Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase

Kira J. Weissman, Matthew Bycroft, Annabel L. Cutter, Ulf Hanefeld, Elizabeth J. Frost, Maire C. Timoney, Rebecca Harris, Sandeep Handa, Marc Roddis, James Staunton, Peter F. Leadlay

Research output: Contribution to journalArticleScientificpeer-review

43 Citations (Scopus)

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 β-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 β-keto diketides in vivo deserves to be explored.

Original languageEnglish
Pages (from-to)743-754
JournalChemistry & Biology
Volume5
Issue number12
DOIs
Publication statusPublished - 1998
Externally publishedYes

Keywords

  • DEBS 1-TE
  • Diketide analogues
  • Erythromycin biosynthesis
  • Polyketide synthase

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