2005-Glucose metabolism at high density growth of

GlucoseMetabolismatHighDensityGrowthofE.coliBandE.coliK:DifferencesinMetabolicPathwaysAreResponsibleforEfficientGlucoseUtilizationinE.coliBasDeterminedbyMicroarraysandNorthernBlotAnalysesJe-NiePhue,1SantoshB.Noronha,2RitabrataHattacharyya,2AlanJ.Wolfe,3JosephShiloach11BiotechnologyUnit,NIHNIDDK,Bethesda,Maryland20892-2715;telephone:[phone];fax:[phone];e-mail:[e-mail]2DepartmentofChemicalEngineeringIIT-Bombay,Powai,Mumbai400076India3DepartmentofMicrobiologyandImmunology,LoyolaUniversityChicago,Maywood,IL60153Received1June2004;accepted18January2005Publishedonline1April2005inWileyInterScience().DOI:10.1002/bit.20478Abstract:Inaseriesofpreviousreportsitwasestablishedbyimplementingmetabolicflux,NMR/MS,andNorthernblotanalysisthattheglyoxylateshunt,theTCAcycle,andacetateuptakebyacetyl-CoAsynthetasearemoreactiveinEscherichiacoliBL21thaninEscherichiacoliJM109.Thesedifferenceswereacceptedasthereasonforthedifferencesintheglucosemetabolismandacetateexcretionofthesetwostrains.ExaminationofthebacterialmetabolismbymicroarraysandtimecourseNorthernblotshowedthatinadditiontotheglyoxylateshunt,theTCAcycleandtheacetateuptake,othermetabolicpathwaysareactivedifferentlyinthetwostrains.Thesearegluconeogenesis,sfcAshunt,ppcshunt,glycogenbiosynthesis,andfattyaciddegradation.ItwasfoundthatinE.coliJM109,acetateisproducedbypyruvateoxidase(poxB)usingpyruvateasasubstrateratherthanbyphosphotransacetylase-acetatekinase(Pta-AckA)systemwhichusesacetyl-CoA.Theinactivationofthegluconeogenesisenzymephosphoenolpyruvatesynthetase(ppsA),theactivationoftheanapleroticsfcAshunt,andlowandstablepyruvatedehydrogenase(aceE,aceF)causepyruvateaccumulationwhichisconvertedtoacetatebypyruvateoxidaseB.ThebehavioroftheppsA,acs,andaceBAKinJM109wasdependentontheglucosesupplystrategy.Whentheglucoseconcentrationwashigh,notranscriptionofthesegeneswasobservedandacetateconcentrationincreased,butatlowglucoseconcentrationsthesegeneswereexpressedandtheacetateconcentrationdecreased.ItispossiblethatthereisamajorregulatorymoleculethatcontrolsnotonlyppsAandaceBAKbutalsoacs.Thegluconeogenesispathway(fbp,pckA,andppsA)whichleadstoglycogenaccumulationisconstitutivelyactiveinE.coliBL21regardlessofglucosefeedingstrategy.Published2005WileyPeriodicals,Inc.{Keywords:Escherichiacoli;acetate;glucose;Northernblot;gluconeogenesisINTRODUCTIONOneofthemajorconsiderationsduringhigh-densitygrowthofE.colionglucoseisacetateaccumulation,particularlyimportantwhenthegrowthisperformedforrecombinantproteinbiosynthesis,sinceacetateconcentrationsabove40mM(2.4g/L)affectgrowthandpossiblyrecombinantproteinproduction(KlemanandStrohl,1994;Koetal.,1995).Themetabolismofglucoseandacetatehasbeenintensivelyinvestigatedduringthepast50yearsandarewellcharacterized(Cozzone,1998).Briefly,whenE.coliisgrownonglucose,acetateisproducedfromacetyl-CoAbyphosphotransacetylase(pta)andacetatekinase(ackA)andfrompyruvatebypyruvateoxidase(poxB)(Abdel-Hamidetal.,2001;El-MansiandHolms,1989;KlemanandStrohl,1994).Theacetateformedcanbeconvertedbacktoacetyl-CoAbyacetyl-CoAsynthetase(acs)andbyreversingthepta-ackApathway(Fig.1).Theacetyl-CoAismetabolizedthroughtheTCAcycleandtheglyoxylateshuntpathwaybyisocitratelyase(aceA)andmalatesynthetase(aceB)(El-MansiandHolms,1989;Kornberg,1966).ThecommonexplanationforacetateaccumulationisthehighcarbonfluxthroughglycolysiswhichexceedstheTCAcyclecapacity,especiallywhenglucoseisinexcess.Gradualadditionofglucosetothecultureusingfed-batchtechniquesandthedevelopmentofmutantstrainswithalteredmetabolicpatternshavebeenimplementedtolimitacetateaccumula-tionsduringhighdensitygrowth(Contieroetal.,2000;Lee,1996;RiesenbergandGuthke,1999).Acetateisutilizedbytheglyoxylateshunt(Kornberg,1966;Ohetal.,2002),awell-studiedpathwaythatisregulatedbytheglyoxylateshuntoperon(aceBAK)thatPublished2005WileyPeriodicals,Inc.{ThisarticleisaUSgovernmentworkand,assuch,isinthepublicdomainintheUnitedStatesofAmerica.Correspondenceto:J.ShiloachcontainsaceK(isocitratedehydrogenasekinase/phospha-tase),aceA(isocitratelyase),andaceB(malatesynthetase)(Chungetal.,1988).Isocitratedehydrogenasehasavitalroleintheregulationoftheglyoxylateshunt(ThorsnessandKoshland,1987),sincetheproteinitselfisregulatedbyphosphorylationanddephosphorylationthroughisocitratedehydrogenasekinase/phosphatase(aceK)(Borthwicketal.,1984;Cozzone,1998;Stuelandetal.,1988).Theexpressionoftheglyoxylateoperonisunderthenegativecontroloftworegulatoryproteins:isocitratelyaserepresor(iclR),whichinhibitstheexpressionoftheglyoxylateoperon,andtheregulatoroffattyacidmetabolism(fadR),whichactivatestheexpressionoftheiclRgene(Guietal.,1996;Sunnarborgetal.,1990).Theglyoxylateoperonexpressionisalsounderthepositivecontroloftworegulatoryproteins:fructoserepressor(fruR)andintegrationhostfactor(IHF),bothstimulatethetranscriptionofaceBAK(Ramseieretal.,1995;Resniketal.,1996).Anotherpathwaythatplaysanessentialrolewhenutiliz-ingacetateisgluconeogenesis.Thispathwaysynthesizesglucosefromnonhexoseprecursors,byconvertingpyruvatetophosphoenolpyruvatethroughphosphoen