Enhanced Incorporation of 3-Hydroxy-4-Methylvalerate Unit into Biosynthetic Polyhydroxyalkanoate Using Leucine as a Precursor.docVIP

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Enhanced Incorporation of 3-Hydroxy-4-Methylvalerate Unit into Biosynthetic Polyhydroxyalkanoate Using Leucine as a Precursor.doc

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Enhanced Incorporation of 3-Hydroxy-4-Methylvalerate Unit into Biosynthetic Polyhydroxyalkanoate Using Leucine as a Precursor

Saikaetal.AMBExpress2011,1:6 /content/1/1/6 ORIGINAL OpenAccess EnhancedIncorporationof3-Hydroxy-4- MethylvalerateUnitintoBiosynthetic PolyhydroxyalkanoateUsingLeucineasa Precursor AzusaSaika1,YorikoWatanabe1,KumarSudesh2,HidekiAbe3andTakeharuTsuge1* Abstract RalstoniaeutrophaPHB-4expressingPseudomonassp.61-3polyhydroxyalkanoate(PHA)synthase1(PhaC1Ps) synthesizesPHAcopolymercontaining3-hydroxybutyrate(3HB)andasmallamount(0.5mol%)of3-hydroxy-4- methylvalerate(3H4MV)fromfructoseasacarbonsource.Inthisstudy,enhancedincorporationof3H4MVinto PHAwasinvestigatedusingbranchedaminoacidleucineasaprecursorof3H4MV.Leucinehasthesamecarbon backboneas3H4MVandisexpectedtobeanaturalandself-producibleprecursor.Wefoundthatthe incorporationof3H4MVwasenhancedbythesupplementationofexcessamount(10g/L)ofleucineintheculture medium.Thisfindingindicatesthat3H4MVcanbederivedfromleucine.Toincreasemetabolicfluxtoleucine biosynthesisinthehoststrainbyeliminatingthefeedbackinhibition,thecellsweresubjectedtoN-methyl-N’-nitro- N-nitrosoguanidine(NTG)mutagenesisandleucineanalogresistantmutantsweregenerated.Themutantsshowed statisticallyhigher3H4MVfractionthantheparentstrainwithoutsupplementingleucine.Additionally,bysupplying excessamountofleucine,themutantssynthesized3HB-basedPHAcopolymercontaining3.1mol%3H4MVand 1.2mol%3-hydroxyvalerate(3HV)asminorconstituents,whichsignificantlyaffectedthethermalpropertiesofthe copolymer.Thisstudydemonstratesthatitispossibletoenhancethemonomersupplyof3H4MVintoPHAby manipulatingleucinemetabolism. Keywords:polyhydroxyalkanoatecopolymer,3H4MVprecursor,leucineanalogresistantmutant Introduction because of its high crystallinity (Sudesh et al. 2000). Polyhydroxyalkanoate(PHA)isakindofaliphaticpolye- Thus, the application of P(3HB) is limited. The stersynthesizedbyawidevarietyofmicroorganismsas mechanical properties of P(3HB) can be effectively intracellular storage and carbon source (Sudesh et al. improved by copolymerization with (R)-3-hydroxyalk- 2000). Itc