title: Chemobiosynthesis of novel 6-deoxyerythronolide B analogues by mutation of the loading module of 6-deoxyerythronolide B synthase 1.
abstract: Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler "monoketide" acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1(o) for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor.
authors.affiliation: Kosan Biosciences Inc., 3832 Bay Center Place, Hayward, CA 94545, USA.
authors.forename: Sumati
authors.initials: S
authors.lastname: Murli
authors.forename: Karen S
authors.initials: KS
authors.lastname: MacMillan
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Hu
authors.forename: Gary W
authors.initials: GW
authors.lastname: Ashley
authors.forename: Steven D
authors.initials: SD
authors.lastname: Dong
authors.forename: James T
authors.initials: JT
authors.lastname: Kealey
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Reeves
authors.forename: Jonathan
authors.initials: J
authors.lastname: Kennedy
mesh_terms: D003470:Culture Media; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005818:Genetic Engineering / Q000379:methods*; D007218:Industrial Microbiology / Q000379:methods; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013864:Thioglycolates / Q000378:metabolism
chemical_list: D003470:Culture Media; D013864:Thioglycolates; C034277:6-deoxyerythronolide B; D004917:Erythromycin; D048630:Polyketide Synthases; C037474:methyl thioglycolate