-based data and thestress resistanceprofile in dis-criminating between rough- and smooth-colony strains of

0.4 –2 –1 0 1 2 3 0.2 0.0 –0.2 –0.2 –2 –1 0 1 2 3 0.0 0.2 PC1 PC2 0.4 52 E 13 12 15 6 G(300)G(400) E(14.5) pH(1.25) PE–2 36 33 CAT–1 pH(1.5) E E(13.5) 4 35 7 19 9 S 8 10 G(500) 16 14 2 SA1 BG1 47 18 3

Fig.3–Principal-ComponentAnalysis(PC1andPC2)oftheyeaststrains(rough-colonystrainsincircles;smooth-colony strainsinsquares)andthephenotypiccharacteristics.Numbersinsidethecirclesandsquaresrefertothestraincodeasin

Table1.Thelegendsinthevectorsdesignatethephenotypiccharacteristicsandthenumberinparenthesesindicatethe values(pH1.25and1.5;Eforethanol,13,13.5,14,14.5%;Gforglucose,300,400and500g/L;Sforsedimentationrate,above 10%)ofthecharacteristic,asdescribedin“Materialandmethods”section.

S.cerevisiae.However,itwasinstrumentalinprovidingnew

insightsintotheprobableoriginsoftherough-colonystrains. Recentstudieshaveindicatedthatdisruptionofasinglegene,

ACE2,isresponsibleforthesnowflakephenotypeoftheyeast

strainY556_{aswellasintheindustrialyeaststrainPE-2.}14

How-ever,fortheroughandsmoothcolonystrainsherestudied, wedidnotobservethisfindingwhenACE2genewasassessed (datanotpublished).

Insummary,thisstudyrevealedthatthetwomajorgroups basedoncolonyphenotype(smoothandroughcolonies)inS

cerevisiaehadcleardifferentiationregardingstressresistance

tohighsugar andethanolconcentrations, lowpHandhigh cellsedimentationrate.Thegeneticprinciples(gene expres-sionpatterns aswellas sequence-levelvariationsingenes regardedtostressresistancetohighethanolandsugar con-centrations) that define and account for the rough-colony phenotypeinS.cerevisiaeposeanextremelyrelevantquestion thatdeservesathoroughanalysis.Itliesattheheartofan importantissueandneedstobeintensivelystudiedinorder toverifytheoriginsofthiscolonyphenotypewithrespectto bioethanolfermentation.Resultsofsuchastudymaydefine principlesthatcouldhelpusavoidingorminimisingthe occur-renceofrough-colonyformationinS.cerevisiae.

Conclusions

Themicrosatelliteanalysiswasnotsuccessfultodistinguish betweenthecolonyphenotypesasphenotypicassays.Unlike thesmoothcolonies,theroughcoloniesofS.cerevisiaeexhibit anenhancedresistancetostressfulconditionsashigh con-centrationsofglucoseand ethanolandhighsedimentation rate.TherelevantindustrialstrainPE-2wasobservedinclose geneticproximitytorough-colonyalthoughitdoesnotdisplay thiscolonymorphology.Auniquegeneticpatternspecifictoa particularphenotyperemainselusive.

Conflicts

of

interest

Theauthorsdeclarethattheyhavenoconflictofinterest.

Acknowledgments

ThisstudywassupportedbyFundac¸ãodeAmparoaPesquisa doEstadodeSãoPaulo(researchsupport2009/14617-4)and

Coordenac¸ãodeAperfeic¸oamentodePessoaldeNivelSuperior (fellowshiptothefirstauthor).

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r

e

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e

s

1. AmorimHV,LopesML,OliveiraJVC,BuckeridgeMS, GoldmanGH.Scientificchallengesofbioethanolproduction inBrazil.ApplBiochemBiotechnol.2011;91:1267–1275.

2. FidalgoM,BarralesRR,IbeasJI,JimenezJ.Adaptiveevolution bymutationsintheFLO11gene.PNAS.2006;103:11228–11233.

3. BassoLC,AmorimHV,OliveiraAJ,LopesML.Yeastselection forfuelethanolproductioninBrazil.FEMSYeastRes.

2008;8:1155–1163.

4. ReisVR,BassiAPG,SilvaJCG,Ceccato-AntoniniSR. CharacteristicsofSaccharomycescerevisiaeyeastsexhibiting roughcoloniesandpseudohyphalmorphologywithrespect toalcoholicfermentation.BrazJMicrobiol.2013;44:

1121–1131.

5. SoaresEV.FlocculationinSaccharomycescerevisiae:areview.J ApplMicrobiol.2010;110:1–18.

6. RatcliffWC,FankhauserJD,RogersDW,GreigD,TravisanoM. Originsofmulticellularevolvabilityinsnowflakeyeast.Nat

Commun.2015;6,http://dx.doi.org/10.1038/ncommms7102.

7. VopalenskáI,HulkováM,JanderováB,PalkováZ.The morphologyofSaccharomycescerevisiaecoloniesisaffectedby celladhesionandthebuddingpattern.ResMicrobiol.

2005;156(9):921–931.

8. VoordeckersK,MaeyerD,vanderZandeE,etal. Identificationofacomplexgeneticnetworkunderlying

Saccharomycescerevisiaecolonymorphology.MolMicrobiol.

2012;86(1):225–239.

9. Ceccato-AntoniniSR.Biotechnologicalimplicationsof filamentationinSaccharomycescerevisiae.BiotechnolLett.

2008;30:1151–1161.

10.RatcliffWC,DenisonRF,BorrelloM,TravisanoM. Experimentalevolutionofmulticellularity.PNAS.

2012;109(5):1595–1600.

11.LopesDD[M.Sc.Dissertation]Estudomolecularemorfológicode levedurasdeprocessosfermentativosdeproduc¸ãodeetanol.

Londrina,Brasil:UniversidadeEstadualdeLondrina;2010,58 pp.

12.KuthanM,DevauxF,JanderováB,SlaninováI,JacqC,Palková Z.DomesticationofwildSaccharomycescerevisiaeis

accompaniedbychangesingeneexpressionandcolony morphology.MolMicrobiol.2003;3:745–754.

13.CavalieriD,TownsendJP,HartlDL.Manifoldanomaliesin geneexpressioninavineyardisolateofSaccharomyces cerevisiaerevealedbyDNAmicroarrayanalysis.PNAS.

2000;97:12369–12374.

14.RodriguesA[Ph.D.thesis]Identificac¸ãoecaracterizac¸ãodegenes efatoresrelacionadosàfloculac¸ãoedesenvolvimentodeuma leveduraindustrialnãofloculante.Campinas,Brasil: UniversidadeEstadualdeCampinas;2013,165pp.

15.González-TecheraA,JubanyS,CarrauFM,GaggeroC. Differentiationofindustrialwineyeaststrainsusing microsatellitemarkers.LettApplMicrobiol.2001;33:71–75.

16.LegrasJL,RuhO,MerdinogluD,KarstF.Selectionof hypervariablemicrosatellitelociforthecharacterizationof

Saccharomycescerevisiaestrains.IntJFoodMicrobiol.

2005;102:73–83.

17.VaudanoE,Garcia-MorunoE.Discriminationof

Saccharomycescerevisiaewinestrainsusingmicrosatellite multiplexPCRandbandpatternanalysis.FoodMicrobiol.

2008;25:56–64.

18.AntonangeloATBF,AlonsoDP,RibollaPEM,ColombiD. Microsatellitemarker-basedassessmentofthebiodiversity ofnativebioethanolyeaststrains.Yeast.2013;30:307–317.

19.PerezMA,GallegoFJ,MartinezI,HidalgoP.Detection, distributionandselectionofmicrosatellites(SSRs)inthe genomeoftheyeastSaccharomycescerevisiaeasmolecular markers.LettApplMicrobiol.2001;33:461–466.

20.YoungET,SloanJS,vanRiperK.Trinucleotiderepeatsare clusteredinregulatorygenesinSaccharomycescerevisiae. Genetics.2000;154:1053–1068.

21.WhiteTJ,BrunsT,LeeS,TaylorJ.Amplificationanddirect sequencingoffungalribosomalRNAgenesfor

phylogenetics.In:InnisMA,GelfaudDH,SninskyJJ,WhiteTJ, eds.PCRProtocols:AGuidetoMethodsandApplications.San Diego:AcademicPress;1990:315–322.

22.NeiM.MolecularEvolutionaryGenetics.NewYork:Columbia UniversityPress;1987.

23.WangFZ,ShenW,RaoZM,FangHY,ZhanXB,ZhugeJ. Constructionofaflocculatingyeastforfuelethanol production.BiotechnolLett.2008;37:97–102.

24.RCoreTeam.R:ALanguageandEnvironmentforStatistical Computing;2012.Vienna.Availableat:

http://www.R-project.org.

25.Della-BiancaBE,HulsterE,PronkJT,MarisAJA,GombertAK. PhysiologyofthefuelethanolstrainSaccharomycescerevisiae

PE-2atlowpHindicatesacontext-dependentperformance relevantforindustrialapplications.FEMSYeastRes.

2014;14:1196–1205.

26.BassiAPG,SilvaJCG,ReisVR,Ceccato-AntoniniSR.Effectsof singleandcombinedcelltreatmentsbasedonlowpHand highconcentrationsofethanolonthegrowthand fermentationofDekkerabruxellensisandSaccharomyces cerevisiae.WorldJMicrobiolBiotechnol.2013;29:1661–1676.

27.JubanyS,TomascoI,PoncedeLeonI,etal.Towardaglobal databaseforthemoleculartypingofSaccharomycescerevisiae

strains.FEMSYeastRes.2008;8:472–484.

28.AntonangeloATBF[Ph.D.thesis]Genotipagemdeleveduras presentesnoprocessoindustrialdeproduc¸ãodeálcoolcombustível eestudodopolimorfismodegenesenvolvidosnoprocesso fermentativoemSaccharomycescerevisiae.Botucatu,Brasil: UniversidadeEstadualPaulistaJuliodeMesquitaFilho;2012, 83pp.

29.Carvalho-NettoOV,CarazolleMF,MofattoLS,etal.

Saccharomycescerevisiaetranscriptionalreprogrammingdue tobacterialcontaminationduringindustrialscale

bioethanolproduction.MicrobCellFact.2015;14:13.

30.FieldD,WillsC.Abundantmicrosatellitepolymorphismin

Saccharomycescerevisiae,andthedifferentdistributionsof microsatellitesineightprokaryotesandS.cerevisiae,result fromstrongmutationpressuresandavarietyofselective forces.PNAS.1998;95(4):1647–1652.

31.RichardsK,GoddardM,GardnerR.Adatabaseof

microsatellitegenotypesforSaccharomycescerevisiae.Antonie vanLeeuwenhoek.2009;96:355–359.