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ChinaBiotechnology,2009,29(5):1201253(350002),:,pH,DO,:(MLFB)(OLFB)(MNLFB)(TLFB):,(qMeOH)0.02-0.03gg-1h-1,,qMeOHQ815:2008209216:20082102173:ljh047@163.com(Pichiapastoris),[1],,,,,22g/L[2],12.2g/L[3],SAM11.63g/L[4],1,321,422D23g/L[5],,:,,,,,1,,InvitrogenBMGY/BMMYBMG/BMMMGY/MMYBSM,,,PTM1BSM,,,,,BSM1.1,,,,,,,,[6],(CMeOH)CMeOH,;CMeOH,0.5%3%(v/v),3%[7],,,CMeOH,,[8]1.2,pH,NH4+,,;,,,NH4+,2009,29(5):[9](NH4)2SO4,NH4+150mmol/L,58.8%Zhang[4]NH4+0.45mol/L,,SAM,43hYang[10]:NH4+0.4mol/L,;0.6mol/L,,;NH4+,0.025mol/L0.6mol/L,0.4mol/L,0.6mol/L,,,1.3PTM1,,,,Surribas[11],30%,20%,Zhao[12]1/4,83.3%48.6%59.2%,25%,HAS2IFN22b92.0%,PTM1,,,,PTM1,,,,,[13]1.4,VitCVitE,;,;EDTA,,Xiao[14]VitC410mmol/L,65.4%,30.2%,56.8%Zhao[12]2%,HAS2IFN22bAyed[15]0.1%EDTA10mmol/L,hIFN2b,,65%2pHpH,pH,,,pH37,rHSA[16],pH,3.07.0,,pH,pH,pH5.54.0,pH6.01.2[17]pH,NH4+,3,,,,,,,,30,,20228,,,[1];,,;AOX,cIFN20307.238.7,,[18]HAS2IFN22b20302.16,44.4%[12]B12301,,66.7%,,AOX2.5[19]203035%,50.1%,10.4%,AOX1.4,[20]4(DO)4.1DO,,,,,,[21],,,DO20%30%,DO,60%,4.2DODO,121ChinaBiotechnologyVol.29No.52009,,,,,,;(VHb),VHb,ATP,[22]5(CWPTI)(PTI)5.1CWPTIPTICWPTIPTI,CWPTIPTICWPTI,[23],;PTI,,,,[24]Wang[23]CWPTI122g/L(),PGL62.5g/L1;Wei[24]CWPTI320g/L(),rljGH160g/L84.0%;PTI0.16/h,0.05/h50.2%,CWPTIPTISAM,CWPTI100g/L()[25];rBoNTE(Hc),CWPTI200g/L()[26];hG2CSF,CWPTI40g/L(),;PTI0.18/h[27]CWPTIPTI:,AOX1;,,,CWPTIPTI5.2CWPTIPTI,PTICWPTI5.2.1,(5g/L),,,CWPTI[28]5.2.2,PTI(0.160.18h21),PTIPTI,,PTI,CWPTI,CWPTI,PTI=0.176/h,34h,CWPTI140g/L(),DO2stat3.4[23],[23],AOX1,,,,Wang[23]PTI=0.176/h10h,98ml/h45ml/h,;Bahrami[27],510g/L,PTI0.21/h0.15/h,0,hG2CSF28%66.16.1.1,,:(methanol2limitedfeedingbatch,MLFB)(oxygen2limitedfeedingbatch,OLFB)(temperature2limitedfeedingbatch,TLFB)(methanol2unlimitedfeedingbatch,MNLFB)[11,12,19,21]MLFB,DOCMeOH,,,,MLFBOLFB,DO,CMeOH,,,OLFB,MLFBOLFB2,MLFB16.1%,,[21];scFV,MLFB,,[29]MNLFBOLFB,DO,MLFB,TLFBMLFB,25%DO,CMeOH,,,,,,MNLFBMLFB,TLFB+MLFB,[19];Surribas[11]MNLFB+TLFB2212009,29(5):,ROLTLFB33.5%,MNLFB+MLFB,6.1.2,CMeOH,,(qMeOH)(Ind)0,qMeOH(1)Ind0,qMeOH0.020.03gg21h21qMeOH0.020.03gg21h21,[30],,MLFB,IndqMeOH(Ind=Yx/sqMeOH,Yx/s1gg21[29,30]),Ind=0.020.03h21Sinha[26]:rBoNTE(Hc)Ind0.0267/h,1.93mgTrinh[31]Ind=0.02/h,,,1OLFB,CMeOHCWPTI,,qMeOH0.02gg21h21Khatri[29,30]:CMeOH,qMeOH0.05gg21h210.02gg21h21,,scFV,3%CMeOH;CWPTI(245g/L())(Ind=0.03/h)qMeOH,6.19(2)Ind0,qMeOHCWPTI,,,CMeOH,qMeOH,,,,,MLFB,CMeOH/CWPTIWang:CMeOH/CWPTI0.1645gg21;CWPTI140g/L,CMeOH/CWPTI0.163gg210.171gg21(qMeOH0.045gg21h21),PGL3.2Zhang[25]:CMeOH0.6%,CWPTI100g/L(),qMeOH0.038gg21h21,SAM6.2,,,;,AOX1,,0,;,,Hu[32]10,10h,6h,4h,DO(30%35%),0.5%0.6%0.06%0.1%,,SAM12.3%34.3%;Wei[24]77:23(v/v),Ind=0.035/h,rljGH,6h;Jungo[33,34]:,6040(C2mol/C2mol)4357(C2mol/C2mol)(),0.06/h0.03/h,0,,,10%30%,28%38%,,:Ind=0.05/h,,,AOX17,,,:NH4+,,PTM1,pH:pH:,:DO20%30%,DO:CWPTIPTI,:MLFBOLFBTLFBMNLFB,,:(1)Ind0,MLFBInd=0.020.03/h,OLFB321ChinaBiotechnologyVol.29No.52009CMeOHCWPTI,,qMeOH0.0220.03gg21h21,,;(2)Ind0,MLFB,CWPTI,CMeOHqMeOH,,,,,10%9,[1]DalyR,HearnMTW.ExpressionofheterologousproteinsinPichiapastoris:ausefulexperimentaltoolinproteinengineeringandproduction.JMolRecognit,2005,18(2):119138[2]HasslacherM,SchallM,HaynM,etal.High2levelintracellularexpressionofhydroxynitrilelyasefromthetropicalrubbertreeHeveabrasiliensisinmicrobialhosts.ProteinExpresPurif,1997,11(1):6171[3]XiongAS,YaoQH,PengRH,etal.HighlevelexpressionofasyntheticgeneencodingPeniophoralyciiphytaseinmethylotrophicyeastPichiapastoris.ApplMicrobiolBiotechnol,2006,72(5):10391047[4]ZhangJG,WangXD,ZhengY,etal.EnhancingyieldofS2adenosylmethionineinPichiapastorisbycontrollingNH4+concentration.BioprocessBiosystEng,2008,31(2):6367[5]HuangHQ,YangPL,LuoHY,etal.High2levelexpressionofatruncated1,321,422D2glucanasefrominFibrobactersuccinogenesPichiapastorisbyoptimizationofcodonsandfermentation.ApplMicrobiolBiotechnol,2008,78:95103[6],,.Pichiapastoris.,2005,26(2):5256SaRB,ShiGY,WangZX.FoodResearchandDevelopent,2005,26(2):5256[7]ReverterD,VenturaS,VillegasV,etal.OverexpressionofhumanprocarboxypeptidaseA2inPichiapastorisanddetailedcharacterizationofitsactivationpathway.TheJournalofBiologicalChemistry,1998,273(6):35353541[8],,..,2002,18(3):348351ZhouXS,FanWM,ZhangYX.ChineseJournalofBiotechnology,2002,18(3):348351[9],,,etal..,2004,30(6):723726XieJL,ZhouQW,DuP,etal.JouranlofEastChinaUniversityofScienceandTechnology,2004,30(6):723726[10]YangJZ,ZhouXS,ZhangYX.ImprovementofrecombinanthirudinproductionbycontrollingNH4+concentrationinPichiapastorisfermentation.BiotechnologyLetters,2004,26(12):10131017[11]SurribasA,StahnR,MontesinosJL,etal.ProductionofaRhizopusoryzaelipasefromPichiapastorisusingalternativeoperationalstrategies.JournalofBiotechnology,2007,130(3):291299[12]ZhaoHL,XueC,WangY,etal.IncreasingthecellviabilityandheterologousproteinexpressionofPichiapastorismutantdeficientinPMR1genebycultureconditionoptimization.ApplMicrobiolBiotechnol,2008,81(2):235241[13],,..,2005,16(2):210212LiHM,WangHN,XuQK.LettersinBiotechnology,2005,16(2):210212[14]XiaoAF,ZhouXS,ZhouL,etal.Improvementofcellviabilityandhirudinproductionbyascorbicaci