Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension

AbstractBackground: Polyketides are a large and structurally diverse group of natural products that include antibiotics, antifungal agents and immunosuppressant compounds. Polyketides are biosynthesised in filamentous bacteria on modular polyketide synthases (PKSs) in which each cycle of chain extension requires a different ‘module’ of enzymatic activities. The recently proposed dimeric model for modular PKSs predicts that even a single-module PKS should be catalytically active in the absence of other PKS components. Researchers are also interested in manipulating the stereochemical outcome of polyketide chain extension using genetic engineering of domains within each module.Results: We have constructed a minimal modular PKS from the erythromycin-producing PKS (DEBS) of Saccharopolyspora erythraea. The diketide synthase (DKS1–2) consists of a single chimaeric extension module, derived from the DEBS module 1 ketoacyl-ACP synthase (KS), sandwiched between a loading module and a chain-terminating thioesterase. When DKS1–2 was expressed in S. erythraea, the strain preferentially accumulated the diketide (2R,3S)-2methyl-3-hydroxy pentanoic acid.Conclusions: These results demonstrate that, as predicted, even a single-module PKS is catalytically active in the absence of other DEBS proteins. In its normal context, the ketosynthase domain KS1 is thought to generate a (2S)-2-methyl-3-hydroxy intermediate by epimerising the initial product of carbon-carbon chain formation, the (2R)-2-methyl-3-ketoester. The observed formation of the alternative (2R)-2-methyl-3-hydroxy product catalysed by DKS1–2 provides strong support for this proposal, and indicates how targeted alteration of stereospecificity can be achieved on a modular PKS