orthogonal surface tags for whole-cell biocatalysis 2017 theo peschke参考.pdf

该【orthogonal surface tags for whole-cell biocatalysis 2017 theo peschke参考 】是由【小舍儿】上传分享，文档一共【5】页，该文档可以免费在线阅读，需要了解更多关于【orthogonal surface tags for whole-cell biocatalysis 2017 theo peschke参考 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。AngewandteCommunicationsChemieInternationalEdition:DOI::DOI:-CellBiocatalysisTheoPeschke,,*Abstract:,suchastheoutermembraneproteinLpp-ompA.[13]thatdisplayorthogonaltagsforimmobilizationontheirWhereascell-surfacedisplayisextensivelyusedforthesurfaceandoverexpressafunctionalheterologous“ination[14,15]content”,[16,17]onlyfewreportsconcernitsapplicationmembraneproteinLpp-ompA,cell-surfacedisplayoftheforthespecificimmobilizationofhostcellsonsolidsub-streptavidin-bindingpeptide,theSpyTag/SpyCatchersystem,-surfaceanchoredcellu-oraHaloTagvariantallowedustogeneratebacterialstrainslaseorcellulose-bindingdomains,[18]chitin-bindingthatcanselectivelybindtosolidsubstrates,asdemonstrateddomains,[19]peptide-taggedamyloidproteins,[20]“sticky”cat--echolaminemoieties,[21]orcucurbituril-bindingpeptides,[22]-,tothebestofourknowledge,cell-surfacedisplayhasnotstrainsgavehighselectivitiesforspecificimmobilizationontoyetbeenusedfororthogonaltaggingandselectiveimmobi-complementarysurfacesandalsointhewhole-cellstereospe-binantbacteriathatcontainenzymesforcifictransformationofaprochiralCS--(Figure1).applicationsinbiocatalysis,[1,2]biosensing,[3]orwastewaterpurification.[4]Numerousmethodshavebeenexploredforbacterial-cellimmobilization,[1,5]whicharebasedoneitherencapsulationintohydrogelsandpolymers[6]oradsorptionontoporousandinertsupportmaterials.[7]Whereastheformerapproachprimarilyaimstoprotectbacteriafromhostileenvironments,-)-expressingfunctionalheterologousproteinsinitscytosolatthesameaswellasstabilizedconditions,highcelldensities,)ThemicroscopyimageshowsabacterialcellthatexpressesreusabilityforefficientandeconomicproductionoffuelsYFP(greenrod)anddisplaystheSBPtagonitssurfacetofacilitate[8,9]--covalentadsorptioncanleadtocelldamageandreducedbiologicalactivitiesorunstablecellattachment,respectively,directionalmethodsarecurrentlyToexperimentallyinvestigateourconcept,,suchinbioconjugationchemistrybuthavenotyetbeenexploitedasantibodies[eneticallyengineeredcell-wall-bindingforwhole-,[11]orrequiretheattachmentofasynthetictagtothestreptavidin-bindingpeptide(SBP)tag[23]bindstotheproteinbacterialcellwall,suchasoligonucleotides.[12]streptavidin(STV)monlyappliedTheso-calledcell-binantproteins.[24]elegantmeansforthedirectionalattachmentandpresenta-TheSpyTag/SpyCatchersystemconsistsofthe113aminoacidtionofpeptidesandproteinsonmicrobialcellwallsbymeanslongSpyCatcher(SC)protein,whichgeneratesacovalenticfusiontogenericmembraneanchormotifsoftheisopeptidebondbetweenoneofitslysineresiduesandanaspartateresidueofthe13aminoacidlongSpyTag(ST)peptide.[25]Theself-labelingHalo-basedoligonucleotide[*],,(HOB)tagprotein(293aminoacids)formsacovalentKarlsruheInstituteofTechnology(KIT)bondwithsmall-moleculechlorohexane(CH)ligandsinInstituteforBiologicalInterfaces(IBG1)Hermann-von-Helmholtz-PlatzasimilarfashionastheregularHaloTagprotein,whichis[26]76344Eggenstein-Leopoldshafen(-mail:******@:DNAoligonucleotidesandDNAnanostructureswithasig-http://dx./.nificantlyhigherefficiencythanHalo.[27],56,1–52017Wiley-VCHVerlagGmbH&,Weinheim1Thesearenotthefinalpagenumbers!üüAngewandteCommunicationsChemiePlasmidsencodingforafusionofthebacterialmembraneproteinLpp-ompAthatisconnectedtotheSBP,ST,SC,orHOBpolypeptidetagsviaaflexibleGGGGSlinkerwereclonedintothepTF16backboneofaplasmidthatcarriesanchloramphenicolresistanceandap15A-oriandforwhichtheproteinexpressionistightlycontrolledwithanarabinose-(DE3)withthebinantbacteriadisplayingeithertheSBP,ST,SC,orHOBtagontheirsurface,-SBP,-ST,-SC,-HOB,respectively,,aselectionoffluorescentprobes,namelyCy3-labeledSTV,SC-eGFP,andST-eGFPfusionproteinoraCH-derivatized22-meroligo-nucleotidelabeledwithCy5(Cy5-F5-CH),wereaddedtoasuspensionoftag-,thecellswerespundown,,thecellswereanalyzedbyfluores-cencemicroscopy(Figure2A),andquantitativedatawereobtainedwithafluorometricmicroplatereader(Figure2B).-,weusedmicrobeadscoatedwithSTV,CHligands,orSCprotein,inthefollowingdenotedasMB-STVs,MB-CHs,andMB-SCs,-merciallyavailable,MB-CHswerepreparedfromMB-STVsusingabiotin–CHlinker,andMB-SCsweregeneratedbycovalentimmobilizationofpurifiedSCproteinontoamino-reactive,epoxide--taggedfluorescentproteins(SBP-,Halo-,andST-taggedeGFPandmKate;,FigureS3).TheresultsclearlyEachofthefourdifferentstrainswasincubatedwiththefourdifferentindicatedthatthebeadswerecapableofspecificimmobiliza-,)Representativefluorescencemicro-Wethengeneratedanumberoftag-(blue:DAPI-stainedbacteria,red:STV-Cy3,purple:CH-F5-Cy5,:SC/ST-eGFP).Scalebars:)QuantitativefluorometricTheseplasmidswerebasedonthepETplasmidbackbone,(RFUs)peramountofcellsnormalizedtothehighestfluorescencesignalwithinpletelyorthogonaltothepFT16-,seeFiguresS1andS2,,acolE1-ori,andaT7promotor,(YFP)asthefirstontheirsurface,******@-SBP,cell–beadaggregates(FigureS4).******@-ST,******@--pounds,[2]weinvestigatedtheSTV,MB-SC,orMB-CHbeads(describedabove).Afteruseoftag-,icseparationplasmidsfortheoverexpressionofstereoselectiveenzymesandthenanalyzedbyfluorescencemicroscopy(Figure3).Itisinwhole-,(KREDs)ple-forthereductionoftheprochiralCS-,asboththebeadsandthe(NDK)1(Scheme1).[28]DependingontheKRED,substratebacteriapresentedmultiplecopiesoftheinteractionpartners1canbereducedoneitheroneorbothofthetwocarbonyl27Wiley-VCHVerlagGmbH&,,56,1–5üüThesearenotthefinalpagenumbers!AngewandteCommunicationsChemieTable1:Biocatalyticactivityofself--******@E.******@[%]STVSBPHOBSTSBPHOBSTR[a]S[b]SD[c]1x>99<1<12x>99<1<13x>99<1<14x<1>99<15x<1>99<16x<1>99<17xxx49648xxxx6941[a]OverallamountofR-configuredhydroxyketones2c/2danddiol3dasdeterminedbyHPLCanalysisonchiralstationaryphase.[b]OnlytheS-configuredhydroxyketone2bwasproducedunderthegivenreactionconditions.[c]Standarddeviation,obtainedfromatleasttwoinde-“functionalcontent”intheircytosolatthesametimecanbeselectivelyimmobilizedonbeadsbearingthechiralHPLCanalysis(Table1,entries1–6andTableS2).-thecaseofcellsexpressingtheenzymeLbADH,NDK1wasingtwoorthogonalplasmids,whichencodeforafusionproteinoftheconvertedintotheR-configuredhydroxyketones2c/2dandmembraneproteinLpp-ompAandtherespectivetag(SBP,HOB,ordiol3dwithadiastereoselectivityof>99%,asdeterminedST)aswellasfortheYFPcontent(visibleasgreenfeatures).Selectivebindingtothebeadsleadstotheformationofcell–(entries1–3).Incontrast,cellsScalebars:-functionalgroups,andallpossibleisomerichydroxy-ketoneanddiolproductscanbeseparatedandiden-tifiedbychiralHPLCanal-ysis.[28]Therefore,thetrans-formationof1wasideallysuitedtostudytheactivityofourself--face-displayedSBP,HOB,binedwitheithertheR-selectivealcoholdehydrogenasefromLactobacillusbrevis14869(LbADH)ortheS-selectivealcoholdehydrogenasefromSac-charomycescerevisiaeYJM193(Gre2p),******@-SBP,******@-HOB,******@-ST,******@-SBP,Gre2-******@-HOB,andGre2-******@-ST,respectively(Table1).)Sequentialbiocatalyticreductionof5-nitrononane-2,8-dione(NDK;1)enablesthestereoselec-)Thetwomodelenzymesusedinthisbiocatalyticproperties,westudy,Gre2pandLbADH,leadtoselectiveformationofhydroxyketones2band2c/2d,-eptshydroxyketonesassubstrates,LbADHcanreducethehydroxyketones2c/2dtoproduce[28]cellenzymaticactivityofthepseudo-C2-(FigureS5).,56,1–52017Wiley-VCHVerlagGmbH&,Weinheim3Thesearenotthefinalpagenumbers!üüAngewandteCommunicationsChemieconfiguredhydroxyketone2bwithanexcellentselectivityofKeywords:bacteria·biocatalysis·cell-surfacedisplay·>99%(entries4–6),asexpectedfromourpreviousstudy.[28]immobilizationtechniques·stereoselectivereactionsOwingtothehighstereoselectivities,the“catalyticcontent”wasalsousedasamarkerfortheselectiveself--******@[1]a),,,BiocatalystsandpetitivelybindontoMB-,2nded.,Wiley-Blackwell,Weinheim,2012;icseparation,b)MethodsinBiotechnology,(Ed.:),2ndregrown,.,Humana,Totowa,2013,[2],..,13,******@-HOBand10086.******@-SBPledtoalmostexclusiveformationofthe[3],