Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli.

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Dayem LC, Carney JR, Santi DV, Pfeifer BA, Khosla C, Kealey JT

Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli.

Biochemistry. 2002 Apr 23;41(16):5193-201.

PubMed ID

11955068 [ View in PubMed

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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.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Hydroxocobalamin	Methylmalonyl-CoA mutase, mitochondrial	Protein	Humans	Yes	Cofactor	Details