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Isobutyryl-CoA and isovaleryl-CoA are important building blocks for many valuable natural compounds.Escherichia coli have been used as a heterologous host for the production of a variety of natural compounds.In the current study,we achieved de novo biosynthesis of the branched-chain acyl-CoAs from glucose in E.coli by integration of the branched-chain α-keto acid dehydrogenase (BCDH) complex from Streptomyces avermitilis.In the presence of chloramphenicol and the chloramphenicol acetyltransferase (cat) gene,both chloramphenicol-3-isobutyrate and chloramphenicol 3-isovalerate were detected in the recombinant E.coli strains,indicating efficient biosynthesis of isobutyryl-CoA and isovaleryl-CoA.Furthermore,we enhanced the production of isobutyryl-CoA and isovaleryl-CoA by overexpressing the α-keto acid module consisting of the alsS gene from Bacillus subtilis,the ilvC and ilvD genes from E.coli.This is the first report of production of isobutyryl-CoA and isovaleryl-CoA in E.coli and opens a way to biosynthesize a variety of valuable natural compounds based on these special building blocks in E.coli from renewable carbon sources.