title: Multi-factorial engineering of heterologous polyketide production in Escherichia coli reveals complex pathway interactions.
abstract: Polyketides represent a significant fraction of all natural products. Many possess pharmacological activity which makes them attractive drug candidates. The production of the parent macrocyclic aglycones is catalyzed by multi-modular polyketide synthases utilizing short-chain acyl-CoA monomers. When producing polyketides through heterologous hosts, one must not only functionally express the synthase itself, but activate the machinery used to generate the required substrate acyl-CoA's. As a result, metabolic engineering of these pathways is necessary for high-level production of heterologous polyketides. In this study, we over-express three different pathways for provision of the two substrates (propionyl-CoA and (2S)-methylmalonyl-CoA) utilized for the biosynthesis of 6-deoxyerythronolide B (6-dEB; the macrolactone precursor of erythromycin): (1) a propionate → propionyl-CoA → (2S)-methylmalonyl-CoA pathway, (2) a methylmalonate → methylmalonyl-CoA → propionyl-CoA pathway, and (3) a succinate → succinyl-CoA → (2R)-methylmalonyl-CoA → (2S)-methylmalonyl-CoA → propionyl-CoA pathway. The current study revealed that propionate is a necessary component for greater than 5 mg L(-1) titers. Deletion of the propionyl-CoA:succinate CoA transferase (ygfH) or over-expression of the transcriptional activator of short chain fatty acid uptake improved titer to over 100 mg L(-1), while the combination of the two improved titer to over 130 mg L(-1). The addition of exogenous methylmalonate could also improve titer to over 100 mg L(-1). Expression of a Streptomyces coelicolor A3(2) methylmalonyl-CoA epimerase, in conjunction with over-expression of Escherichia coli's native methylmalonyl-CoA mutase, allowed for the incorporation of exogenously fed succinate into the 6-dEB core.
authors.affiliation: Department of Chemical and Biological Engineering, Science and Technology Center, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, USA.
authors.forename: Brett A
authors.initials: BA
authors.lastname: Boghigian
authors.forename: Haoran
authors.initials: H
authors.lastname: Zhang
authors.forename: Blaine A
authors.initials: BA
authors.lastname: Pfeifer
mesh_terms: D001483:Base Sequence; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D018942:Macrolides / Q000378:metabolism*; D008764:Methylmalonic Acid / Q000378:metabolism; D008765:Methylmalonyl-CoA Mutase / Q000235:genetics / Q000378:metabolism; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism; D011422:Propionates / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D018942:Macrolides; D011422:Propionates; C034277:6-deoxyerythronolide B; D004917:Erythromycin; D048630:Polyketide Synthases; D008764:Methylmalonic Acid; D008765:Methylmalonyl-CoA Mutase