Metabolic Engineering of a Methylmalonyl-CoA Mutase−Epimerase Pathway for Complex Polyketide Biosynthesis in Escherichia coli

• Published in: Biochemistry (Easton) 2002-04, Vol.41 (16), p.5193-5201
• Main Authors: Dayem, Linda C, Carney, John R, Santi, Daniel V, Pfeifer, Blaine A, Khosla, Chaitan, Kealey, James T
• Format: Article
• Language: English
• Subjects: Cloning, Molecular; Enzyme Activation - genetics; Escherichia coli - enzymology; Escherichia coli - genetics; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genetic Vectors - chemical synthesis; Genetic Vectors - metabolism; Methylmalonyl-CoA Mutase - chemistry; Methylmalonyl-CoA Mutase - genetics; Methylmalonyl-CoA Mutase - metabolism; Molecular Sequence Data; Multienzyme Complexes - chemistry; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Polymerase Chain Reaction - methods; Propionibacterium - enzymology; Propionibacterium - genetics; Protein Engineering - methods
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi015593k

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 B12-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 ∼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 [13C]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.