EnzymaticSynthesisofa Bacterial Polyketide fromAcetyl and MalonylCoenzyme A

Microorganisms andplants manufacture a large collection ofmedically andcommercially useful natural products called polyketides bya processthat resembles fatty acid biosynthesis. Genetically engineered microorganisms withmodified polyketide synthase (PKS) genescanproduce new metabolites that may havenew orimproved pharmacological activity. A potentially general method topreparecell-free systems forstudying bacterial type11PKSenzymeshasbeendeveloped that facilitates thepurification andreconstitution oftheir constituent proteins. Selective expression ofdifferent combinations oftheStreptomyces glaucescens tetracenomycin (Tcm)tcmJKLMN genesina tcmGHIJKLMNO null background hasbeenusedtoshowthat theTcmPKSconsists ofatleast theTcmKLMN proteins. Addition oftheTcmJprotein tothelatter four enzymesresulted inagreater than fourfold increase ofoverall activity andthusrepresents theoptimal TcmPKS.Polyclonal antibodies raised against eachoftheTcmKLMNproteins strongly inhibit theTcmPKS, as doknowninhibitors targeted totheactive site CysandSerresidues ofafatty acid synthase. Thissystemexhibits a strict starter unit specificity because neither propionyl, butyryl, or isobutyryl coenzymeA substitute foracetyl coenzymeA inassembly oftheTcmdecaketide. Because theTcmPKSactivity issignificantly diminished byremoval oftheTcmM acylcarrier protein andcanberestored byaddition ofseparately purified TcmMtotwo different typesofTcmM-deficient PKS,itshould bepossible tousesuchpreparations to assayforeachoftheconstituents oftheTcmPKS.