Metabolic Engineering of a Methylmalonyl-CoA Mutase-Epimerase Pathway for Complex Polyketide Biosynthesis in Escherichia coli ²,‡ Linda C. Dayem, § John R. Carney, § Daniel V. Santi, § Blaine A. Pfeifer, | Chaitan Khosla, | and James T. Kealey* ,§ Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, and Departments of Chemical Engineering, Chemistry, and Biochemistry, Stanford UniVersity, Stanford, California 94305 ReceiVed August 2, 2001; ReVised Manuscript ReceiVed January 8, 2002 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 ∼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. Polyketides are complex natural products that are particu- larly abundant in soil microorganisms (1). Although fewer than 10 000 polyketides have been identified to date, they include a large number of major pharmaceuticals that span a broad range of therapeutic areas, including cancer (adria- mycin), infectious disease (tetracyclines, erythromycin), cardiovascular (mevacor, lovastatin), and immunosuppression (rapamycin, tacrolimus). Complex polyketides are produced by modular polyketide synthases (PKSs) 1 slarge, multifunctional enzymes that are organized into multiple catalytic “modules”, each containing a set of 3-6 functional domains that determine the identity of a 2-carbon unit of the polyketide. As illustrated in Figure 1 for the prototypical deoxyerythronolide B synthase (DEBS) (2), modules are linearly arranged, beginning with a starter module, followed by a number of extender modules, and terminating with a releasing domain. Each of the modules recognizes the acyl group of a specific acyl-CoA, catalyzes its condensation with an acyl-group tethered to the preceding module to form a nascent polyketide chain, and modifies the -carbon atom of the growing chain. This extension/ modification process occurs in an assembly line fashion through the entire sequence of modules, resulting in a one- to-one correlation between the constituent enzymatic activi- ties of a PKS module and the corresponding 2-carbon ketide unit of a polyketide. Owing to the modular nature of polyketide synthesis, genetic engineering can be used to create specific structural modifications of polyketides in a predictable fashion and to produce new libraries of these natural products. In theory, by modifying the genes that encode PKS modules, a specific 2-carbon unit of a polyketide may be changed to one of about 20 others, reflecting the combinations of chain extender units (malonate, methylmalonate, or rarer units), the fate of the incoming -keto group at each step of chain extension (keto, hydroxyl, enoyl, or methylene), and the stereochemistry of methyl and hydroxyl branches. In practice, such modifica- tions are complicated by several factors, including the facts ² This research was supported in part by NIH Grant R01-CA66736 to C.K. ‡ The P. shermanii methylmalonyl-CoA epimerase DNA sequence has been deposited into the GenBank database under accession number AY046899. * Address correspondence to this author. Telephone: 510-732-8400, x224. Fax: 510-732-8401. Email: kealey@kosan.com. § Kosan Biosciences, Inc. | Stanford University. 1 Abbreviations: CoA, coenzyme A; PCR, polymerase chain reac- tion; HPLC, high-performance liquid chromatography; DEBS, 6-deoxy- erythronolide B synthase; IPTG, -isopropyl-thiogalactoside; EDTA, ethylenediaminetetraacetic acid; TCA, trichloroacetic acid; LB, Luria- Bertani medium; DTT, dithiothreitol; NADH, nicotinamide adenine dinucleotide; TLC, thin-layer chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TKL, (2R,3S,4S,5R)- 2,4-dimethyl-3,5-dihydroxy-n-heptanoic acid ∂-lactone (triketide lac- tone); 6-dEB, 6-deoxyerythronolide B; ELSD, evaporative light- scattering detector; ESI-TOF, electrospray ionization time-of-flight. 5193 Biochemistry 2002, 41, 5193-5201 10.1021/bi015593k CCC: $22.00 © 2002 American Chemical Society Published on Web 03/29/2002