Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase |
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Authors: | Kira J Weissman Matthew Bycroft Annabel L Cutter Ulf Hanefeld Elizabeth J Frost Máire C Timoney Rebecca Harris Sandeep Handa Marc Roddis James Staunton Peter F Leadlay |
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Institution: | Kira J. Weissman , Matthew Bycroft , Annabel L. Cutter , Ulf Hanefeld , Elizabeth J. Frost , Máire C. Timoney , Rebecca Harris , Sandeep Handa , Marc Roddis , James Staunton ,Peter F. Leadlay |
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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. |
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Keywords: | Author Keywords: DEBS 1-TE diketide analogues erythromycin biosynthesis polyketide synthase |
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