title: Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli.
abstract: A barrier to heterologous production of complex polyketides in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common extender substrate for the biosynthesis of complex polyketides by modular polyketide synthases. One biosynthetic route to (2S)-methylmalonyl-CoA involves the sequential actions of two enzymes, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase, which convert succinyl-CoA to (2R)- and then to (2S)-methylmalonyl-CoA. As reported [McKie, N., et al. (1990) Biochem. J. 269, 293-298; Haller, T., et al. (2000) Biochemistry 39, 4622-4629], when genes encoding coenzyme B(12)-dependent methylmalonyl-CoA mutases were expressed in E. coli, the inactive apo-enzyme was produced. However, when cells harboring the mutase genes from Propionibacterium shermanii or E. coli were treated with the B12 precursor hydroxocobalamin, active holo-enzyme was isolated, and (2R)-methylmalonyl-CoA represented approximately 10% of the intracellular CoA pool. When the E. coli BAP1 cell line [Pfeifer, B. A., et al. (2001) Science 291, 1790-1792] harboring plasmids that expressed P. shermanii methylmalonyl-CoA mutase, Streptomyces coelicolor methylmalonyl-CoA epimerase, and the polyketide synthase DEBS (6-deoxyerythronolide B synthase) was fed propionate and hydroxocobalamin, the polyketide 6-deoxyerythronolide B (6-dEB) was produced. Isotopic labeling studies using [(13)C]propionate showed that the starter unit for polyketide synthesis was derived exclusively from exogenous propionate, while the extender units stemmed from methylmalonyl-CoA via the mutase-epimerase pathway. Thus, the introduction of an engineered mutase-epimerase pathway in E. coli enabled the uncoupling of carbon sources used to produce starter and extender units of polyketides.
authors.affiliation: Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, and Departments of Chemical Engineering, Chemistry, and Biochemistry, Stanford University, Stanford, California 94305.
authors.forename: Linda C
authors.initials: LC
authors.lastname: Dayem
authors.forename: John R
authors.initials: JR
authors.lastname: Carney
authors.forename: Daniel V
authors.initials: DV
authors.lastname: Santi
authors.forename: Blaine A
authors.initials: BA
authors.lastname: Pfeifer
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
authors.forename: James T
authors.initials: JT
authors.lastname: Kealey
mesh_terms: D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D008765:Methylmalonyl-CoA Mutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011424:Propionibacterium / Q000201:enzymology / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*
chemical_list: D009097:Multienzyme Complexes; D008765:Methylmalonyl-CoA Mutase