title: 6-Deoxyerythronolide B analogue production in Escherichia coli through metabolic pathway engineering.
abstract: The erythromycin precursor polyketide 6-deoxyerythronolide B (6-dEB) is produced from one propionyl-CoA starter unit and six (2S)-methylmalonyl-CoA extender units. In vitro studies have previously demonstrated that the loading module of 6-deoxyerythronolide B synthase (DEBS) exhibits relaxed substrate specificity and is able to accept butyryl-CoA, leading to the production of polyketides with butyrate starter units. We have shown that we can produce butyryl-CoA at levels of up to 50% of the total CoA pool in Escherichia coli cells that overexpress the acetoacetyl-CoA:acetyl-CoA transferase, AtoAD (EC 2.8.3.8), in media supplemented with butyrate. The DEBS polyketide synthase (PKS) used butyryl-CoA and methylmalonyl-CoA supplied in vivo by the AtoAD and methylmalonyl-CoA mutase pathways, respectively, to produce 15-methyl-6-dEB. Priming DEBS with endogenous butyryl-CoA affords an alternative and more direct route to 15-Me-6-dEB than that provided by the chemobiosynthesis method [Jacobsen, J. R., et al. (1997) Science 277, 367-369], which relies on priming a mutant DEBS with an exogenously fed diketide thioester. The approach described here demonstrates the utility of metabolic engineering in E. coli to introduce precursor pathways for the production of novel polyketides.
authors.affiliation: Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Kennedy
authors.forename: Sumati
authors.initials: S
authors.lastname: Murli
authors.forename: James T
authors.initials: JT
authors.lastname: Kealey
mesh_terms: D000090:Acetoacetates / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001430:Bacteriocins; D002087:Butyrates / Q000378:metabolism; D045647:DNA Glycosylases / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D009097:Multienzyme Complexes / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D010957:Plasmids; D019998:Racemases and Epimerases / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000090:Acetoacetates; D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D001430:Bacteriocins; D002087:Butyrates; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010455:Peptides; C110490:mutacin II prepeptide, bacteria; C015357:methylmalonyl-coenzyme A; C034277:6-deoxyerythronolide B; C024343:butyryl-coenzyme A; C009061:propionyl-coenzyme A; C016635:acetoacetic acid; D004917:Erythromycin; D045647:DNA Glycosylases; C079122:MutB protein, Salmonella typhimurium; D019998:Racemases and Epimerases; C436308:NDP-4-keto-6-deoxy-D-glucose 3',5'-epimerase