| 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 bymodular polyketide synthases. One biosynthetic route to (2S)-methylmalonyl-CoA involves the sequentialactions of two enzymes, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase, which convertsuccinyl-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 coenzymeB12-dependent methylmalonyl-CoA mutases were expressed in E. coli, the inactive apo-enzyme wasproduced. However, when cells harboring the mutase genes from Propionibacterium shermanii or E. coliwere 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) Science291, 1790−1792] harboring plasmids that expressed P. shermaniimethylmalonyl-CoA mutase, Streptomyces coelicolor methylmalonyl-CoA epimerase, and the polyketidesynthase DEBS (6-deoxyerythronolide B synthase) was fed propionate and hydroxocobalamin, thepolyketide 6-deoxyerythronolide B (6-dEB) was produced. Isotopic labeling studies using [13C]propionateshowed 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 carbonsources used to produce starter and extender units of polyketides.
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