Investigating the role of native propionyl‐CoA and methylmalonyl‐CoA metabolism on heterologous polyketide production in Escherichia coli

Abstract

6-Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over-expression of a native prpE gene (encoding a propionyl-CoA synthetase) and heterologous pcc genes (encoding a propionyl-CoA carboxylase) to supply the needed propionyl-CoA and (2S)-methylmalonyl-CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl-CoA mutase), YgfG (a methylmalonyl-CoA decarboxylase), and YgfH (a propionyl-CoA:succinate CoA transferase), also involved in propionyl-CoA and methylmalonyl-CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over-expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over-expression of sbm did not influence 6dEB production; ygfG over-expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L.

Engineering an iterative polyketide pathway in Escherichia coli results in single-form alkene and alkane overproduction

“…While in the first report of erythromycin production in E. coli, a yield of only $ 20 mg/L of 6-deoxyerythronolide B (6-dEB) was obtained (Pfeifer et al, 2001). Through engineering and optimization of the 6-dEB biosynthesis pathway, a final titer of over 100 mg/L was obtained in E. coli (Boghigian et al, 2011;Pfeifer et al, 2002;Zhang et al, 2009). Given 6.…”

Construction of a novel anaerobic pathway in Escherichia coli for propionate production

“…There were also researches concerning products involved Sbm cycle. Zhang et al studied the role of propionyl-CoA and methylmalonyl-CoA metabolism, and found propionyl-CoA could be supplied for polyketide production by E. coli in complex media, but the Sbm cycle might not be directly involved [20]. In the research for propanol production, Sbm cycle was also employed to convert succinyl-CoA to propionyl-CoA.…”

Heterologous expression of diverse propionyl-CoA carboxylases affects polyketide production in Escherichia coli

“…8 For many assembly-line PKS pathways, the extender unit for the polyketide chain is (2 S )-methylmalonyl-CoA (mmCoA), a metabolite not naturally produced by E. coli. 9 Murli et al 10 compared three routes of heterologous mmCoA production in E. coli : (1) carboxylation of propionyl-CoA by the propionyl-CoA carboxylase (PCC) from Streptomyces coelicolor , (2) conversion of succinyl-CoA to (2 R )-mmCoA and then to (2 S )-mmCoA by the mmCoA mutase/epimerase from Propionibacterium shermanii and (3) synthesis of (2 S )-mmCoA from methylmalonate by the malonyl/methylmalonyl-CoA ligase from S. coelicolor . The 6-dEB titers varied depending on the means of mmCoA production, with the PCC system consistently proving the most productive.…”