Combining Promiscuous Acyl-CoA Oxidase and Enoyl-CoA Carboxylase/Reductases for Atypical Polyketide Extender Unit Biosynthesis

Abstract

The incorporation of different extender units generates structural diversity in polyketides. There is significant interest in engineering substrate specificity of polyketide synthases (PKSs) to changetheir chemical structure. Efforts to change extenderunit selectivity are hindered by the lack of simple screening methods and easily available atypical extender units. Here, we present a chemo-biosyntheticstrategy that employs biocatalytic proofreading and allows access to a large variety of extender units. First, saturated acids are chemically coupled to free coenzyme A (CoA). The corresponding acyl-CoAs are then converted to alkylmalonyl-CoAs in a "one-pot" reaction through the combinedaction of an acyl-CoA oxidase and enoyl-CoA carboxylase/reductase. We synthesized six different extender units and used them in invitro competition screens to investigate active site residues conferring extender unit selectivity. Our results show the importance of an uncharacterized glutamine in extender unit selectivity and openthe possibility for comprehensive studies on extender incorporation in PKSs.