title: Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase.
abstract: BACKGROUND
  Modular polyketide synthases (PKSs) catalyse the biosynthesis of complex polyketides using a different set of enzymes for each successive cycle of chain extension. Directed biosynthesis starting from synthetic diketides is a potentially valuable route to novel polyketides. We have used a purified bimodular derivative of the erythromycin-producing polyketide synthase (DEBS 1-TE) to study chain extension starting from a variety of diketide analogues and, in some cases, from the alternative acyl-CoA thioester substrates.
  
  
  RESULTS
  Chain initiation in vitro by DEBS 1-TE module 2 using a synthetic diketide analogue as a substrate was tolerant of significant structural variation in the starter unit of the synthetic diketide, but other changes completely abolished activity. Interestingly, a racemic beta-keto diketide was found to be reduced in situ on the PKS and utilised in place of its more complex hydroxy analogue as a substrate for chain extension. The presence of a diketide analogue strongly inhibited chain initiation via the loading module. Significantly higher concentrations of diketide N-acetylcysteamine analogues than their corresponding acyl-CoA thioesters are required to achieve comparable yields of triketide lactones.
  
  
  CONCLUSIONS
  Although a broad range of variation in the starter residue is acceptable, the substrate specificity of module 2 of a typical modular PKS in vitro is relatively intolerant of changes at C-2 and C-3. This will restrict the usefulness of approaches to synthesise novel erythromycins using synthetic diketides in vivo. The use of synthetic beta-keto diketides in vivo deserves to be explored.
authors.affiliation: Cambridge Centre for Molecular Recognition Department of Organic Chemistry University of Cambridge Cambridge CB2 1EW UK.
authors.forename: K J
authors.initials: KJ
authors.lastname: Weissman
authors.forename: M
authors.initials: M
authors.lastname: Bycroft
authors.forename: A L
authors.initials: AL
authors.lastname: Cutter
authors.forename: U
authors.initials: U
authors.lastname: Hanefeld
authors.forename: E J
authors.initials: EJ
authors.lastname: Frost
authors.forename: M C
authors.initials: MC
authors.lastname: Timoney
authors.forename: R
authors.initials: R
authors.lastname: Harris
authors.forename: S
authors.initials: S
authors.lastname: Handa
authors.forename: M
authors.initials: M
authors.lastname: Roddis
authors.forename: J
authors.initials: J
authors.lastname: Staunton
authors.forename: P F
authors.initials: PF
authors.lastname: Leadlay
mesh_terms: D002384:Catalysis; D004917:Erythromycin / Q000138:chemical synthesis* / Q000737:chemistry; D007783:Lactones / Q000378:metabolism; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D007783:Lactones; D009097:Multienzyme Complexes; D004917:Erythromycin