Metabolic engineering of Escherichia coli for improved 6-deoxyerythronolide B production

Abstract

Escherichia coli is an attractive candidate as a host for polyketide production and has been engineered to produce the erythromycin precursor polyketide 6-deoxyerythronolide B (6dEB). In order to identify and optimize parameters that affect polyketide production in engineered E. coli, we first investigated the supply of the extender unit ( 2S)-methylmalonyl-CoA via three independent pathways. Expression of the Streptomyces coelicolor malonyl/methylmalonyl-CoA ligase ( matB) pathway in E. coli together with methylmalonate feeding resulted in the accumulation of intracellular methylmalonyl-CoA to as much as 90% of the acyl-CoA pool. Surprisingly, the methylmalonyl-CoA generated from the matB pathway was not converted into 6dEB. In strains expressing either the S. coelicolor propionyl-CoA carboxylase (PCC) pathway or the Propionibacteria shermanii methylmalonyl-CoA mutase/epimerase pathway, methylmalonyl-CoA accumulated up to 30% of the total acyl-CoA pools, and 6dEB was produced; titers were fivefold higher when strains contained the PCC pathway rather than the mutase pathway. When the PCC and mutase pathways were expressed simultaneously, the PCC pathway predominated, as indicated by greater flux of (13)C-propionate into 6dEB through the PCC pathway. To further optimize the E. coli production strain, we improved 6dEB titers by integrating the PCC and mutase pathways into the E. coli chromosome and by expressing the 6-deoxyerythronolide B synthase (DEBS) genes from a stable plasmid system.

A Single Module Type I Polyketide Synthase Directs de Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

“…5 and Table 3). Propionate can be converted to propionyl-CoA and subsequently to (2S)-methylmalonyl-CoA by endogenous propionate-CoA synthetase and propionyl-CoA carboxylase (20). Thus, intact incorporation of propionate into the polyketide backbone is indicative of a methylmalonyl-CoA origin.…”

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

“…Presently, although the molecular genetic tools have been developed for most of the important antibiotic-producing organisms and much useful information has been gained describing the genetic control of antibiotic biosynthesis, the application of this new technology to strain improvement of secondary metabolites has met with limited commercial success. Nevertheless, many new innovative approaches towards secondary metabolite strain improvement are now available or under development using a variety of different approaches, some of which show promise for general commercial application to the field (Askenazi et al, 2003;Tang et al, 2000;Murli et al, 2003;Zhang et al, 2002;Lum et al, 2004, Brunker et al, 1998.…”

A specific role of the Saccharopolyspora erythraea thioesterase II gene in the function of modular polyketide synthases

“…Samples of the supernatants were analysed by online extraction by LC-MS. For LC-MS analyses, 20 ml clarified broth was chromatographed on a Phenomenex Develosil column (5 mm, 4?66150 mm) with a mobile phase of 39 % 3 : 2 (v/v) MeCN 15-nor-6-deoxyerythromycin B: Co-expression of ery-ORF5 TEII and eryA DEBS genes in E. coli. The E. coli K207-3 host strain for 6dEB production has been described previously (Murli et al, 2003). Briefly, this strain has four T7-promoter-regulated genes integrated in the chromosome: sfp (required to pantetheinylate the DEBS proteins), prpE (required to convert propionate to propionyl-CoA) and accA1/pccB [required to convert propionyl-CoA to (2S)-methylmalonyl-CoA)].…”