菌种改造新方法及新设备的产学研合作研发-邢新会-清

面向生物产业的高效菌种改造新方法及新设备的产学研合作研发邢新会1，李和平2，王立言1,3，张翀11清华大学化工系生物化工研究所Email:[e-mail]2清华大学工程物理系3思清源生物科技有限公司全国发酵工程技术与工艺装备科技创新与产业化发展论坛2013年3月23-24日于上海发酵微生物育种技术重要性优良微生物的高效选育或育种技术是生物产业的核心。菌株——生物产业的生命！生物产业优良微生物的基本性能要求发酵生产效率高、菌种鲁棒性强、稳定性高、GRAS如何获取优良工业微生物？直接富集筛选：耗时，获得优良菌种的几率小；微生物育种：在基因层面对微生物的遗传性状进行改变，从而获取性状优良、适合工业生产的菌株食品发酵工业及农业生物技术育种的基本要求：从安全性出发，大多需要非基因改良生物（GMO），因此，需要高效非GMO定向进化育种技术快速、高效微生物进化育种技术国是生物技术领域的重要方向生命的本质：智能多功能体系（目的论）获取途径：进化及组织化•多样性diversity•高效性Efficiency•可持续性Sustainability分子单细胞组织个体群落尺度scale效率efficiency生命的工程学原理：功能分化与功能集成生物进化过程肇庆西江虾(腿不一样长）•突变是生命世界的自然现象•进化是偶然中的必然，像中彩票•能按照期待的方向控制生物进化吗?LotteryTicket彩票•DNA自然复制突变率(1个碱基置换错误率):1/107-108•突变率可以通过外界力量增加•突变导致进化，进而使生命获得新功能、适应新的环境条件加速突变MutationAccelerationAdaptiveevolutionSub-OptimalWildtypeOptimalEvolvedStrains~60days~60daysNature2002,420(6912):186-9;JBacteriol.2003,185(21):6400-8ChemostatcultureEnrichmentcultureAdvantage:Selectionpressure,“directed”cellevolutionDisadvantage:needinglongtime,timeandlaborconsuming,lowefficiency如何实现高效微生物进化？Howtomakeeffectivemicrobialevolution?MicrobialEvolutionEnvironmentalstressesGenomemutationFitness•Adaptationbyalteringphenotypesoremergingnewfunctions(evolution)？？PotentiationActualizationRefinementmakingatraitpossiblemakingthetraitmanifestmakingthetraiteffective(newfunctions)JE.Barricketal.,Nature,461,1243-1249(2009)ZacharyD.Blountetal.,Nature,doi:10.1038/nature11514(2012)Totalmutations450400350300Mutator250evolves200150100500Cit+evolves010,00020,00030,00040,000GenerationsRelativefitnessMutations504540353025208001560010400200500010K20K30K40K1.91.81.71.61.51.41.31.21.11.002,5005,0007,50010,00012,50015,00017,50020,000GenerationFigure2|Ratesofgenomicevolutionandfitnessimprovement.Bluecircles微生物进化育种高效微生物进化育种•基因组水平突变•能够形成大突变库•突变快速、有方向性•适宜的高通量定向筛选方法yhdm10y表型自然突变进化育种MicrobialEvolutionDrivenbyMutationIoninjectionTechnology•Succeededforplantandmicrobialmutation•Molecularmechanismunclear•Operationneedsanti-radiationMutationPhysicalToolsUVIrradiationIonInjectionPlasma•Feasibility•Rapidness•NonGMO•RandommutationChemicalMutagens等离子体面向进化育种的微生物突变方法•Basedonthermodynamicequilibrium:Thermalplasmas(hightemperature)Nonthermalplasmas(coldplasma,lowtemperature)等离子体Plasma大气压介质阻挡放电APDBD(Atmospheric-PressureDielectricBarrierDischarge）:需要的放电电压较高，很难获得均匀、温和的辉光放电等离子体，含有臭氧和紫外线;放电区的丝状放电通道温度可达万度量级；微生物突变的效率低和可控性差大气压射频辉光放电RFAPNEDs(Radio-FrequencyAtmospheric-PressureNon-EquilibriumDischarges):•气体温度：可控、室温范围、无热效应•工作压力：1atm，操作方便•活性粒子：种类丰富、浓度高、均匀，可控性高；不产生臭氧和紫外线•微生物突变效率高、突变操作快，突变多样性大，可控性高，易实现高通量诱变SolidIceLiquidWaterGasVaporPlasmaIonizedGasTemperature0℃100℃10000℃清华大学ARTP及其生物学效应研究平台RFAPNEDs：气体温度：可控、室温范围、无热效应工作压力：1atm，操作方便活性粒子：种类丰富、浓度高、可控不产生臭氧和紫外线适合生物技术应用/生物技术ARTP:AtmosphericandRoomTemperaturePlasma(1atm,20-40˚C)UniquePropertiesofARTPforMutationHeARTPspectrum•ActivatedchemicalspeciesnoUV,noozone•Mildconditionsatmosphericpressure,roomtemperatureAPPLIEDPHYSICSLETTERS,89,161504(2006)APPLIEDPHYSICSLETTERS,89,161502(2006)PLASMASOURCESSCIENCE&TECHNOLOGY,16,290-296(2007)APPLIEDPHYSICSLETTERS,92,221504(2008)JournalofPhysicsD:AppliedPhysics,2008.41(20):202001PlasmaChemistryandPlasmaProcessing,2007.27(5):p.529-545AppliedPhysicsLetters,2009.95(20):p.201501JournalofAppliedPhysics,2010.107(10):103304李果：清华大学硕士论文（2009）ARTP:AtmosphericandRoomTemperaturePlasmas•ARTPgenerateshighlyhomogenousplasmajet•He2*speciesaremorestable0.0000.0020.0041E121E131E141E151E161E17BAB101710161015101410131012024粒子数密度（m-3）He2*eHe*135x(mm)SpatialdistributionofHe2*NumberdensityUnpublisheddata,2013AmporiMCSgfplacZpMD-18T-phoC3600bpDNADamagebyARTPMaldi-TOFspectrumofPolyAandG.(8nucleotides)0min1min2min4minTreatingTimePolyA5’-AAAAAAA-3’PolyG5’-gggggggg-3’PolyC5’-CCCCCCCC-3’PolyT5’-tttttttt-3’ΔA=313.2ΔA1=134.2901005040302010080010001200140024001600%IntM/CΔA2=118.1ΔA3=17.1ΔA1ΔA5'3'NNNNNH2OHHHHHHONNNNNH2OHOHHHHHOPHOOO(AMP)n,n=1~6.ΔA3(1)(2)28009010050403020100%Int80010001200140024001600M/C2800ΔG=329.2ΔG1=150.1ΔG2=118.1ΔG3=17.1NHNNONH2NOHHHHHOHONHNNONH2NOHHHHHOHOP-OOO(GMP)n,n=1~6.ΔGΔG1ΔG25'3'NNH2ONOHOHHHHHONNH2ONOHOHHHHHOP-OOO(CMP)n,n=1~6.9010050403020100%IntM/C800100012001400240016003'2800ΔC=289.2ΔCΔC1=110.1ΔC1ΔC25'ΔC2=95.0PolyA5’-AAAAAAA-3’PolyG5’-gggggggg-3’PolyC5’-CCCCCCCC-3’InsubmissiontoPPPQuantitativeDetectionofDNADamagebyDouble-strandsLabeledMicro-particles20μm03060901202.5x1043.0x1043.5x1044.0x1044.5x104Fluorecentintensity(-)ARTP-treatmenttime(s)Unpublisheddata,2013HomogenousARTPexposuretomicrobialcells?POumuDumuC''lacZlacYoriAPEcoRIEcoRILexARepressorProtein(Uninduced-fusiongene)(Induced-fusiongene)UmuC'-'LacZfusionprotein(Hybrid-galactosidase)PrincipleofumutestActivatedfilamentRecAProteinSOSSignalDNADamageTranscriptionTranslationm-RNASplitsinglestrandDNALexAself-autodigestionXUnpublisheddata,2013NM2009LB（25μg/mlAmp,5μg/mlCm）37℃,180rpm,15h1:50inoculatedtoTGA，culturetoOD0.3-0.5ARTPoperation37℃,180rpm,2hforβ-galactosidaseexpression200μlcultures+200μl1mMFDG,37℃,1min3.6mlPI(icecold,1μg/mlinPBS)wasaddedFDG:di-β-D-galactoppyranosideβ-galactosidaseFluoresceinFDG:SOSResponseRI:DeadCellsUniformDNAdamageincellsb