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2000-Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isopr

大肠杆菌的代谢研究

2000-Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isopr

ApplMicrobiolBiotechnol(2000)53:396±400ÓSpringer-Verlag2000

P.D.MatthewsáE.T.Wurtzel

MetabolicengineeringofcarotenoidaccumulationinEscherichiacolibymodulationoftheisoprenoidprecursorpoolwithexpressionofdeoxyxylulosephosphatesynthase

Received:6August1999/Receivedrevision:25October1999/Accepted:5November1999

AbstractTherecentlydiscoverednon-mevalonatepathwaytoisoprenoids,whichusesglycolyticinterme-diates,hasbeenmodulatedbyoverexpressionofEsc-herichiacoliD-1-deoxyxylulose5-phosphatesynthase(DXS)toincreasedeoxyxylulose5-phosphateand,consequently,increasetheisoprenoidprecursorpoolinE.coli.Carotenoidsarealargeclassofbiologicallyimportantcompoundssynthesizedfromisoprenoidprecursorsandofinterestformetabolicengineering.However,carotenoidsarenotordinarilypresentinE.coli.Co-overexpressionofE.colidxswithErwiniauredovorageneclustersencodingcarotenoidbiosyn-theticenzymesledtoanincreasedaccumulationofthecarotenoidslycopeneorzeaxanthinovercontrolsnotexpressingDXS.Thus,rate-controllingenzymesencod-edbythecarotenogenicgeneclustersareresponsivetoanincreaseinisoprenoidprecursorpools.Levelsofac-cumulatedcarotenoidswereincreasedupto10.8timesthelevelsofcontrolsnotoverexpressingDXS.Lycopeneaccumulatedtoalevelashighas1333lg/gdwandzeaxanthinaccumulatedtoalevelashighas592lg/gdw,whenpigmentswereextractedfromcolonies.Zeaxanthin-producingcoloniesgrewabouttwiceasfastaslycopene-producingcoloniesthroughoutatimecourseof11days.Metabolicengineeringofcarbon¯owfromsimpleglucosemetabolitestorepresentativesofthelargestclassofnaturalproductswasdemonstratedinthismodelsystem.

Introduction

Isoprenoidsareadiversegroupofnaturalproductsfoundinallorganisms.TheyarederivedfromtheC5skeletonofisopentenylpyrophosphate(IPP).IPPwasthoughttobestrictlyderivedfromthemevalonatepathway,andtheenzymeHMGCoAreductasewasconsideredtobearate-limitingenzymeforthisbiosyn-theticroute(Dimster-Denketal.1994;Chappell1995).Recently,anovelnon-mevalonatebiosyntheticpathwaywasdiscoveredthatoperatesspeci®callyinplastidsofplants(reviewedinLichtenthaler1999)aswellasinbacteriaandincyanobacteria;inplants,themevalonatepathwayoperatesinthecytoplasminparallelwiththeplastid-localizednon-mevalonateIPPpathway(Rohmeretal.1993;Schwenderetal.1996).Inthenon-mevalo-nateroute,IPPisderivedfromdeoxyxylulose5-phos-phate(DXP),whichinEscherichiacolihasalsobeenfoundtobeacommonprecursorinthebiosynthesisofvitaminsB1(thiamin)andB6(pyridoxal)(Sprengeretal.1997).TheenzymeD-1-deoxyxylulose5-phosphatesyn-thase(DXS)isresponsibleforcatalyzingthesynthesisofDXPfrompyruvateandGAP(glyceraldehyde3-phos-phate)(Sprengeretal.1997;Langeetal.1998;Loisetal.1998).Inplantsandbacteria,competitionforDXPmighta ectend-productaccumulationfordi erentisoprenoid-derivedbiosyntheticpathways.TheenzymeresponsibleforDXPsynthesismightrepresentanovelrate-controllingenzymewhoseexpressioncouldbemodi®edtoincreasesubstrateavailabilityforpathwaysunderconsiderationformetabolicengineering.

Carotenoidsareoneexampleofisoprenoidsforwhichinterestinmetabolicengineeringrelatestotheiruseful-nessascoloringagentsandasprecursorstovitaminAandtoretinoids,compoundsessentialtogrowthanddevelopment(Misawaetal.1991,1993;Yamanoetal.1994;Burkhardtetal.1997;Rutheretal.1997;Wangetal.1999).Carotenoidsaresynthesizedincertainbac-teria,fungi,andinplastidsofplants(asreviewedinArmstrongandHearst1996;CunninghamandGantt

P.D.MatthewsáE.T.Wurtzel(&)

DepartmentofBiologicalSciences,LehmanCollegeandTheGraduateSchoolandUniversityCenter,TheCityUniversityofNewYork,250BedfordParkBoulevardWest,Bronx,NY10468,USA

e-mail:etwlc@cunyvm.cuny.eduTel.:+1-718-9608643

Fax:+1-718-9608236/9607348

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