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

Background: Modular polyketide synthases (PKSs) catalyse the biosynthesis o f complex polyketides using a different set of enzymes for each successive cycle of chain extension. Directed biosynthesis starting from synthetic dik etides is a potentially valuable route to novel polyketides. We have used a purified bimodular derivative of the erythromycin-producing polyketide syn thase (DEBS 1-TE) to study chain extension starting from a variety of diket ide analogues and, in some cases, from the alternative acyl-CoA thioester s ubstrates. Results: Chain initiation in vitro by DEBS 1-TE module 2 using a synthetic diketide analogue as a substrate was tolerant of significant structural var iation in the starter unit of the synthetic diketide, but other changes com pletely 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 c omplex hydroxy analogue as a substrate for chain extension. The presence of a diketide analogue strongly inhibited chain initiation via the loading mo dule. Significantly higher concentrations of diketide N-acetylcysteamine an alogues than their corresponding acyl-CoA thioesters are required to achiev e 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 rest rict the usefulness of approaches to synthesise novel erythromycins using s ynthetic diketides in vivo. The use of synthetic beta-keto diketides in viv o deserves to be explored.