inivoranswww.avtt3.com
www.avtt3.com 时间:2021-04-09 阅读:()
BioMedCentralPage1of10(pagenumbernotforcitationpurposes)MicrobialCellFactoriesOpenAccessReviewProcessdevelopmentinHansenulapolymorphaandArxulaadeninivorans,are-assessmentChristophStckmann1,MarcoScheidle1,BarbaraDittrich2,ArminMerckelbach3,GritHehmann3,GeorgMelmer3,DorisKlee2,JochenBüchs1,HyunAhKang4andGerdGellissen*3Address:1InstituteofBiochemicalEngineering,RWTHAachenUniversity,WorringerWeg1,52074Aachen,Germany,2InstituteofTextileandMacromolecularChemistry,RWTHAachenUniversity,Pauwelsstr.
8,52074Aachen,Germany,3PharmedArtisGmbH,Forckenbeckstr.
6,52074Aachen,Germanyand4DepartmentofLifeSciences,ChungAngUniversity,221Heukseok-dong,Dongjak-gu,156-756Seoul,KoreaEmail:ChristophStckmann-stoeckmann@biovt.
rwth-aachen.
de;MarcoScheidle-marco.
scheidle@avt.
rwth-aachen.
de;BarbaraDittrich-dittrich@itmc.
rwth-aachen.
de;ArminMerckelbach-armin.
merckelbach@pharmedartis.
de;GritHehmann-grit.
hehmann@pharmedartis.
de;GeorgMelmer-georg.
melmer@pharmedartis.
de;DorisKlee-klee@itmc.
rwth-aachen.
de;JochenBüchs-jochen.
buechs@avt.
rwth-aachen.
de;HyunAhKang-hyunkang@cau.
ac.
kr;GerdGellissen*-gerd.
gellissen@pharmedartis.
de*CorrespondingauthorAbstractArangeofindustrialH.
polymorpha-basedprocessesexist,mostofthemfortheproductionofpharmaceuticals.
TheestablishedindustrialprocessesleanontheuseofpromotersderivedfromMOXandFMD,genesofthemethanolmetabolismpathway.
InHansenulapolymorphathesepromotersarede-repressedupondepletionofarangeofcarbonsourceslikeglucoseandglycerolinsteadofbeinginducedbymethanolasreportedforothermethylotrophs.
Duetothesecharacteristicsscreeningandfermentationmodeshavebeendefinedforstrainsharbouringsuchexpressioncontrolelementsthatleanonalimitedsupplementationofglycerolorglucosetoaculturemedium.
ForfermentationofH.
polymorphaasyntheticminimalmedium(SYN6)hasbeendeveloped.
NoindustrialprocesseshavebeendevelopedsofarbasedonArxulaadeninivoransandonlyalimitedrangeofstrongpromoterelementsexists,suitableforheterologousgeneexpression.
SYN6originallydesignedforH.
polymorphaprovidedasuitablebasisfortheinitialdefinitionoffermentationconditionsforthisdimorphicyeast.
Characteristicslikeosmo-andthermotolerancecanbeaddressedforthedefinitionofcultureconditions.
HansenulapolymorphaandArxulaadeninivoransandtheircompetitiveenvironmentInthelastthreedecadesawiderangeofrecombinantpro-teins,especiallypharmaceuticals,havebeenproducedbasedonheterologousgeneexpressioninbacterialorgan-isms,mammaliancellsandseveralyeastsandfungi[1-3].
Productionprocesseshadtobedevelopedthatemployplatformswhichmeetboth,thedemandforefficientmassproductionandcriteriaofsafetyandauthenticityoftheproducedcompounds.
Inthisrespectyeastsofferconsid-erableadvantagesoveralternativemicrobialandmamma-liancellsystemsinprovidinglow-costscreeningandproductionsystemsforauthenticallyprocessedandmod-ifiedproteins.
Theorganismsmeetsafetyprerequisitesinthattheydonotharbourpyrogens,pathogensorviralinclusions[4,5].
RecentengineeringofyeasthostswithPublished:15April2009MicrobialCellFactories2009,8:22doi:10.
1186/1475-2859-8-22Received:7January2009Accepted:15April2009Thisarticleisavailablefrom:http://www.
microbialcellfactories.
com/content/8/1/222009Stoeckmannetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page2of10(pagenumbernotforcitationpurposes)thecapabilitytoaddhumanizedN-glycansoftheinter-mediatemannosetype[6]oreventhecomplextype[7]providestheoptiontoproducebiopharmaceuticalswithhumanproteinmodifications.
Therecognitionofyeastsasattractiveexpressionplatformsforbiopharmaceuticalsismetbygenomeanalysisofanincreasingnumberofyeastspecies,amongothersthatofSaccharomycescerevisiae[8]andHansenulapolymorpha[9].
AsaconsequencesomeearlyexamplesofFDA-approvedbiopharmaceuticalslikeinsulin[2]andhepatitisBvac-cines[10,11]havebeenproducedinthebaker'syeastS.
cerevisiae.
However,certainlimitationsanddrawbacksareencounteredwhenusingthissystem:S.
cerevisiaetendstohyperglycosylaterecombinantproteins;N-linkedcarbo-hydratechainsareterminatedbymannoseattachedtothechainviaanα1,3bond,whichisconsideredtobealler-genic.
Thelimitedcarbonsourceutilizationimposesrestrictionsonthedesignoffermentationprocesses;duetothepreferentialuseofepisomalvectorsinstabilitiesofrecombinantstrainsandasaresultbatchinconsistenciesofproductionrunsareofmajorconcern[12].
Thereforeanincreasingnumberofalternativeyeastsys-temshavebeendefinedthatcanpotentiallyovercomethedescribedlimitationsofthetraditionalbaker'syeast.
Theavailabilityofawide-rangeyeastvectorsystem(CoMed)enablestheassessmentofseveralyeastsinparallelfortheircapabilitytoproduceaparticularproteinindesiredqualitywithasinglevectortoidentifyanoptimalhostatthebeginningofaproductandprocessdevelopment.
Forexpressioncontrolthewide-rangevectorcontainsacon-stitutiveTEF1promoterderivedfromvarioussourcesthatisactiveinallyeastspeciesanalyzedsofar.
Ifneededthispromoterelementcaneasilybesubstitutedduringfurtherstraindevelopmentbyapromoteroptimalforthedefinedplatform[[13],Additionalfile1].
Outoftheplethoraofaddressablespecieswedescribeinthisarticlemethylo-trophicH.
polymorpha,arecognizedproducerofbiophar-maceuticalsandotherrecombinantproteins,anddimorphicArxulaadeninivorans,anovelplatformthathasyettoestablishitselfforindustrialapplications.
FirstexperimentsindicatethatscreeningandfermentationconditionsbasedonminimalSYN6medium(SYN6)withglucosesupplementationasdescribedinthisarticlecanalsobeappliedtoyeastplatformsothersthanH.
polymor-phaandA.
adeninivorans.
H.
polymorpha(Pichiaangusta)belongstoalimitednumberofyeastspeciesthatareabletoutilizemethanolasasoleenergyandcarbonsource.
Twooutofthreebasicstrainswithunclearrelationships,differentfeatures,andindependentoriginsarebiotechnologicallyapplied:strainCBS4732(CCY38-22-2,ATCC34438,NRRL-Y-5445)andDL-1(NRRL-Y-7560,ATCC26012)andauxotrophicderivativesthereof[14,15].
AmicrographofaH.
polymor-phacellpreparedfromachemostatwithamethanolfeedisshowninFig.
1a.
TherangeofbiotechnologicallyappliedmethylotrophicyeastsfurthermoreincludesCandidaboidinii,Pichiameth-anolica,andPichiapastoris[16].
Inallinstancesmostexamplesofheterologousgeneexpressionarelinkedtostrongandadjustablepromotersderivedfromgenesofthemethanolutilizationpathway[12,17],mostcom-monlytheelementsderivedfromthealcoholoxidasegenes,namelyAOX1fromP.
pastoris[16-18],MOXfromH.
polymorpha[14,17];AOD1fromC.
boidinii[16,17,19]andAUG1(nowdesignatedMOD1)fromP.
methanolica[16,17,20].
InH.
polymorphatheFMD(formatedehydro-genase)promoter,derivedfromanothermethanolutiliza-tionpathwaygeneofsimilarregulation,hasfoundpreferentialapplicationtoestablishedindustrialprocesses[14,16].
A.
adeninivorans(Blastobotrysadeninivorans)isayeastwithunusualcharacteristics.
Itisadimorphicspeciesandcanutilizeadenine,xanthine,uricacid,putrescineandn-alkylaminesascarbon,nitrogenorenergysourcesinaddi-tiontoglucose.
LikeH.
polymorphaitisanitrate-assimilat-ing,thermo-andosmotolerantorganism.
Adistinctivefeatureisatemperature-dependentdimorphismwithmycelialstructuresformedattemperaturesabove42°C[5,12][Fig.
1b,c].
ForFe(II)-oxidaseAfet3p,O-glycosyla-tionwasobservedtoberestrictedtothebuddingcellsta-tus[21].
ItremainstobeshownwhetherthisdifferentialO-glycosylationpatternincorrelationtothemorphologi-calstatusisalsopresentinrecombinantandotherhostproteins.
Again,severalstrainshavebeenidentifiedafteritsfirstdescriptionasTrichosporonadeninovorans[22].
Mostoftheresearchandthebiotechnologicalapplica-tionshavebeenperformedwithstrainLS3(PAR-4),iso-latedinSibiriabyKapultsevich,andarangeofauxotrophicmutantshavebeengenerated[5,12].
Strain135isamutantthatformsmycelialstructuresat30°C[23].
RecentlyauxotrophichoststrainsforheterologousgeneexpressionhavebeengeneratedbasedonstrainsCBS7350andCBS1738(seeTab.
1).
Sofar,noindustrialA.
adeninivorans-basedprocessexists.
ForexpressionandfermentationstudiesonalaboratoryscaleheterologousgenesweremostlyexpressedundercontrolofTEF1,acon-stitutiveA.
adeninivorans-derivedpromoterofappropriatestrength[5].
FordescriptionweselectedestablishedH.
polymorpha-basedprocesseswithstrainsexpressingaheterologousgeneundercontroloftheadjustableFMDandMOXpro-motersandA.
adeninivorans-basedlabscaleprocesseswithstrainsexpressingaheterologousgeneundercontroloftheconstitutiveA.
adeninivorans-derivedTEF1promoter,MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page3of10(pagenumbernotforcitationpurposes)withculturingconditionsthatcanpossiblybeappliedtotheassessmentofotheryeastswithconstitutiveheterolo-gousgeneexpression.
MicrographsofthetwoselectedplatformsareshowninFig.
1.
H.
polymorpha-basedprocessesundercontrolofMOXandFMDpromoters–are-assessmentMOXandFMDaregenesencodingenzymesofthemeth-anolutilizationpathwaythatissharedbyallmethylo-trophicyeasts.
Theenzymecomponentsofthispathwayandtheircontrolhavebeenreviewedextensivelyintherecentpast[4,17,19].
Thegenesofthispathwayaredescribedtobetightlyregulated;theyarehighlyrepressedinthepresenceofnon-limitingconcentrationsofglucoseandstronglyinducedifmethanolisusedasacarbonsource[17].
Methylotrophicgrowthisfurthermoreaccompaniedbyamassiveproliferationofperoxisomesinwhichseveralmethanol-metabolizingenzymesarecom-partmentalized[19,24].
However,itsoonbecameevidentthatactivationofmethanolpathwaypromotersdidnotdependonthepresenceofmethanolinH.
polymorphaincontrasttothesituationintheothermethylotrophs[16].
Forallothermethylotrophicyeastspeciesaninductiveactivationofsuchpromotershasbeenstatedthatisstrictlydependentonthepresenceofmethanol[17].
Asaconse-quenceseveralH.
polymorpha-basedindustrialfermenta-tionprocesseshavebeendefinedthatleanonglucoseorglycerolsupplementationinsuitableconcentrationstoaculturebrothwithoutanymethanoladditions[1,25,26].
ThedistinctfeatureoftheH.
polymorpha-derivedmetha-nolpathwaypromoterswaselucidated,whennewtoolsofgenomicsandpostgenomicanalysisbecameavailable.
AftersequencingtheentiregenomeofstrainCBS4732[9]acDNAmicroarraywasconstructedthatallowedcompre-hensivegeneexpressionprofiling[27,28].
Whenanalyz-ingthetranscriptomeofH.
polymorphastrainsofglucose-supplementedgrowthandaftertransitiontomethanol-supplementedgrowthitbecameevidentthatthemetha-noldissimilationgenesincludingMOXandFMDareacti-vatedbyde-repressionuponcarbonsourcelimitationanddepletionandnotuponinductionbymethanol.
Incon-trastgenesofperoxisomebiogenesisandproliferationareinducedbymethanol[14,15].
Withrespecttothesefindings,recombinantH.
polymorphastrainsexpressingaGFPreportergeneundercontroloftheFMDpromoterwerescreenedapplyingglucose-orglyc-erol-supplementedmediatostrainculturing.
Highthroughputscreeningexperimentsareusuallyper-formedinabatch-mode[29].
Jeudeetal.
describedasys-temfortheslowreleaseofglucosefromasiliconeelastomerematrixinshakeflask[30].
Theyshowedtheadvantageofusingfed-batchcultivationsincontrasttoMicroscopyanalysisofHansenulapolymorphaandArxulaaden-inivoranscellsFigure1MicroscopyanalysisofHansenulapolymorphaandArxulaadeninivoranscells.
aH.
polymorphacellgrowninamethanol-supplementedchemostat(courtesybyM.
Veen-huis,Groningen).
Underthesegrowthconditionsprolifera-tionofperoxisomesisinduced.
bA.
adeninivoranscellsgrownat37°C.
cA.
adeninivoranscellsgrownat42°C.
DimorphicA.
adeninivoransgrowsinfilamentousformsabove42°C.
abcMicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page4of10(pagenumbernotforcitationpurposes)batchcultivationsinsmallscale,whereupto420-foldincreasedGFPproductionwasreached[30].
Theslow-releasetechniquewastransferredtoadeep-well-platefor-mat.
Intheseplatestheglucose-containingsiliconematrixisfixedatthebottomofeachwell.
Hence,96parallelfed-batch-cultivationscanbeperformed.
AcomparativescreeningwithdifferentH.
polymorphapC10-FMD(PFMD-GFP)strains(strainsofaCBS4732background)[31]wasperformedinbatchmodewithglycerolandglucosesup-plementationandinfed-batchmodewithglucosesupple-mentation[Fig.
2a].
ThecomparisonatteststhatcloneswithanFMDpromoter-controlledgeneexpressioncanbescreenedinsmallvolumesofmediasupplementedwithglycerolorglucose.
Thissupportsthatmethanolpathwaypromotersareactivateduponglycerolandglucosedeple-tionassuggestedbythetranscriptomeanalysis.
Interest-inglysomeclonesofmaximalproductivityidentifiedunderconditionsofglucosestarvation(fed-batchcondi-tions)arenotidenticaltothoseidentifiedundercondi-tionofglycerolstarvation(batchconditions).
Thereasonsforthisphenomenonremainobscure.
MOXpromoter-drivenGFPexpressionunderglucosestarvationcondi-tionswascorroboratedforrecombinantstrainsofaDL-1backgroundculturedinadifferentmediumandonalargerscaleasshownbyfluorescenceanalysisinFig.
2b.
Thecommercialsuccessofarecombinantproductdoesnotdependonlyonthecharacteristicsofthemicrobialhostbutalsotoalargeextentonthedefinitionofefficientfermentationprocesses.
IncaseofH.
polymorpha,adefinedminimalmineralmediumhasbeendevelopeddesignatedSYN6[25,32].
Itiscomposedofsalts,vitaminsandtraceelementstosupportgrowthtohighcelldensitiesandithastobeadjustedtoappropriatepHconditionsandhastobesupplementedwithsuitablecarbonsources(fordetailsofcomponentssee[32]).
ThefermentationstrategyinpreviousestablishedprocessdevelopmentswithFMDorMOXpromoter-drivenproductionreliedongrowthusingeitherglucoseorglycerolinthebeginningoffermentation,followedbycarbonsourcelimitationsinasecondphase.
Therespectivefermentationparametersforthesupplementationofglucoseorglycerolhavebeendefinedpriortotheelucidationofthetranscriptomepro-filedescribedbefore.
Inasingleprocessexamplethefer-mentationbrothwassupplementedinalatephasewithmethanol.
ThethreedifferentfermentationmodesareschematicallydepictedinFig.
3a–c.
Anearlyexampleforaglycerolstarvationprocessistheproductionprocessforhirudin[33-35][Fig.
3a].
Asinallotherdescribedprocessesexpressionvectorswerecon-structedtotransformtheuracil-auxotrophicstrainRB11,likestrainMedHp1(Tab.
1)aderivativeofstrainLR9[36-38].
ThevectorscontainanexpressioncassettewithahirudinsequenceundercontroloftheMOXpromoter.
Forsecretionitwasfusedtoasecretionleader,derivedfromthepheromoneprecursorMFα1p[33].
Fermenta-tionona35literscalewascarriedoutat30°CinSYN6atpH5.
Itwasstartedwith30g/Lofglycerol.
Aftercon-sumptionofthecarbonsourceafter35hoursafeedwasinitiatedthataddedglycerolbyapO2-controlledfeedingdevice.
Hirudinproductionstartedaftersome20hourswhentheMOX-promoterwasactivatedbyde-repressionunderglycerollimitation.
Thesubsequentfeedingcondi-tionssupportedthede-repressedstatusofthepromoterbymaintainingglycerolconcentrationsbetween0.
5and3.
0g/L.
Thesefeedingconditionsresultedinanincreasingaccumulationoftheproductinthemedium[33,34,39][Fig.
3a].
Similarfermentationconditionswereappliedtoculturingofproductionstrainsfortheanticoagulantsaratin[35,39]orforaprotinin[37],nowapplyingtheFMDpromotertoexpressioncontrol.
Thisfermentationdesignwasmodi-fiedwhendevelopingaproductionprocessforthecytokineIFNalpha-2a[40,41].
IFNalpha-2aformsadisulfidebondbetweenaminoacidsCys1andCys98.
BondformationofthefirstaminoacidCys1ofthematuresequenceprovidesasterichindranceforcorrectmatura-tionwhenprocessedfromanMFα1/IFNalpha-2aprecur-sor.
AccordinglyalargeshareofsecretedrecombinanthirudinconsistedofincorrectlyprocessedmoleculeswithN-terminalextensions.
Thiscouldbeovercomebyco-pro-ductionoftheprocessingenzymeKex2p,howeverattheexpenseofamorepronouncedproteolyticdegradation.
TominimizethisdegradationpHwasloweredfrompH5(asappliedtostandardfermentations)topH2–3.
TheglycerolstarvationconditionsforFMDpromoterde-repressionremainedunchanged.
Fortheproductionofphytaseanextremelyefficientpro-ductionprocesshasbeendeveloped.
Inthisprocessallstepsandcomponentsoftheprocessfollowedarationaleofefficiencyandcost-effectiveness.
Thisrationalepro-vokedanassessmentofglucoseassolecarbonsourceforfermentation[42].
Inafermentationofaphytaseproduc-tionstrainwithFMD-controlledexpressionon2000Lscaleglucosewassupplementedas20g/L.
Upondeple-tion,aglucose-limitingfeedwasinitiatedthataddedthecarbonsourcewithastepwiseincreasingfeedingrateincorrelationtothecellmass.
Inthisglucosestarvationprocessafinalyieldof13.
5g/Lphytasewasobserved[42,43][Fig.
3b].
TheonlyestablishedindustrialfermentationprocesswithmethanolsupplementationisthatfortheproductionofthehepatitisBsurfaceantigenHBsAg,thefirstbiophar-maceuticalproducedinH.
polymorpha.
Severalprocessesforthisvaccinehavebeendescribedthatarebasedonboth,MOXorFMD-controlledexpression[11].
InFig.
3cMicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page5of10(pagenumbernotforcitationpurposes)atypicalfermentationprocessisschematicallyshown.
Thebatchphaseandafirstfed-batchphaseissimilartotheexamplesofglycerolstarvationdescribedbefore[Fig.
3a].
Incontrastamixtureofglycerolandmethanolisfedduringthelasthoursoffermentation.
ObviouslythisresultsinaninductiveincreaseofHBsAgproduction.
However,inviewofthetranscriptomeprofilingitseemsthatmethanolsupplementationdoesnotinducetheFMD-controlledexpressionoftheheterologousantigengene,butitinducesmembraneproliferation.
AstheHBsAgisproducedasparticleswiththerecombinantanti-geninsertedintohost-derivedmembranes,methanolisconsideredtoprovideabalancedco-productionofbothparticlecomponentsinhightiters[10,11].
TheselectedprocessexamplesdemonstratethepossibilitytodevelopefficientscreeningandfermentationprocessesforstrainswithMOX-orFMD-drivenheterologousgeneexpressionwithoutmethanolsupplementationtoamedium.
CulturingofArxulaadeninivoransstrainsForA.
adeninivoransindustrialprocessparametershavenotbeendefinedyetandmainlycultivationsonashakeflaskscalehavebeencarriedoutsofar.
MostofthecurrentexpressionstudiesarebasedonwildtypestrainLS3[44]oritsleucine-auxotrophicderivativeG1211.
Additionalstrainsandleucine-auxotrophicmutantsthereofhavebeenestablishedmorerecently(seeTab.
1).
Acidphosphataseproductionwascharacterizedinfer-mentationsofbothstrainLS3andarecombinantstrainexpressingtheAPHO1geneundercontrolofthestrongTEF1promoter[45].
UsingthePlackett-Burmandesignthreevariables(pH,sucroseconcentrations,andpeptoneconcentration)wereoptimizedformediumcomposition,aroughlyfourtimesenhancementwasobservedinmediacontaining39g/Lsucroseand16g/LpeptoneatpH3.
8[46].
ShakeflaskculturesofA.
adeninivoransstrainswereana-lyzedusingadeviceforonlinemeasurementoftherespi-rationrates(RAMOS,respiratoryactivitymonitoringsystem)[47,48].
ThisdevicehadpreviouslybeenappliedtotheanalysisofH.
polymorphaculturesandtoalternativeplatforms[49-52].
Inafirstseriesyeastminimalmedium(YMM)wasassessed.
Priortoanypracticalexperiment,YMMammo-niumconcentrationofthestandardmediumwasraisedfrom2.
2mmolN/gglucoseto4.
6mmolN/gglucosesincetheoreticalmaterialbalancingrevealedaseverelackofnitrogenwithregardtotheaveragenitrogencontentofyeast.
TheculturebrothwasadditionallysupplementedwithcalciumandironinhigherconcentrationsaswellasMethanolpathwaypromoter-controlledGFPproductioninrecombinantH.
polymorphastrainsFigure2Methanolpathwaypromoter-controlledGFPproductioninrecombinantH.
polymorphastrains.
A.
ScreeningofCBS4732-derivedtransformantsproducingGFPundercontroloftheFMDpromoter.
Screeningof44differentH.
polymorphapC10-FMD(PFMD-GFP)clonesculturedunderdifferentcultivationmodes;screeningexperimentswereperformedindeep-well-platesforbatchandinFeedBead96-platesforfed-batchcultiva-tion.
Gaspermeablesealingswereusedassterileclosures.
Toreducetheevaporationduringthecultivation,water-saturatedairwasusedforgassing.
Tocomparetheresultsfromthedifferentcultivationmodesanidenticalcarbonsourceconcentrationof16.
63g/Lwereappliedtoeachfermentation(batchwithglycerol,batchwithglucose,fed-batchwithglucose).
Thebatchcultivationswerestoppedwhenthestationarygrowthphase(glycerol21h,glucose16h)wasreachedandthefed-batchcultivationswerestoppedwhen16.
63g/LglucosewerereleasedfromtheFeadBeat96-plate(16h).
Drycellweight(DCW)wascalculatedfromopticaldensitymeasurementsinaPowerWave*340microtiterplatereader.
GreenFluorescentProtein(GFP)wasmeas-uredat485nmexcitationand520nmemissionwavelengths.
Cul-tivationconditions:shakingfrequency400rpm,shakingdiameter50mm,temperature37°C,fillingvolumes300μLperwell;media:SYN6-MESwith16.
63g/Lofdifferentcarbonsources.
Carbonsources:batchmodewithglycerol(blackline),batchmodewithglucose(greybars),fed-batchmodewithglucose(blackbars);inocularatio1:30.
B.
GFPproductioninaDL-1-derivedtrans-formantundercontroloftheMOXpromoter.
H.
polymorphatransformantsproducingGFPundercontrolofMOXpromoterwereculturedfor12honYPmediumcontaining10g/Lmethanol(YP+1%M)or0.
5g/Lglucose(YP+0.
05%D)andthenanalyzedbyconfocalmicroscopy.
aScreeningof44FMD-clones036DCW[g/L]024GFP[g/L]0481216202428323640440.
00.
40.
8FMD-clonesspec.
productyieldGFP/DCW[g/g]b12hrYP+1%M12hrYP+0.
05%DGFPMOX(p)HARSAmprReportervectorMicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page6of10(pagenumbernotforcitationpurposes)A,B,C:SchematicdepictionofH.
polymorpha-basedfermentationprocessesFigure3A,B,C:SchematicdepictionofH.
polymorpha-basedfermentationprocesses.
Thefiguresaremodifiedversionsoffiguresfrompreviouspublications.
Forfurtherdetailsseetext.
afermentationofahirudin-producingstrain(MOXpromoter-controlledexpression,glycerolstarvation),bfermentationofaphytase-producingstrain(FMDpromoter-controlledgeneexpression,glucosestarvation),cfermentationofaHBsAg-producingstrain(FMDpromoter-controlledexpression,glycerolstarvation,followedbyaglycerol/methanolfeed).
dottedline:drycellweight,solidline:glycerol(a,c)orglucose(b),chaindot-tedline:glucosefeedingrate(b)ormethanol(c),dashedline:productofa,bandcrespectively.
atime[h]drycellweightorglycerol[g/L]hirudin[%]110100020406080100growth01224364860de-repressiontime[h]drycellweightorglycerol[g/L]hirudin[%]110100020406080100growth01224364860de-repressionbglucosefeedingrate[g/h]orphytase[%]drycellweightorglucose[g/L]time[h]0244872961201441680startfeeding10100204060801001growthde-repressionglucosefeedingrate[g/h]orphytase[%]drycellweightorglucose[g/L]time[h]0244872961201441680startfeeding10100204060801001growthde-repressionctime[h]drycellweightorglycerolormethanol[g/L]hepatitisBsurfaceantigen[%]110100020406080100growthinductionde-repression01224364860time[h]drycellweightorglycerolormethanol[g/L]hepatitisBsurfaceantigen[%]110100020406080100growthinductionde-repression01224364860MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page7of10(pagenumbernotforcitationpurposes)withMES(2-[N-morpholino]ethanesulfonicacid)forbufferingtoresultinYMM*[25].
Despitenitrogenaddi-tiontoYMM,therespectiveculturesremainedlimitedasshownrepresentativelyforA.
adeninivoransLS3inFig.
4,blackcircles.
Initially,culturerespirationincreasedexpo-nentially,butwasthenlimitedto9mmol/(L*h)after13hours,andcontinuedtodeclineoverfermentationtime.
Finallyonlyca.
6.
6gdrycellweight(DCW)/Lwasobtained.
InfermentationsofstrainLS3inMES-bufferedYMM*therespirationrateexponentiallyincreasedfollowedbyalin-earincreasepresumablyindicatinganutrientdeficiency.
After24hours,respirationratedroppeduponglucosedepletion.
[Fig.
4,greycircles].
Subsequentlyglucose-supplementedSYN6wasassessedforapplicabilitytoshakeflaskculturesofA.
adeninivorans.
ThehighnutrientconcentrationsofstandardSYN6remainedunchanged.
Again,thepHofSYN6hadtobebufferedwithMES(SYN6-MES,see[25]fordetaileddescription)forpHstabilizationbetween6.
4and5.
3.
Fig.
4,opencirclesshowstherespirationrateofashakeflaskculturewithstrainLS3inSYN6-MES.
Thedescribedcul-turecourseadvertstothenon-limitedgrowthofA.
aden-inivoransintherespectiveshakeflaskculture.
Thus,favourablenon-limitinggrowthconditionsforA.
adenini-voransinshakeflaskswereproventobedeveloped.
Additionallysuitablefed-batchconditionsforhighcelldensityfermentations(HCDF)inSYN6werefound.
TheseconditionswereassessedforculturingarecombinantA.
adeninivoransstrainproducingphytaseundercontroloftheTEF1promoter[5,53].
Duringthefeedingunderglu-cose-limitationphytasewassecretedtomaximaltitresofca.
900FTU/mL(oneFTUequatestothephytaseamountliberating1μmolofinorganicphosphateperminuteatpH5.
5and37°C)[25].
Thus,thegrowthconditionsdefinedforshakeflaskculturesandHCDFofA.
adeninivo-ranswildtypestrainLS3,provedtobeapplicableforthephytase-producingrecombinantA.
adeninivoransstrain.
Finallythephytase-producingstrainwasculturedunderpressurizedconditionsina50LSTR,againusingaSYN6-derivedmediumforculturing.
Duringthefed-batchphasethereactorpressurewasincreasedstepwiseupto5bar.
After42hcellshadgrownupto224g/L.
PhytaseamountCoMedvectorsystemFigure5CoMedvectorsystem.
Thebasicvectorsarederivedfromstandardvectorswithanengineeredmultiplecloningsite(MCS)fortheuptakeofvariousmodules.
Initsbasicformallmodulesarefunctionalinallyeaststestedsofar.
Module1:ARS/CENsequencesfromvarioussources(optional);Module2:rDNAtargetingsequences(NTS2-ETS-18SrDNA-ITS1fromvarioussources);Module3:selectionmarkers(i.
e.
kanMX,hph,leu2,ura3andcombinationsthereof);Module4:expressioncassettesconsistingofaTEF1promoterfromvarioussources–cloningsite–terminator.
Thewide-rangeTEF1promotercaneasilybereplacedbyalternativestrongspecies-specificpromoterslikeMOXorFMD.
Forfurtherdetailsseetext.
Module1Module2Module3Module4Eco47IIISacIINotIBcuI(SphI)SalI(BsiWI)ApaIAmp(r)pCoMedTMbasicvectorII.
ColE1oriI.
f1(-)originAssessmentofminimalmediaforArxulaadeninivoransLS3bymeasuringrespirationratesinshakeflaskcultures(foroper-atingconditionsandmediaformulationsee[25])Figure4AssessmentofminimalmediaforArxulaadeninivo-ransLS3bymeasuringrespirationratesinshakeflaskcultures(foroperatingconditionsandmediaformu-lationsee[25]).
OriginalYMM(blackcircles),containingpoorconcentrationsofcalcium(0.
338mg/L)andiron(0.
041mg/L),ledtopoorculturerespirationratesbelow10mmol/(L*h).
ModifiedYMM*(greycircles)ispH-bufferedwithMESandcontainshigherconcentrationsofcalcium(272.
8mg/L)andiron(2.
1mg/L),thusleadingtoamorevitalcultivationasshownbythehigherrespirationrateofupto25mmol/(L*h)andacondensedcultivationtimeof24hours(seedropofrespiration,signallingthedepletionofthecarbonsourceglu-cose).
SYN6-MES(opencircles),containingexceedingnutri-entquantities,providednon-limitedgrowthofthecultureasshownbythedistinctexponentialincreaseofrespirationto45mmol/(L*h)andanearlyceaseofrespirationafteronly15hours,againsignallingthedepletionofcarbonsource.
01020304050012243time[h]respirationrate[mmol/(L*h)]6MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page8of10(pagenumbernotforcitationpurposes)increasedupto10*106FTU.
Fermentationsunderpres-surizedconditionsmayresultinincreasedproductyieldsandshorterfermentationtime[54].
Theconditionsofculturingcanpotentiallybeappliedtoscreeningandculturingofotheryeastsexpressingafor-eigngeneundercontrolofaconstitutiveTEF1promoter–akeyelementoftheCoMedsystemdescribedinthefol-lowingsection.
TheCoMedsystemWhileallestablishedexpressionsystemsaredistinguishedbycertainfavorablecharacteristics,itisevidentthatnosinglesystemisoptimalforallproteins.
Aninitialselec-tioncanresultincostlytime-andresource-consumingfailures.
Itisthusadvisabletoassessseveralplatformcan-didatesinparallelforcriteriasuchasproductivity,appro-priateprocessingandmodification.
ProductionofinterleukinIL-6invariousyeastplatformshasrecentlybeendescribedasastrikingexampleforthenecessityofacomparativeevaluationofseveralyeasts.
Correctprocess-ingfromanMFα1/IL-6precursorwasobservedinA.
aden-inivoranswhereasN-terminallytruncatedmoleculesweresecretedfromS.
cerevisiaeandH.
polymorphahosts[55].
Anovelyeast/vectorsystemprovidesaversatiletooltoaddresssimultaneouslywithasinglevectorarangeofyeastslikethetwodescribedbefore,namelyHansenulapolymorphaandArxulaadeninivorans,andotherslikeSac-charomycescerevisiae,Pichiapastoris,Kluyveromyceslactis.
Initsbasicformthevectoriscomposedofgeneticmodulesthatarefunctionalinallyeasts,namelyanrDNAtargetingsequence,anappropriateselectionmarkerandanexpres-sioncassetteundercontrolofaTEF1promoterfromvari-oussources.
TheCoMedsystemhasrecentlybeendescribedandsomeapplicationexampleshavebeenpro-vided[13,55-57].
ThebasicdesignofthevectorandaselectionofaddressableyeastspeciesareshowninFig.
5andAdditionalfile1.
Itisdesirablethattherangeofyeastsaddressedinparallelcanbealsoassessedinparallelforoptimalperformanceinagivencase.
FirstexperimentsindicatethatSYN6andderivativesthereofaresuitableminimalmediaforyeastsothersthanA.
adeninivoransandH.
polymorpha.
Thegen-eraluseoftheconstitutiveTEF1promoterisexpectedtoensurescreeningandfermentationconditionssimilartothosedescribedforH.
polymorphaandA.
adeninivorans.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsCS,MS,BD,DKandJBcontributedtothereviewedandnewdataonscreeningunderglucoselimitationsandonthefermentationdesignforArxulaadeninivoranscultures,HKistheprincipalscientistfortheHansenulapolymorphamicroarrayandshecontributedthedataonGFP-produc-tioninDL-1underglucosefermentation.
AM,GH,GMandGGperformedtheworkontheCoMedsystem.
GGwasaprojectpartnerinthemicroarrayworkandpartici-patedintheprojectsandthepublicationsonHansenulapolymorphareviewedinthismanuscript.
Allauthorsreadandapprovedthemanuscript.
AdditionalmaterialAcknowledgementsHAKangwassupportedbyagrantfromtheKoreanMinistryofScienceandTechnology(MicrobialGenomicsandApplicationsResearchandDevelop-mentProgram).
ThecontentofthisreviewhasbeenpresentedattheconferenceBiotech2008:Biopharmaceuticals:WhyyeastsWdenswil,Switzerland.
May232008.
References1.
GellissenG:Productionofrecombinantproteins:NovelmicrobialandeukaryoticexpressionsystemsWeinheim:Wiley-VCH;2005.
2.
MelmerG:Biopharmaceuticalsandtheindustrialenviron-ment.
InProductionofrecombinantproteins:novelmicrobialandeukary-oticexpressionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:361-383.
3.
YinJ,LiG,ReuX,HerlerG:Selectwhatyouneed:acompara-tiveevaluationoftheadvantagesandlimitationsoffre-quentlyusedexpressionsystemsforforeigngenes.
JBiotechnol2007,127:335-347.
4.
GellissenG,KunzeG,GaillardinC,CreggJM,BerardiE,VeenhuisM,KleiIvander:Newyeastexpressionplatformsbasedonmeth-ylotrophicHansenulapolymorphaandPichiapastorisanddimorphicArxulaadeninivoransandYarrowialipolytica–acomparison.
FEMSYeastRes2005,5:1079-1096.
5.
BerE,GellissenG,KunzeG:Arxulaadeninivorans.
InProductionofrecombinantproteins:novelmicrobialandeukaryoticexpressionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:89-110.
6.
KimMW,KimEJ,KimJY,ParkJS,OhDB,ShimmaJI,ChibaY,JigamiY,RheeSK,KangHA:FunctionalcharacterizationoftheHansenulapolymorphaHOC1,OCH1,andOCR1genesasmembersoftheyeastOCH1mannosyltransferasefamilyinvolvedinproteinglycosylation.
JBiolChem2006,281:6261-6272.
7.
HamiltonSR,DavidsonRC,SethuramanN,NettJH,JiangY,RiosS,BobrowiczP,StadheimTA,LiH,ChoiBK,HopkinsD,WischnewskiH,RoserJ,MitchellT,StrawbridgeRR,HoopesJ,WildtS,GerngrossTU:Humanizationofyeasttoproducecomplexterminallysialylatedglycoproteins.
Science2006,313:1441-1443.
8.
GoffeauA,BarrellRG,BuseyH,DavisRW,DujonB,FeldmannH,GalibertF,HoheiselJD,JacqC,JohnstonM,LouisEJ,MewesHW,MurakamiY,PhilippsenP,TettelinH,OliverSG:Lifewith6000genes.
Science1996,274:563-567.
9.
Ramezani-RadM,HollenbergCP,LauberJ,WedlerH,GriessE,Wag-nerC,AlbermannK,HaniJ,PiontekM,DahlemsU,GellissenG:TheAdditionalfile1Table1ComponentsoftheCoMedsystem.
Thetablecontainsaselec-tionofgeneticcomponentsandyeaststrainsoftheCoMedstrain/vectorsystem.
Clickhereforfile[http://www.
biomedcentral.
com/content/supplementary/1475-2859-8-22-S1.
doc]MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page9of10(pagenumbernotforcitationpurposes)Hansenulapolymorpha(strainCBS4732)genome–sequenc-ingandanalysis.
FEMSYeastRes2003,4:207-215.
10.
BrockeP,SchaeferS,MelberK,JenzelewskiV,MuellerF,DahlemsU,BartelsenO,ParkKN,JanowiczZA,GellissenG:HepatitisBvac-cines–diseasecharacterizationandvaccineproduction.
InProductionofrecombinantproteins:novelmicrobialandeukaryoticexpres-sionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:319-60.
11.
MelmerG,KunzeG,GellissenG:Recombinantvaccineproduc-tioninyeast.
BiopharmInternJanuarySuppl2008:8-13.
12.
BerE,SteinbornG,GellissenG,KunzeG:Productionofinter-leukin-6inArxulaadeninivorans,HansenulapolymorphaandSaccharomycescerevisiaebyapplyingawide-rangeyeastvec-tor(CoMed)systemtosimultaneouscomparativeassess-ment.
FEMSYeastRes2007,7:1181-1187.
13.
SteinbornG,BerE,ScholzA,TagK,KunzeG,GellissenG:Appli-cationofawide-rangeyeastvector(CoMed)systemtorecombinantproteinproductionindimorphicArxulaadenin-ivorans,methylotrophicHansenulapolymorphaandotheryeasts.
MicrobialCellFactories2006,5:33.
14.
KangHA,GellissenG:Hansenulapolymorpha.
InProductionofrecombinantproteins:novelmicrobialandeukaryoticexpressionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:111-142.
15.
KunzeG,KangHA,GellissenG:Hansenulapolymorpha–biologyandapplications.
InYeastBiotechnology:DiversityandapplicationsEditedby:SatyaranayanaT,KunzeG.
Berlin:Springerinpress.
16.
GellissenG:Heterologousproteinproductioninmethylo-trophicyeasts.
ApplMicrobiolBiotechnol2000,54:741-750.
17.
HartnerFS,GliederA:Regulationofmethanolutilisationpath-waygenesinyeasts.
MicrobialCellFactories2006,5:39.
18.
TschoppJF,BrustTF,CreggJM,StillmanCA,GingerasTR:Expres-sionofthelacZgenefromtwomethanol-regulatedpromot-ersinPichiapastoris.
NucleicAcidsRes1987,15:3859-3876.
19.
YurimotoH,SakaiY,KatoN:Methanolmetabolism.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Wein-heim:Wiley-VCH;2002:61-75.
20.
NakagawaT,InagakiA,ItoT,FujimuraS,MijajiT,YurimotoH,KatoN,SakaiY,TomizukaN:Regulationoftwodistinctalcoholoxi-dasepromotersinthemethylotrophicyeastPichiamethano-lica.
Yeast2006,23:15-22.
21.
WartmannT,StephanUW,BubeI,BerE,MelzerM,ManteuffelR,StoltenburgR,GuengerichL,GellissenG,KunzeG:Post-transla-tionalmodificationsoftheAFET3geneproduct–acompo-nentoftheirontransportsysteminbuddingcellsandmyceliaoftheyeastArxulaadeninivorans.
Yeast2002,19:849-862.
22.
MiddelhovenWJ,Hoogkamer-TeNietC,KregerVanRijNWJ:Tri-chosporonadeninovoranssp.
nov.
,ayeastspeciesutilizingadenine,xanthine,uricacid,putrescineandprimaryn-alkylaminesassolesourceofcarbon,nitrogenandenergy.
AntonievanLeeuwenhoek1984,50:369-378.
23.
WartmannT,ErdmannJ,KunzeI,KunzeG:Morphology-relatedeffectsongeneexpressionandproteinaccumulationoftheyeastArxulaadeninivoransLS3.
ArchMicrobiol2000,173:253-261.
24.
KleiIJvander,VeenhuisM:Hansenulapolymorpha–aversatilemodelinperoxisomeresearch.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:76-94.
25.
HellwigS,StckmannC,GellissenG,BüchsJ:Comparativefer-mentation.
InProductionofrecombinantproteins:novelmicrobialandeukaryoticexpressionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:287-317.
26.
GellissenG:Hansenulapolymorpha–biologyandapplicationsWein-heim:Wiley-VCH;2002.
27.
OhKS,KwonO,OhYW,SohnMJ,JungS,KimYK,KimMG,RheeSK,GellissenG,KangHA:FabricationofapartialgenomemicroarrayofthemethylotrophicyeastHansenulapolymor-pha:Optimizationandevaluationfortranscriptprofiling.
JMicrobiolBiotechnol2004,14:1239-1248.
28.
ParkJN,SohnMJ,OhDB,KwonO,RheeSK,HurCG,LeeSY,Gel-lissenG,KangHA:Identificationofthecadmium-inducibleHansenulapolymorphaSEO1genepromoterbytranscrip-tomeanalysisanditsapplicationtowhole-cellheavy-metaldetectionsystems.
ApplEnvironmMicrobiol2007,73:5990-6000.
29.
BerrowNS,BüssowK,CoutardB,DiproseJ,EkbergM,FolkersGE,LevyN,LieuV,OwensRJ,PelegY,PinagliaC,Quevillon-CheruelS,SalimL,ScheichC,VinventelliR,BussoD:RecombinantproteinexpressionandsolubilityscreeninginEscherichiacoli:acom-parativestudy.
ActaCrystallogrDBiolCrystallogr2006,62:1218-1226.
30.
JeudeM,DittrichB,NiederschulteH,AnderleiT,KnockeC,KleeD,BüchsJ:Fed-batchmodeinshakeflasksbyslow-releasetech-nique.
BiotechnolBioeng2006,95:433-445.
31.
AmuelC,GellissenG,HollenbergCP,SuckowM:AnalysisofheatshockpromotersinHansenulapolymorpha:theTPS1pro-moter,anovelelementforheterologousgeneexpression.
BiotechnolBioprocessEng2000,5:247-252.
32.
JenzelewskiV:Fermentationandprimaryproductrecovery.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:156-174.
33.
WeydemannU,KeupP,PiontekM,StrasserAWM,SchwedenJ,Gel-lissenG:High-levelproductionofhirudinbyHansenulapoly-morpha–authenticprocessingofthreedifferentpreprohirudins.
ApplMicrobiolBiotechnol1995,44:377-385.
34.
AvgerinosGC,TurnerBG,GorelickKJ,PapendieckA,WeydemannU,GellissenG:ProductionandpreclinicalanalysisofH.
poly-morpha-derivedPEG-hirudin.
SemThrombHemostas2001,27:357-371.
35.
BarnesCS,KrafftB,FrechM,HoffmannUR,PapendieckA,DahlemsU,GellissenG,HoylartsMF:Productionandcharacterizationofsaratin,aninhibitorofvonWillebrandfactor-dependentplateletadhesiontocollagen.
SeminThrombHemost2001,27:337-347.
36.
RoggenkampR,HansenH,EckartM,JanowiczZA,HollenbergCP:TransformationofthemethylotrophicyeastHansenulapoly-morphabyautonomousreplicationandintegrationvectors.
MolGenGenet1988,202:302-308.
37.
ZurekC,KubisE,KeupP,HrleinD,BeuninkJ,ThmmesJ,KulaMR,HollenbergCP,GellissenG:Productionoftwoaprotininvari-antsinHansenulapolymorpha.
ProcBiochem1996,31:679-689.
38.
SuckowM,GellissenG:TheexpressionplatformbasedonHansenulapolymorphaRB11–history,statusandperspec-tives.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:105-123.
39.
BartelsenO,BarnesCS,GellissenG:Productionofanticoagu-lantsinHansenulapolymorpha.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:211-228.
40.
MüllerFII,TiekeA,WaschkD,MühleC,MüllerFI,SeigelchiferM,PesceA,JenzelewskiV,GellissenG:ProductionofIFNα-2ainHansenulapolymorpha.
ProcBiochem2002,38:15-25.
41.
GellissenG,MüllerF,SieberH,TiekeA,JenzelewskiV,DegelmannA,StrasserAWM:ProductionofcytokinesinHansenulapolymor-pha.
InHansenulapolymorpha–biologyandapplicationsEditedby:Gel-lissenG.
Weinheim:Wiley-VCH;2002:229-254.
42.
MayerAF,HellmuthK,SchliekerH,Lopez-UlibarriR,OertelS,Dahl-emsU,StrasserAWM,vanLoonAPGM:Anexpressionsystemmatures:ahighlyefficientandcost-effectiveprocessforphytaseproductionbyrecombinantstrainsofHansenulapol-ymorpha.
BiotechnolBioeng1999,63:373-381.
43.
PapendieckA,DahlemsU,GellissenG:Technicalenzymepro-ductionandwhole-cellbiocatalysis:applicationofHansenulapolymorpha.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:255-271.
44.
KunzeG,KunzeI:CharacterizationofArxulaadeninivoransfromdifferenthabitats.
AntonievanLeeuwenhoek1994,65:29-34.
45.
KaurP,LingnerA,SinghB,BerE,PolajevaJ,SteinbornG,BodeR,GellissenG,SatyanarayanaT,KunzeG:APHO1fromtheyeastArxulaadeninivoransencodesanacidphosphataseofbroadsubstratespecificity.
AvanLeeuwenhoek2007,91:45-55.
46.
MinochaN,KaurP,SatyanarayanaT,KunzeG:AcidphosphataseproductionbyrecombinantArxulaadeninivorans.
ApplMicro-biolBiotechnol2007,76:387-393.
47.
AnderleiT,BüchsJ:Deviceforsterileonlinemeasurementoftheoxygentransferrateinshakingflasks.
BiochemEngJ2001,7:157-162.
48.
AnderleiT,ZangW,PapaspyrouM,BüchsJ:Onlinerespirationactivitymeasurement(OTR,CTR,RQ)inshakeflasks.
Bio-chemEngJ2004,17:187-194.
PublishwithBioMedCentralandeveryscientistcanreadyourworkfreeofcharge"BioMedCentralwillbethemostsignificantdevelopmentfordisseminatingtheresultsofbiomedicalresearchinourlifetime.
"SirPaulNurse,CancerResearchUKYourresearchpaperswillbe:availablefreeofchargetotheentirebiomedicalcommunitypeerreviewedandpublishedimmediatelyuponacceptancecitedinPubMedandarchivedonPubMedCentralyours—youkeepthecopyrightSubmityourmanuscripthere:http://www.
biomedcentral.
com/info/publishing_adv.
aspBioMedcentralMicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page10of10(pagenumbernotforcitationpurposes)49.
SilberbachM,MaierB,ZimmermannM,BüchsJ:GlucoseoxidationbyGluconobacteroxydans:characterizationinshakingflasks,scale-upandoptimizationofthepHprofile.
ApplMicrobiolBio-technol2003,62:92-98.
50.
StckmannC,LosenM,DahlemsU,KnockeC,GellissenG,BüchsJ:Effectofoxygensupplyonpassaging,stabilisationandscreeningofrecombinantH.
polymorphaproductionstrainsintesttubescultures.
FEMSYeastRes2003,4:195-205.
51.
StckmannC,MaierU,AnderleiT,KnockeC,GellissenG,BüchsJ:Theoxygentransferrateaskeyparameterforthecharac-terizationofHansenulapolymorphascreeningcultures.
JIndMicrobiolBiotechnol2003,30:613-622.
52.
LosenM,FroehlichB,PohlM,BüchsJ:EffectofoxygenlimitationandmediumcompositiononEscherichiacolifermentationinshake-flaskcultures.
BiotechnologyProgress2004,20:1062-1068.
53.
RselH,KunzeG:CloningandcharacterizationofaTEF1geneforelongationfactor1αfromtheyeastArxulaadeninivorans.
CurrGenet1995,28:360-366.
54.
KnollA,BartschS,HusemannB,EngelP,SchroerK,RibeiroB,Stck-mannC,SeletzkyJ,BüchsJ:Highcelldensitycultivationofrecombinantyeastsandbacteriaundernon-pressurizedandpressurizedconditionsinstirredtankreactors.
JBiotechnol2007,132:167-179.
55.
BerE,SteinbornG,MatrosA,MockHP,GellissenG,KunzeG:Pro-ductionofinterleukin-6inArxulaadeninivorans,HansenulapolymorphaandSaccharomycescerevisiaebyapplyingthewide-rangeyeastvector(CoMed)systemtosimultaneouscomparativeassessment.
FEMSYeastRes2007,5:1181-1187.
56.
BerE,SteinbornG,KunzeG,GellissenG:Yeastexpressionplat-forms.
ApplMicrobiolBiotechnol2007,77:13-523.
57.
SteinbornG,BerE,KunzeG,GellissenG:ApplicationoftheCoMedsystem.
InYeastBiotechnology:DiversityandapplicationsEditedby:SatyaranayanaT,KunzeG.
Berlin:Springerinpress.
8,52074Aachen,Germany,3PharmedArtisGmbH,Forckenbeckstr.
6,52074Aachen,Germanyand4DepartmentofLifeSciences,ChungAngUniversity,221Heukseok-dong,Dongjak-gu,156-756Seoul,KoreaEmail:ChristophStckmann-stoeckmann@biovt.
rwth-aachen.
de;MarcoScheidle-marco.
scheidle@avt.
rwth-aachen.
de;BarbaraDittrich-dittrich@itmc.
rwth-aachen.
de;ArminMerckelbach-armin.
merckelbach@pharmedartis.
de;GritHehmann-grit.
hehmann@pharmedartis.
de;GeorgMelmer-georg.
melmer@pharmedartis.
de;DorisKlee-klee@itmc.
rwth-aachen.
de;JochenBüchs-jochen.
buechs@avt.
rwth-aachen.
de;HyunAhKang-hyunkang@cau.
ac.
kr;GerdGellissen*-gerd.
gellissen@pharmedartis.
de*CorrespondingauthorAbstractArangeofindustrialH.
polymorpha-basedprocessesexist,mostofthemfortheproductionofpharmaceuticals.
TheestablishedindustrialprocessesleanontheuseofpromotersderivedfromMOXandFMD,genesofthemethanolmetabolismpathway.
InHansenulapolymorphathesepromotersarede-repressedupondepletionofarangeofcarbonsourceslikeglucoseandglycerolinsteadofbeinginducedbymethanolasreportedforothermethylotrophs.
Duetothesecharacteristicsscreeningandfermentationmodeshavebeendefinedforstrainsharbouringsuchexpressioncontrolelementsthatleanonalimitedsupplementationofglycerolorglucosetoaculturemedium.
ForfermentationofH.
polymorphaasyntheticminimalmedium(SYN6)hasbeendeveloped.
NoindustrialprocesseshavebeendevelopedsofarbasedonArxulaadeninivoransandonlyalimitedrangeofstrongpromoterelementsexists,suitableforheterologousgeneexpression.
SYN6originallydesignedforH.
polymorphaprovidedasuitablebasisfortheinitialdefinitionoffermentationconditionsforthisdimorphicyeast.
Characteristicslikeosmo-andthermotolerancecanbeaddressedforthedefinitionofcultureconditions.
HansenulapolymorphaandArxulaadeninivoransandtheircompetitiveenvironmentInthelastthreedecadesawiderangeofrecombinantpro-teins,especiallypharmaceuticals,havebeenproducedbasedonheterologousgeneexpressioninbacterialorgan-isms,mammaliancellsandseveralyeastsandfungi[1-3].
Productionprocesseshadtobedevelopedthatemployplatformswhichmeetboth,thedemandforefficientmassproductionandcriteriaofsafetyandauthenticityoftheproducedcompounds.
Inthisrespectyeastsofferconsid-erableadvantagesoveralternativemicrobialandmamma-liancellsystemsinprovidinglow-costscreeningandproductionsystemsforauthenticallyprocessedandmod-ifiedproteins.
Theorganismsmeetsafetyprerequisitesinthattheydonotharbourpyrogens,pathogensorviralinclusions[4,5].
RecentengineeringofyeasthostswithPublished:15April2009MicrobialCellFactories2009,8:22doi:10.
1186/1475-2859-8-22Received:7January2009Accepted:15April2009Thisarticleisavailablefrom:http://www.
microbialcellfactories.
com/content/8/1/222009Stoeckmannetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page2of10(pagenumbernotforcitationpurposes)thecapabilitytoaddhumanizedN-glycansoftheinter-mediatemannosetype[6]oreventhecomplextype[7]providestheoptiontoproducebiopharmaceuticalswithhumanproteinmodifications.
Therecognitionofyeastsasattractiveexpressionplatformsforbiopharmaceuticalsismetbygenomeanalysisofanincreasingnumberofyeastspecies,amongothersthatofSaccharomycescerevisiae[8]andHansenulapolymorpha[9].
AsaconsequencesomeearlyexamplesofFDA-approvedbiopharmaceuticalslikeinsulin[2]andhepatitisBvac-cines[10,11]havebeenproducedinthebaker'syeastS.
cerevisiae.
However,certainlimitationsanddrawbacksareencounteredwhenusingthissystem:S.
cerevisiaetendstohyperglycosylaterecombinantproteins;N-linkedcarbo-hydratechainsareterminatedbymannoseattachedtothechainviaanα1,3bond,whichisconsideredtobealler-genic.
Thelimitedcarbonsourceutilizationimposesrestrictionsonthedesignoffermentationprocesses;duetothepreferentialuseofepisomalvectorsinstabilitiesofrecombinantstrainsandasaresultbatchinconsistenciesofproductionrunsareofmajorconcern[12].
Thereforeanincreasingnumberofalternativeyeastsys-temshavebeendefinedthatcanpotentiallyovercomethedescribedlimitationsofthetraditionalbaker'syeast.
Theavailabilityofawide-rangeyeastvectorsystem(CoMed)enablestheassessmentofseveralyeastsinparallelfortheircapabilitytoproduceaparticularproteinindesiredqualitywithasinglevectortoidentifyanoptimalhostatthebeginningofaproductandprocessdevelopment.
Forexpressioncontrolthewide-rangevectorcontainsacon-stitutiveTEF1promoterderivedfromvarioussourcesthatisactiveinallyeastspeciesanalyzedsofar.
Ifneededthispromoterelementcaneasilybesubstitutedduringfurtherstraindevelopmentbyapromoteroptimalforthedefinedplatform[[13],Additionalfile1].
Outoftheplethoraofaddressablespecieswedescribeinthisarticlemethylo-trophicH.
polymorpha,arecognizedproducerofbiophar-maceuticalsandotherrecombinantproteins,anddimorphicArxulaadeninivorans,anovelplatformthathasyettoestablishitselfforindustrialapplications.
FirstexperimentsindicatethatscreeningandfermentationconditionsbasedonminimalSYN6medium(SYN6)withglucosesupplementationasdescribedinthisarticlecanalsobeappliedtoyeastplatformsothersthanH.
polymor-phaandA.
adeninivorans.
H.
polymorpha(Pichiaangusta)belongstoalimitednumberofyeastspeciesthatareabletoutilizemethanolasasoleenergyandcarbonsource.
Twooutofthreebasicstrainswithunclearrelationships,differentfeatures,andindependentoriginsarebiotechnologicallyapplied:strainCBS4732(CCY38-22-2,ATCC34438,NRRL-Y-5445)andDL-1(NRRL-Y-7560,ATCC26012)andauxotrophicderivativesthereof[14,15].
AmicrographofaH.
polymor-phacellpreparedfromachemostatwithamethanolfeedisshowninFig.
1a.
TherangeofbiotechnologicallyappliedmethylotrophicyeastsfurthermoreincludesCandidaboidinii,Pichiameth-anolica,andPichiapastoris[16].
Inallinstancesmostexamplesofheterologousgeneexpressionarelinkedtostrongandadjustablepromotersderivedfromgenesofthemethanolutilizationpathway[12,17],mostcom-monlytheelementsderivedfromthealcoholoxidasegenes,namelyAOX1fromP.
pastoris[16-18],MOXfromH.
polymorpha[14,17];AOD1fromC.
boidinii[16,17,19]andAUG1(nowdesignatedMOD1)fromP.
methanolica[16,17,20].
InH.
polymorphatheFMD(formatedehydro-genase)promoter,derivedfromanothermethanolutiliza-tionpathwaygeneofsimilarregulation,hasfoundpreferentialapplicationtoestablishedindustrialprocesses[14,16].
A.
adeninivorans(Blastobotrysadeninivorans)isayeastwithunusualcharacteristics.
Itisadimorphicspeciesandcanutilizeadenine,xanthine,uricacid,putrescineandn-alkylaminesascarbon,nitrogenorenergysourcesinaddi-tiontoglucose.
LikeH.
polymorphaitisanitrate-assimilat-ing,thermo-andosmotolerantorganism.
Adistinctivefeatureisatemperature-dependentdimorphismwithmycelialstructuresformedattemperaturesabove42°C[5,12][Fig.
1b,c].
ForFe(II)-oxidaseAfet3p,O-glycosyla-tionwasobservedtoberestrictedtothebuddingcellsta-tus[21].
ItremainstobeshownwhetherthisdifferentialO-glycosylationpatternincorrelationtothemorphologi-calstatusisalsopresentinrecombinantandotherhostproteins.
Again,severalstrainshavebeenidentifiedafteritsfirstdescriptionasTrichosporonadeninovorans[22].
Mostoftheresearchandthebiotechnologicalapplica-tionshavebeenperformedwithstrainLS3(PAR-4),iso-latedinSibiriabyKapultsevich,andarangeofauxotrophicmutantshavebeengenerated[5,12].
Strain135isamutantthatformsmycelialstructuresat30°C[23].
RecentlyauxotrophichoststrainsforheterologousgeneexpressionhavebeengeneratedbasedonstrainsCBS7350andCBS1738(seeTab.
1).
Sofar,noindustrialA.
adeninivorans-basedprocessexists.
ForexpressionandfermentationstudiesonalaboratoryscaleheterologousgenesweremostlyexpressedundercontrolofTEF1,acon-stitutiveA.
adeninivorans-derivedpromoterofappropriatestrength[5].
FordescriptionweselectedestablishedH.
polymorpha-basedprocesseswithstrainsexpressingaheterologousgeneundercontroloftheadjustableFMDandMOXpro-motersandA.
adeninivorans-basedlabscaleprocesseswithstrainsexpressingaheterologousgeneundercontroloftheconstitutiveA.
adeninivorans-derivedTEF1promoter,MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page3of10(pagenumbernotforcitationpurposes)withculturingconditionsthatcanpossiblybeappliedtotheassessmentofotheryeastswithconstitutiveheterolo-gousgeneexpression.
MicrographsofthetwoselectedplatformsareshowninFig.
1.
H.
polymorpha-basedprocessesundercontrolofMOXandFMDpromoters–are-assessmentMOXandFMDaregenesencodingenzymesofthemeth-anolutilizationpathwaythatissharedbyallmethylo-trophicyeasts.
Theenzymecomponentsofthispathwayandtheircontrolhavebeenreviewedextensivelyintherecentpast[4,17,19].
Thegenesofthispathwayaredescribedtobetightlyregulated;theyarehighlyrepressedinthepresenceofnon-limitingconcentrationsofglucoseandstronglyinducedifmethanolisusedasacarbonsource[17].
Methylotrophicgrowthisfurthermoreaccompaniedbyamassiveproliferationofperoxisomesinwhichseveralmethanol-metabolizingenzymesarecom-partmentalized[19,24].
However,itsoonbecameevidentthatactivationofmethanolpathwaypromotersdidnotdependonthepresenceofmethanolinH.
polymorphaincontrasttothesituationintheothermethylotrophs[16].
Forallothermethylotrophicyeastspeciesaninductiveactivationofsuchpromotershasbeenstatedthatisstrictlydependentonthepresenceofmethanol[17].
Asaconse-quenceseveralH.
polymorpha-basedindustrialfermenta-tionprocesseshavebeendefinedthatleanonglucoseorglycerolsupplementationinsuitableconcentrationstoaculturebrothwithoutanymethanoladditions[1,25,26].
ThedistinctfeatureoftheH.
polymorpha-derivedmetha-nolpathwaypromoterswaselucidated,whennewtoolsofgenomicsandpostgenomicanalysisbecameavailable.
AftersequencingtheentiregenomeofstrainCBS4732[9]acDNAmicroarraywasconstructedthatallowedcompre-hensivegeneexpressionprofiling[27,28].
Whenanalyz-ingthetranscriptomeofH.
polymorphastrainsofglucose-supplementedgrowthandaftertransitiontomethanol-supplementedgrowthitbecameevidentthatthemetha-noldissimilationgenesincludingMOXandFMDareacti-vatedbyde-repressionuponcarbonsourcelimitationanddepletionandnotuponinductionbymethanol.
Incon-trastgenesofperoxisomebiogenesisandproliferationareinducedbymethanol[14,15].
Withrespecttothesefindings,recombinantH.
polymorphastrainsexpressingaGFPreportergeneundercontroloftheFMDpromoterwerescreenedapplyingglucose-orglyc-erol-supplementedmediatostrainculturing.
Highthroughputscreeningexperimentsareusuallyper-formedinabatch-mode[29].
Jeudeetal.
describedasys-temfortheslowreleaseofglucosefromasiliconeelastomerematrixinshakeflask[30].
Theyshowedtheadvantageofusingfed-batchcultivationsincontrasttoMicroscopyanalysisofHansenulapolymorphaandArxulaaden-inivoranscellsFigure1MicroscopyanalysisofHansenulapolymorphaandArxulaadeninivoranscells.
aH.
polymorphacellgrowninamethanol-supplementedchemostat(courtesybyM.
Veen-huis,Groningen).
Underthesegrowthconditionsprolifera-tionofperoxisomesisinduced.
bA.
adeninivoranscellsgrownat37°C.
cA.
adeninivoranscellsgrownat42°C.
DimorphicA.
adeninivoransgrowsinfilamentousformsabove42°C.
abcMicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page4of10(pagenumbernotforcitationpurposes)batchcultivationsinsmallscale,whereupto420-foldincreasedGFPproductionwasreached[30].
Theslow-releasetechniquewastransferredtoadeep-well-platefor-mat.
Intheseplatestheglucose-containingsiliconematrixisfixedatthebottomofeachwell.
Hence,96parallelfed-batch-cultivationscanbeperformed.
AcomparativescreeningwithdifferentH.
polymorphapC10-FMD(PFMD-GFP)strains(strainsofaCBS4732background)[31]wasperformedinbatchmodewithglycerolandglucosesup-plementationandinfed-batchmodewithglucosesupple-mentation[Fig.
2a].
ThecomparisonatteststhatcloneswithanFMDpromoter-controlledgeneexpressioncanbescreenedinsmallvolumesofmediasupplementedwithglycerolorglucose.
Thissupportsthatmethanolpathwaypromotersareactivateduponglycerolandglucosedeple-tionassuggestedbythetranscriptomeanalysis.
Interest-inglysomeclonesofmaximalproductivityidentifiedunderconditionsofglucosestarvation(fed-batchcondi-tions)arenotidenticaltothoseidentifiedundercondi-tionofglycerolstarvation(batchconditions).
Thereasonsforthisphenomenonremainobscure.
MOXpromoter-drivenGFPexpressionunderglucosestarvationcondi-tionswascorroboratedforrecombinantstrainsofaDL-1backgroundculturedinadifferentmediumandonalargerscaleasshownbyfluorescenceanalysisinFig.
2b.
Thecommercialsuccessofarecombinantproductdoesnotdependonlyonthecharacteristicsofthemicrobialhostbutalsotoalargeextentonthedefinitionofefficientfermentationprocesses.
IncaseofH.
polymorpha,adefinedminimalmineralmediumhasbeendevelopeddesignatedSYN6[25,32].
Itiscomposedofsalts,vitaminsandtraceelementstosupportgrowthtohighcelldensitiesandithastobeadjustedtoappropriatepHconditionsandhastobesupplementedwithsuitablecarbonsources(fordetailsofcomponentssee[32]).
ThefermentationstrategyinpreviousestablishedprocessdevelopmentswithFMDorMOXpromoter-drivenproductionreliedongrowthusingeitherglucoseorglycerolinthebeginningoffermentation,followedbycarbonsourcelimitationsinasecondphase.
Therespectivefermentationparametersforthesupplementationofglucoseorglycerolhavebeendefinedpriortotheelucidationofthetranscriptomepro-filedescribedbefore.
Inasingleprocessexamplethefer-mentationbrothwassupplementedinalatephasewithmethanol.
ThethreedifferentfermentationmodesareschematicallydepictedinFig.
3a–c.
Anearlyexampleforaglycerolstarvationprocessistheproductionprocessforhirudin[33-35][Fig.
3a].
Asinallotherdescribedprocessesexpressionvectorswerecon-structedtotransformtheuracil-auxotrophicstrainRB11,likestrainMedHp1(Tab.
1)aderivativeofstrainLR9[36-38].
ThevectorscontainanexpressioncassettewithahirudinsequenceundercontroloftheMOXpromoter.
Forsecretionitwasfusedtoasecretionleader,derivedfromthepheromoneprecursorMFα1p[33].
Fermenta-tionona35literscalewascarriedoutat30°CinSYN6atpH5.
Itwasstartedwith30g/Lofglycerol.
Aftercon-sumptionofthecarbonsourceafter35hoursafeedwasinitiatedthataddedglycerolbyapO2-controlledfeedingdevice.
Hirudinproductionstartedaftersome20hourswhentheMOX-promoterwasactivatedbyde-repressionunderglycerollimitation.
Thesubsequentfeedingcondi-tionssupportedthede-repressedstatusofthepromoterbymaintainingglycerolconcentrationsbetween0.
5and3.
0g/L.
Thesefeedingconditionsresultedinanincreasingaccumulationoftheproductinthemedium[33,34,39][Fig.
3a].
Similarfermentationconditionswereappliedtoculturingofproductionstrainsfortheanticoagulantsaratin[35,39]orforaprotinin[37],nowapplyingtheFMDpromotertoexpressioncontrol.
Thisfermentationdesignwasmodi-fiedwhendevelopingaproductionprocessforthecytokineIFNalpha-2a[40,41].
IFNalpha-2aformsadisulfidebondbetweenaminoacidsCys1andCys98.
BondformationofthefirstaminoacidCys1ofthematuresequenceprovidesasterichindranceforcorrectmatura-tionwhenprocessedfromanMFα1/IFNalpha-2aprecur-sor.
AccordinglyalargeshareofsecretedrecombinanthirudinconsistedofincorrectlyprocessedmoleculeswithN-terminalextensions.
Thiscouldbeovercomebyco-pro-ductionoftheprocessingenzymeKex2p,howeverattheexpenseofamorepronouncedproteolyticdegradation.
TominimizethisdegradationpHwasloweredfrompH5(asappliedtostandardfermentations)topH2–3.
TheglycerolstarvationconditionsforFMDpromoterde-repressionremainedunchanged.
Fortheproductionofphytaseanextremelyefficientpro-ductionprocesshasbeendeveloped.
Inthisprocessallstepsandcomponentsoftheprocessfollowedarationaleofefficiencyandcost-effectiveness.
Thisrationalepro-vokedanassessmentofglucoseassolecarbonsourceforfermentation[42].
Inafermentationofaphytaseproduc-tionstrainwithFMD-controlledexpressionon2000Lscaleglucosewassupplementedas20g/L.
Upondeple-tion,aglucose-limitingfeedwasinitiatedthataddedthecarbonsourcewithastepwiseincreasingfeedingrateincorrelationtothecellmass.
Inthisglucosestarvationprocessafinalyieldof13.
5g/Lphytasewasobserved[42,43][Fig.
3b].
TheonlyestablishedindustrialfermentationprocesswithmethanolsupplementationisthatfortheproductionofthehepatitisBsurfaceantigenHBsAg,thefirstbiophar-maceuticalproducedinH.
polymorpha.
Severalprocessesforthisvaccinehavebeendescribedthatarebasedonboth,MOXorFMD-controlledexpression[11].
InFig.
3cMicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page5of10(pagenumbernotforcitationpurposes)atypicalfermentationprocessisschematicallyshown.
Thebatchphaseandafirstfed-batchphaseissimilartotheexamplesofglycerolstarvationdescribedbefore[Fig.
3a].
Incontrastamixtureofglycerolandmethanolisfedduringthelasthoursoffermentation.
ObviouslythisresultsinaninductiveincreaseofHBsAgproduction.
However,inviewofthetranscriptomeprofilingitseemsthatmethanolsupplementationdoesnotinducetheFMD-controlledexpressionoftheheterologousantigengene,butitinducesmembraneproliferation.
AstheHBsAgisproducedasparticleswiththerecombinantanti-geninsertedintohost-derivedmembranes,methanolisconsideredtoprovideabalancedco-productionofbothparticlecomponentsinhightiters[10,11].
TheselectedprocessexamplesdemonstratethepossibilitytodevelopefficientscreeningandfermentationprocessesforstrainswithMOX-orFMD-drivenheterologousgeneexpressionwithoutmethanolsupplementationtoamedium.
CulturingofArxulaadeninivoransstrainsForA.
adeninivoransindustrialprocessparametershavenotbeendefinedyetandmainlycultivationsonashakeflaskscalehavebeencarriedoutsofar.
MostofthecurrentexpressionstudiesarebasedonwildtypestrainLS3[44]oritsleucine-auxotrophicderivativeG1211.
Additionalstrainsandleucine-auxotrophicmutantsthereofhavebeenestablishedmorerecently(seeTab.
1).
Acidphosphataseproductionwascharacterizedinfer-mentationsofbothstrainLS3andarecombinantstrainexpressingtheAPHO1geneundercontrolofthestrongTEF1promoter[45].
UsingthePlackett-Burmandesignthreevariables(pH,sucroseconcentrations,andpeptoneconcentration)wereoptimizedformediumcomposition,aroughlyfourtimesenhancementwasobservedinmediacontaining39g/Lsucroseand16g/LpeptoneatpH3.
8[46].
ShakeflaskculturesofA.
adeninivoransstrainswereana-lyzedusingadeviceforonlinemeasurementoftherespi-rationrates(RAMOS,respiratoryactivitymonitoringsystem)[47,48].
ThisdevicehadpreviouslybeenappliedtotheanalysisofH.
polymorphaculturesandtoalternativeplatforms[49-52].
Inafirstseriesyeastminimalmedium(YMM)wasassessed.
Priortoanypracticalexperiment,YMMammo-niumconcentrationofthestandardmediumwasraisedfrom2.
2mmolN/gglucoseto4.
6mmolN/gglucosesincetheoreticalmaterialbalancingrevealedaseverelackofnitrogenwithregardtotheaveragenitrogencontentofyeast.
TheculturebrothwasadditionallysupplementedwithcalciumandironinhigherconcentrationsaswellasMethanolpathwaypromoter-controlledGFPproductioninrecombinantH.
polymorphastrainsFigure2Methanolpathwaypromoter-controlledGFPproductioninrecombinantH.
polymorphastrains.
A.
ScreeningofCBS4732-derivedtransformantsproducingGFPundercontroloftheFMDpromoter.
Screeningof44differentH.
polymorphapC10-FMD(PFMD-GFP)clonesculturedunderdifferentcultivationmodes;screeningexperimentswereperformedindeep-well-platesforbatchandinFeedBead96-platesforfed-batchcultiva-tion.
Gaspermeablesealingswereusedassterileclosures.
Toreducetheevaporationduringthecultivation,water-saturatedairwasusedforgassing.
Tocomparetheresultsfromthedifferentcultivationmodesanidenticalcarbonsourceconcentrationof16.
63g/Lwereappliedtoeachfermentation(batchwithglycerol,batchwithglucose,fed-batchwithglucose).
Thebatchcultivationswerestoppedwhenthestationarygrowthphase(glycerol21h,glucose16h)wasreachedandthefed-batchcultivationswerestoppedwhen16.
63g/LglucosewerereleasedfromtheFeadBeat96-plate(16h).
Drycellweight(DCW)wascalculatedfromopticaldensitymeasurementsinaPowerWave*340microtiterplatereader.
GreenFluorescentProtein(GFP)wasmeas-uredat485nmexcitationand520nmemissionwavelengths.
Cul-tivationconditions:shakingfrequency400rpm,shakingdiameter50mm,temperature37°C,fillingvolumes300μLperwell;media:SYN6-MESwith16.
63g/Lofdifferentcarbonsources.
Carbonsources:batchmodewithglycerol(blackline),batchmodewithglucose(greybars),fed-batchmodewithglucose(blackbars);inocularatio1:30.
B.
GFPproductioninaDL-1-derivedtrans-formantundercontroloftheMOXpromoter.
H.
polymorphatransformantsproducingGFPundercontrolofMOXpromoterwereculturedfor12honYPmediumcontaining10g/Lmethanol(YP+1%M)or0.
5g/Lglucose(YP+0.
05%D)andthenanalyzedbyconfocalmicroscopy.
aScreeningof44FMD-clones036DCW[g/L]024GFP[g/L]0481216202428323640440.
00.
40.
8FMD-clonesspec.
productyieldGFP/DCW[g/g]b12hrYP+1%M12hrYP+0.
05%DGFPMOX(p)HARSAmprReportervectorMicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page6of10(pagenumbernotforcitationpurposes)A,B,C:SchematicdepictionofH.
polymorpha-basedfermentationprocessesFigure3A,B,C:SchematicdepictionofH.
polymorpha-basedfermentationprocesses.
Thefiguresaremodifiedversionsoffiguresfrompreviouspublications.
Forfurtherdetailsseetext.
afermentationofahirudin-producingstrain(MOXpromoter-controlledexpression,glycerolstarvation),bfermentationofaphytase-producingstrain(FMDpromoter-controlledgeneexpression,glucosestarvation),cfermentationofaHBsAg-producingstrain(FMDpromoter-controlledexpression,glycerolstarvation,followedbyaglycerol/methanolfeed).
dottedline:drycellweight,solidline:glycerol(a,c)orglucose(b),chaindot-tedline:glucosefeedingrate(b)ormethanol(c),dashedline:productofa,bandcrespectively.
atime[h]drycellweightorglycerol[g/L]hirudin[%]110100020406080100growth01224364860de-repressiontime[h]drycellweightorglycerol[g/L]hirudin[%]110100020406080100growth01224364860de-repressionbglucosefeedingrate[g/h]orphytase[%]drycellweightorglucose[g/L]time[h]0244872961201441680startfeeding10100204060801001growthde-repressionglucosefeedingrate[g/h]orphytase[%]drycellweightorglucose[g/L]time[h]0244872961201441680startfeeding10100204060801001growthde-repressionctime[h]drycellweightorglycerolormethanol[g/L]hepatitisBsurfaceantigen[%]110100020406080100growthinductionde-repression01224364860time[h]drycellweightorglycerolormethanol[g/L]hepatitisBsurfaceantigen[%]110100020406080100growthinductionde-repression01224364860MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page7of10(pagenumbernotforcitationpurposes)withMES(2-[N-morpholino]ethanesulfonicacid)forbufferingtoresultinYMM*[25].
Despitenitrogenaddi-tiontoYMM,therespectiveculturesremainedlimitedasshownrepresentativelyforA.
adeninivoransLS3inFig.
4,blackcircles.
Initially,culturerespirationincreasedexpo-nentially,butwasthenlimitedto9mmol/(L*h)after13hours,andcontinuedtodeclineoverfermentationtime.
Finallyonlyca.
6.
6gdrycellweight(DCW)/Lwasobtained.
InfermentationsofstrainLS3inMES-bufferedYMM*therespirationrateexponentiallyincreasedfollowedbyalin-earincreasepresumablyindicatinganutrientdeficiency.
After24hours,respirationratedroppeduponglucosedepletion.
[Fig.
4,greycircles].
Subsequentlyglucose-supplementedSYN6wasassessedforapplicabilitytoshakeflaskculturesofA.
adeninivorans.
ThehighnutrientconcentrationsofstandardSYN6remainedunchanged.
Again,thepHofSYN6hadtobebufferedwithMES(SYN6-MES,see[25]fordetaileddescription)forpHstabilizationbetween6.
4and5.
3.
Fig.
4,opencirclesshowstherespirationrateofashakeflaskculturewithstrainLS3inSYN6-MES.
Thedescribedcul-turecourseadvertstothenon-limitedgrowthofA.
aden-inivoransintherespectiveshakeflaskculture.
Thus,favourablenon-limitinggrowthconditionsforA.
adenini-voransinshakeflaskswereproventobedeveloped.
Additionallysuitablefed-batchconditionsforhighcelldensityfermentations(HCDF)inSYN6werefound.
TheseconditionswereassessedforculturingarecombinantA.
adeninivoransstrainproducingphytaseundercontroloftheTEF1promoter[5,53].
Duringthefeedingunderglu-cose-limitationphytasewassecretedtomaximaltitresofca.
900FTU/mL(oneFTUequatestothephytaseamountliberating1μmolofinorganicphosphateperminuteatpH5.
5and37°C)[25].
Thus,thegrowthconditionsdefinedforshakeflaskculturesandHCDFofA.
adeninivo-ranswildtypestrainLS3,provedtobeapplicableforthephytase-producingrecombinantA.
adeninivoransstrain.
Finallythephytase-producingstrainwasculturedunderpressurizedconditionsina50LSTR,againusingaSYN6-derivedmediumforculturing.
Duringthefed-batchphasethereactorpressurewasincreasedstepwiseupto5bar.
After42hcellshadgrownupto224g/L.
PhytaseamountCoMedvectorsystemFigure5CoMedvectorsystem.
Thebasicvectorsarederivedfromstandardvectorswithanengineeredmultiplecloningsite(MCS)fortheuptakeofvariousmodules.
Initsbasicformallmodulesarefunctionalinallyeaststestedsofar.
Module1:ARS/CENsequencesfromvarioussources(optional);Module2:rDNAtargetingsequences(NTS2-ETS-18SrDNA-ITS1fromvarioussources);Module3:selectionmarkers(i.
e.
kanMX,hph,leu2,ura3andcombinationsthereof);Module4:expressioncassettesconsistingofaTEF1promoterfromvarioussources–cloningsite–terminator.
Thewide-rangeTEF1promotercaneasilybereplacedbyalternativestrongspecies-specificpromoterslikeMOXorFMD.
Forfurtherdetailsseetext.
Module1Module2Module3Module4Eco47IIISacIINotIBcuI(SphI)SalI(BsiWI)ApaIAmp(r)pCoMedTMbasicvectorII.
ColE1oriI.
f1(-)originAssessmentofminimalmediaforArxulaadeninivoransLS3bymeasuringrespirationratesinshakeflaskcultures(foroper-atingconditionsandmediaformulationsee[25])Figure4AssessmentofminimalmediaforArxulaadeninivo-ransLS3bymeasuringrespirationratesinshakeflaskcultures(foroperatingconditionsandmediaformu-lationsee[25]).
OriginalYMM(blackcircles),containingpoorconcentrationsofcalcium(0.
338mg/L)andiron(0.
041mg/L),ledtopoorculturerespirationratesbelow10mmol/(L*h).
ModifiedYMM*(greycircles)ispH-bufferedwithMESandcontainshigherconcentrationsofcalcium(272.
8mg/L)andiron(2.
1mg/L),thusleadingtoamorevitalcultivationasshownbythehigherrespirationrateofupto25mmol/(L*h)andacondensedcultivationtimeof24hours(seedropofrespiration,signallingthedepletionofthecarbonsourceglu-cose).
SYN6-MES(opencircles),containingexceedingnutri-entquantities,providednon-limitedgrowthofthecultureasshownbythedistinctexponentialincreaseofrespirationto45mmol/(L*h)andanearlyceaseofrespirationafteronly15hours,againsignallingthedepletionofcarbonsource.
01020304050012243time[h]respirationrate[mmol/(L*h)]6MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page8of10(pagenumbernotforcitationpurposes)increasedupto10*106FTU.
Fermentationsunderpres-surizedconditionsmayresultinincreasedproductyieldsandshorterfermentationtime[54].
Theconditionsofculturingcanpotentiallybeappliedtoscreeningandculturingofotheryeastsexpressingafor-eigngeneundercontrolofaconstitutiveTEF1promoter–akeyelementoftheCoMedsystemdescribedinthefol-lowingsection.
TheCoMedsystemWhileallestablishedexpressionsystemsaredistinguishedbycertainfavorablecharacteristics,itisevidentthatnosinglesystemisoptimalforallproteins.
Aninitialselec-tioncanresultincostlytime-andresource-consumingfailures.
Itisthusadvisabletoassessseveralplatformcan-didatesinparallelforcriteriasuchasproductivity,appro-priateprocessingandmodification.
ProductionofinterleukinIL-6invariousyeastplatformshasrecentlybeendescribedasastrikingexampleforthenecessityofacomparativeevaluationofseveralyeasts.
Correctprocess-ingfromanMFα1/IL-6precursorwasobservedinA.
aden-inivoranswhereasN-terminallytruncatedmoleculesweresecretedfromS.
cerevisiaeandH.
polymorphahosts[55].
Anovelyeast/vectorsystemprovidesaversatiletooltoaddresssimultaneouslywithasinglevectorarangeofyeastslikethetwodescribedbefore,namelyHansenulapolymorphaandArxulaadeninivorans,andotherslikeSac-charomycescerevisiae,Pichiapastoris,Kluyveromyceslactis.
Initsbasicformthevectoriscomposedofgeneticmodulesthatarefunctionalinallyeasts,namelyanrDNAtargetingsequence,anappropriateselectionmarkerandanexpres-sioncassetteundercontrolofaTEF1promoterfromvari-oussources.
TheCoMedsystemhasrecentlybeendescribedandsomeapplicationexampleshavebeenpro-vided[13,55-57].
ThebasicdesignofthevectorandaselectionofaddressableyeastspeciesareshowninFig.
5andAdditionalfile1.
Itisdesirablethattherangeofyeastsaddressedinparallelcanbealsoassessedinparallelforoptimalperformanceinagivencase.
FirstexperimentsindicatethatSYN6andderivativesthereofaresuitableminimalmediaforyeastsothersthanA.
adeninivoransandH.
polymorpha.
Thegen-eraluseoftheconstitutiveTEF1promoterisexpectedtoensurescreeningandfermentationconditionssimilartothosedescribedforH.
polymorphaandA.
adeninivorans.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsCS,MS,BD,DKandJBcontributedtothereviewedandnewdataonscreeningunderglucoselimitationsandonthefermentationdesignforArxulaadeninivoranscultures,HKistheprincipalscientistfortheHansenulapolymorphamicroarrayandshecontributedthedataonGFP-produc-tioninDL-1underglucosefermentation.
AM,GH,GMandGGperformedtheworkontheCoMedsystem.
GGwasaprojectpartnerinthemicroarrayworkandpartici-patedintheprojectsandthepublicationsonHansenulapolymorphareviewedinthismanuscript.
Allauthorsreadandapprovedthemanuscript.
AdditionalmaterialAcknowledgementsHAKangwassupportedbyagrantfromtheKoreanMinistryofScienceandTechnology(MicrobialGenomicsandApplicationsResearchandDevelop-mentProgram).
ThecontentofthisreviewhasbeenpresentedattheconferenceBiotech2008:Biopharmaceuticals:WhyyeastsWdenswil,Switzerland.
May232008.
References1.
GellissenG:Productionofrecombinantproteins:NovelmicrobialandeukaryoticexpressionsystemsWeinheim:Wiley-VCH;2005.
2.
MelmerG:Biopharmaceuticalsandtheindustrialenviron-ment.
InProductionofrecombinantproteins:novelmicrobialandeukary-oticexpressionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:361-383.
3.
YinJ,LiG,ReuX,HerlerG:Selectwhatyouneed:acompara-tiveevaluationoftheadvantagesandlimitationsoffre-quentlyusedexpressionsystemsforforeigngenes.
JBiotechnol2007,127:335-347.
4.
GellissenG,KunzeG,GaillardinC,CreggJM,BerardiE,VeenhuisM,KleiIvander:Newyeastexpressionplatformsbasedonmeth-ylotrophicHansenulapolymorphaandPichiapastorisanddimorphicArxulaadeninivoransandYarrowialipolytica–acomparison.
FEMSYeastRes2005,5:1079-1096.
5.
BerE,GellissenG,KunzeG:Arxulaadeninivorans.
InProductionofrecombinantproteins:novelmicrobialandeukaryoticexpressionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:89-110.
6.
KimMW,KimEJ,KimJY,ParkJS,OhDB,ShimmaJI,ChibaY,JigamiY,RheeSK,KangHA:FunctionalcharacterizationoftheHansenulapolymorphaHOC1,OCH1,andOCR1genesasmembersoftheyeastOCH1mannosyltransferasefamilyinvolvedinproteinglycosylation.
JBiolChem2006,281:6261-6272.
7.
HamiltonSR,DavidsonRC,SethuramanN,NettJH,JiangY,RiosS,BobrowiczP,StadheimTA,LiH,ChoiBK,HopkinsD,WischnewskiH,RoserJ,MitchellT,StrawbridgeRR,HoopesJ,WildtS,GerngrossTU:Humanizationofyeasttoproducecomplexterminallysialylatedglycoproteins.
Science2006,313:1441-1443.
8.
GoffeauA,BarrellRG,BuseyH,DavisRW,DujonB,FeldmannH,GalibertF,HoheiselJD,JacqC,JohnstonM,LouisEJ,MewesHW,MurakamiY,PhilippsenP,TettelinH,OliverSG:Lifewith6000genes.
Science1996,274:563-567.
9.
Ramezani-RadM,HollenbergCP,LauberJ,WedlerH,GriessE,Wag-nerC,AlbermannK,HaniJ,PiontekM,DahlemsU,GellissenG:TheAdditionalfile1Table1ComponentsoftheCoMedsystem.
Thetablecontainsaselec-tionofgeneticcomponentsandyeaststrainsoftheCoMedstrain/vectorsystem.
Clickhereforfile[http://www.
biomedcentral.
com/content/supplementary/1475-2859-8-22-S1.
doc]MicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page9of10(pagenumbernotforcitationpurposes)Hansenulapolymorpha(strainCBS4732)genome–sequenc-ingandanalysis.
FEMSYeastRes2003,4:207-215.
10.
BrockeP,SchaeferS,MelberK,JenzelewskiV,MuellerF,DahlemsU,BartelsenO,ParkKN,JanowiczZA,GellissenG:HepatitisBvac-cines–diseasecharacterizationandvaccineproduction.
InProductionofrecombinantproteins:novelmicrobialandeukaryoticexpres-sionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:319-60.
11.
MelmerG,KunzeG,GellissenG:Recombinantvaccineproduc-tioninyeast.
BiopharmInternJanuarySuppl2008:8-13.
12.
BerE,SteinbornG,GellissenG,KunzeG:Productionofinter-leukin-6inArxulaadeninivorans,HansenulapolymorphaandSaccharomycescerevisiaebyapplyingawide-rangeyeastvec-tor(CoMed)systemtosimultaneouscomparativeassess-ment.
FEMSYeastRes2007,7:1181-1187.
13.
SteinbornG,BerE,ScholzA,TagK,KunzeG,GellissenG:Appli-cationofawide-rangeyeastvector(CoMed)systemtorecombinantproteinproductionindimorphicArxulaadenin-ivorans,methylotrophicHansenulapolymorphaandotheryeasts.
MicrobialCellFactories2006,5:33.
14.
KangHA,GellissenG:Hansenulapolymorpha.
InProductionofrecombinantproteins:novelmicrobialandeukaryoticexpressionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:111-142.
15.
KunzeG,KangHA,GellissenG:Hansenulapolymorpha–biologyandapplications.
InYeastBiotechnology:DiversityandapplicationsEditedby:SatyaranayanaT,KunzeG.
Berlin:Springerinpress.
16.
GellissenG:Heterologousproteinproductioninmethylo-trophicyeasts.
ApplMicrobiolBiotechnol2000,54:741-750.
17.
HartnerFS,GliederA:Regulationofmethanolutilisationpath-waygenesinyeasts.
MicrobialCellFactories2006,5:39.
18.
TschoppJF,BrustTF,CreggJM,StillmanCA,GingerasTR:Expres-sionofthelacZgenefromtwomethanol-regulatedpromot-ersinPichiapastoris.
NucleicAcidsRes1987,15:3859-3876.
19.
YurimotoH,SakaiY,KatoN:Methanolmetabolism.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Wein-heim:Wiley-VCH;2002:61-75.
20.
NakagawaT,InagakiA,ItoT,FujimuraS,MijajiT,YurimotoH,KatoN,SakaiY,TomizukaN:Regulationoftwodistinctalcoholoxi-dasepromotersinthemethylotrophicyeastPichiamethano-lica.
Yeast2006,23:15-22.
21.
WartmannT,StephanUW,BubeI,BerE,MelzerM,ManteuffelR,StoltenburgR,GuengerichL,GellissenG,KunzeG:Post-transla-tionalmodificationsoftheAFET3geneproduct–acompo-nentoftheirontransportsysteminbuddingcellsandmyceliaoftheyeastArxulaadeninivorans.
Yeast2002,19:849-862.
22.
MiddelhovenWJ,Hoogkamer-TeNietC,KregerVanRijNWJ:Tri-chosporonadeninovoranssp.
nov.
,ayeastspeciesutilizingadenine,xanthine,uricacid,putrescineandprimaryn-alkylaminesassolesourceofcarbon,nitrogenandenergy.
AntonievanLeeuwenhoek1984,50:369-378.
23.
WartmannT,ErdmannJ,KunzeI,KunzeG:Morphology-relatedeffectsongeneexpressionandproteinaccumulationoftheyeastArxulaadeninivoransLS3.
ArchMicrobiol2000,173:253-261.
24.
KleiIJvander,VeenhuisM:Hansenulapolymorpha–aversatilemodelinperoxisomeresearch.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:76-94.
25.
HellwigS,StckmannC,GellissenG,BüchsJ:Comparativefer-mentation.
InProductionofrecombinantproteins:novelmicrobialandeukaryoticexpressionsystemsEditedby:GellissenG.
Weinheim:Wiley-VCH;2005:287-317.
26.
GellissenG:Hansenulapolymorpha–biologyandapplicationsWein-heim:Wiley-VCH;2002.
27.
OhKS,KwonO,OhYW,SohnMJ,JungS,KimYK,KimMG,RheeSK,GellissenG,KangHA:FabricationofapartialgenomemicroarrayofthemethylotrophicyeastHansenulapolymor-pha:Optimizationandevaluationfortranscriptprofiling.
JMicrobiolBiotechnol2004,14:1239-1248.
28.
ParkJN,SohnMJ,OhDB,KwonO,RheeSK,HurCG,LeeSY,Gel-lissenG,KangHA:Identificationofthecadmium-inducibleHansenulapolymorphaSEO1genepromoterbytranscrip-tomeanalysisanditsapplicationtowhole-cellheavy-metaldetectionsystems.
ApplEnvironmMicrobiol2007,73:5990-6000.
29.
BerrowNS,BüssowK,CoutardB,DiproseJ,EkbergM,FolkersGE,LevyN,LieuV,OwensRJ,PelegY,PinagliaC,Quevillon-CheruelS,SalimL,ScheichC,VinventelliR,BussoD:RecombinantproteinexpressionandsolubilityscreeninginEscherichiacoli:acom-parativestudy.
ActaCrystallogrDBiolCrystallogr2006,62:1218-1226.
30.
JeudeM,DittrichB,NiederschulteH,AnderleiT,KnockeC,KleeD,BüchsJ:Fed-batchmodeinshakeflasksbyslow-releasetech-nique.
BiotechnolBioeng2006,95:433-445.
31.
AmuelC,GellissenG,HollenbergCP,SuckowM:AnalysisofheatshockpromotersinHansenulapolymorpha:theTPS1pro-moter,anovelelementforheterologousgeneexpression.
BiotechnolBioprocessEng2000,5:247-252.
32.
JenzelewskiV:Fermentationandprimaryproductrecovery.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:156-174.
33.
WeydemannU,KeupP,PiontekM,StrasserAWM,SchwedenJ,Gel-lissenG:High-levelproductionofhirudinbyHansenulapoly-morpha–authenticprocessingofthreedifferentpreprohirudins.
ApplMicrobiolBiotechnol1995,44:377-385.
34.
AvgerinosGC,TurnerBG,GorelickKJ,PapendieckA,WeydemannU,GellissenG:ProductionandpreclinicalanalysisofH.
poly-morpha-derivedPEG-hirudin.
SemThrombHemostas2001,27:357-371.
35.
BarnesCS,KrafftB,FrechM,HoffmannUR,PapendieckA,DahlemsU,GellissenG,HoylartsMF:Productionandcharacterizationofsaratin,aninhibitorofvonWillebrandfactor-dependentplateletadhesiontocollagen.
SeminThrombHemost2001,27:337-347.
36.
RoggenkampR,HansenH,EckartM,JanowiczZA,HollenbergCP:TransformationofthemethylotrophicyeastHansenulapoly-morphabyautonomousreplicationandintegrationvectors.
MolGenGenet1988,202:302-308.
37.
ZurekC,KubisE,KeupP,HrleinD,BeuninkJ,ThmmesJ,KulaMR,HollenbergCP,GellissenG:Productionoftwoaprotininvari-antsinHansenulapolymorpha.
ProcBiochem1996,31:679-689.
38.
SuckowM,GellissenG:TheexpressionplatformbasedonHansenulapolymorphaRB11–history,statusandperspec-tives.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:105-123.
39.
BartelsenO,BarnesCS,GellissenG:Productionofanticoagu-lantsinHansenulapolymorpha.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:211-228.
40.
MüllerFII,TiekeA,WaschkD,MühleC,MüllerFI,SeigelchiferM,PesceA,JenzelewskiV,GellissenG:ProductionofIFNα-2ainHansenulapolymorpha.
ProcBiochem2002,38:15-25.
41.
GellissenG,MüllerF,SieberH,TiekeA,JenzelewskiV,DegelmannA,StrasserAWM:ProductionofcytokinesinHansenulapolymor-pha.
InHansenulapolymorpha–biologyandapplicationsEditedby:Gel-lissenG.
Weinheim:Wiley-VCH;2002:229-254.
42.
MayerAF,HellmuthK,SchliekerH,Lopez-UlibarriR,OertelS,Dahl-emsU,StrasserAWM,vanLoonAPGM:Anexpressionsystemmatures:ahighlyefficientandcost-effectiveprocessforphytaseproductionbyrecombinantstrainsofHansenulapol-ymorpha.
BiotechnolBioeng1999,63:373-381.
43.
PapendieckA,DahlemsU,GellissenG:Technicalenzymepro-ductionandwhole-cellbiocatalysis:applicationofHansenulapolymorpha.
InHansenulapolymorpha–biologyandapplicationsEditedby:GellissenG.
Weinheim:Wiley-VCH;2002:255-271.
44.
KunzeG,KunzeI:CharacterizationofArxulaadeninivoransfromdifferenthabitats.
AntonievanLeeuwenhoek1994,65:29-34.
45.
KaurP,LingnerA,SinghB,BerE,PolajevaJ,SteinbornG,BodeR,GellissenG,SatyanarayanaT,KunzeG:APHO1fromtheyeastArxulaadeninivoransencodesanacidphosphataseofbroadsubstratespecificity.
AvanLeeuwenhoek2007,91:45-55.
46.
MinochaN,KaurP,SatyanarayanaT,KunzeG:AcidphosphataseproductionbyrecombinantArxulaadeninivorans.
ApplMicro-biolBiotechnol2007,76:387-393.
47.
AnderleiT,BüchsJ:Deviceforsterileonlinemeasurementoftheoxygentransferrateinshakingflasks.
BiochemEngJ2001,7:157-162.
48.
AnderleiT,ZangW,PapaspyrouM,BüchsJ:Onlinerespirationactivitymeasurement(OTR,CTR,RQ)inshakeflasks.
Bio-chemEngJ2004,17:187-194.
PublishwithBioMedCentralandeveryscientistcanreadyourworkfreeofcharge"BioMedCentralwillbethemostsignificantdevelopmentfordisseminatingtheresultsofbiomedicalresearchinourlifetime.
"SirPaulNurse,CancerResearchUKYourresearchpaperswillbe:availablefreeofchargetotheentirebiomedicalcommunitypeerreviewedandpublishedimmediatelyuponacceptancecitedinPubMedandarchivedonPubMedCentralyours—youkeepthecopyrightSubmityourmanuscripthere:http://www.
biomedcentral.
com/info/publishing_adv.
aspBioMedcentralMicrobialCellFactories2009,8:22http://www.
microbialcellfactories.
com/content/8/1/22Page10of10(pagenumbernotforcitationpurposes)49.
SilberbachM,MaierB,ZimmermannM,BüchsJ:GlucoseoxidationbyGluconobacteroxydans:characterizationinshakingflasks,scale-upandoptimizationofthepHprofile.
ApplMicrobiolBio-technol2003,62:92-98.
50.
StckmannC,LosenM,DahlemsU,KnockeC,GellissenG,BüchsJ:Effectofoxygensupplyonpassaging,stabilisationandscreeningofrecombinantH.
polymorphaproductionstrainsintesttubescultures.
FEMSYeastRes2003,4:195-205.
51.
StckmannC,MaierU,AnderleiT,KnockeC,GellissenG,BüchsJ:Theoxygentransferrateaskeyparameterforthecharac-terizationofHansenulapolymorphascreeningcultures.
JIndMicrobiolBiotechnol2003,30:613-622.
52.
LosenM,FroehlichB,PohlM,BüchsJ:EffectofoxygenlimitationandmediumcompositiononEscherichiacolifermentationinshake-flaskcultures.
BiotechnologyProgress2004,20:1062-1068.
53.
RselH,KunzeG:CloningandcharacterizationofaTEF1geneforelongationfactor1αfromtheyeastArxulaadeninivorans.
CurrGenet1995,28:360-366.
54.
KnollA,BartschS,HusemannB,EngelP,SchroerK,RibeiroB,Stck-mannC,SeletzkyJ,BüchsJ:Highcelldensitycultivationofrecombinantyeastsandbacteriaundernon-pressurizedandpressurizedconditionsinstirredtankreactors.
JBiotechnol2007,132:167-179.
55.
BerE,SteinbornG,MatrosA,MockHP,GellissenG,KunzeG:Pro-ductionofinterleukin-6inArxulaadeninivorans,HansenulapolymorphaandSaccharomycescerevisiaebyapplyingthewide-rangeyeastvector(CoMed)systemtosimultaneouscomparativeassessment.
FEMSYeastRes2007,5:1181-1187.
56.
BerE,SteinbornG,KunzeG,GellissenG:Yeastexpressionplat-forms.
ApplMicrobiolBiotechnol2007,77:13-523.
57.
SteinbornG,BerE,KunzeG,GellissenG:ApplicationoftheCoMedsystem.
InYeastBiotechnology:DiversityandapplicationsEditedby:SatyaranayanaT,KunzeG.
Berlin:Springerinpress.
- inivoranswww.avtt3.com相关文档
- capacitorswww.avtt3.com
- WXtwww.avtt3.com
- 模型www.avtt3.com
- profileswww.avtt3.com
- columnswww.avtt3.com
- truncatedwww.avtt3.com
企鹅小屋：垃圾服务商有跑路风险，站长注意转移备份数据！
企鹅小屋：垃圾服务商有跑路风险！企鹅不允许你二次工单的，二次提交工单直接关服务器，再严重就封号，意思是你提交工单要小心，别因为提交工单被干了账号！前段时间，就有站长说企鹅小屋要跑路了，站长不太相信，本站平台已经为企鹅小屋推荐了几千元的业绩，CPS返利达182.67CNY。然后，站长通过企鹅小屋后台申请提现，提现申请至今已经有20几天，企鹅小屋也没有转账。然后，搞笑的一幕出现了：平台账号登录不上提示...
小欢互联19元/月起, 即日起至10月底 美国CERA 促销活动 美国/香港八折
小欢互联成立于2019年10月，主打海外高性价比云服务器、CDN和虚拟主机服务。近期上线了自营美国CERA机房高速VPS，进行促销活动，为客户奉上美国/香港八折优惠码：Xxc1mtLB优惠码适用于美国CERA一区/二区以及香港一区/二区优惠时间：即日起至10月底优惠码可无限次使用，且续费同价！官网：https://idc.xh-ws.com购买地址：美国CERA一区：https://idc.xh-...
易探云美国云服务器评测，主机低至33元/月,336元/年
美国服务器哪家平台好?美国服务器无需备案，即开即用，上线快。美国服务器多数带防御，且有时候项目运营的时候，防御能力是用户考虑的重点，特别是网站容易受到攻击的行业。现在有那么多美国一年服务器，哪家的美国云服务器好呢？美国服务器用哪家好?这里推荐易探云，有美国BGP、美国CN2、美国高防、美国GIA等云服务器，线路优化的不错。易探云刚好就是做香港及美国云服务器的主要商家之一，我们来看一下易探云美国云服...
www.avtt3.com为你推荐
-
sonicchatwe chat和微信区别留学生认证留学生前阶段双认证认证什么内容？冯媛甑冯媛甄多大啊?sss17.com为什么GAO17.COM网站打不开了haokandianyingwang谁给个好看的电影网站看看。www.vtigu.com如图所示的RT三角形ABC中,角B=90°(初三二次根式）30 如图所示的RT三角形ABC中,角B=90°,点p从点B开始沿BA边以1厘米每秒的速度向A移动；同时，点Q也从点B开始沿BC边以2厘米每秒的速度向点C移动。问：几秒后三角形PBQ的面积为35平方厘米？PQ的距离是多少ip查询器怎么样查看自己电脑上的IP地址杨丽晓博客杨丽晓今年高考了吗？m88.comwww.m88.com现在的官方网址是哪个啊 ？www.m88.com怎么样？铂金血痕手上出现这种血痕是什么情况。有谁知道能告诉下吗？ 怎么治疗！