Combining Promiscuous Acyl-CoA Oxidase and Enoyl-CoA Carboxylase/Reductases for Atypical Polyketide Extender Unit Biosynthesis. Issue 7 (19th July 2018)

• Record Type: Journal Article
• Authors: Vögeli, Bastian; Geyer, Kyra; Gerlinger, Patrick D.; Benkstein, Sarah; Cortina, Niña Socorro; Erb, Tobias J.
• Is Part Of: Cell chemical biology. Volume 25:Issue 7(2018)
• Page Start: 833
• Page End: 839.e4
• Publication Date: 2018-07-19
• Subjects: polyketides -- extender units -- acyl-CoA oxidase -- enoyl-CoA carboxylase/reductase -- substrate selectivity; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2018.04.009
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: The incorporation of different extender units generates structural diversity in polyketides. There is significant interest in engineering substrate specificity of polyketide synthases (PKSs) to change their chemical structure. Efforts to change extender unit selectivity are hindered by the lack of simple screening methods and easily available atypical extender units. Here, we present a chemo-biosynthetic strategy 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 combined action of an acyl-CoA oxidase and enoyl-CoA carboxylase/reductase. We synthesized six different extender units and used them in in vitro 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 open the possibility for comprehensive studies on extender incorporation in PKSs. Graphical Abstract: Highlights: Kinetic characterization of six promiscuous ECRs for various substrates Characterization of a promiscuous acyl-CoA oxidase functioning as proofreading enzyme One-pot synthesis method production of various atypical alkylmalonyl-CoA extender units In vitro competition screens for incorporation of atypical extender units in DEBS Abstract : Vögeli et al. present a chemo-biosynthetic strategy that employs biocatalytic proofreading and allows production of a large variety of alkylmalonyl-CoA extender units by utilizing a promiscuous acyl-CoA oxidase and an engineered promiscuous enoyl-CoA carboxylase/reductase. The extender units are used to test the substrate specificity of the polyketide synthase DEBS.