Modular α-hydroxylation in trans-AT PKSs Franziska Hemmerling ETH Zürich, Switzerland
Trans -acyltransferase polyketide synthases ( trans -AT PKSs) are multimodular megaenzymes with a great architectural diversity. They have evolved a functional repertoire of catalytic domains to furnish complex chemical scaffolds from simple malonyl-CoA building blocks. In phylogenetic analyses, ketosynthase (KS) domains form distinct clades correlating to the chemical structure of the incoming substrate. This feature can therefore be suggestive of the enzyme-catalyzed reactions upstream of a particular KS domain, and the analysis of unassigned clades can provide clues for novel enzymology. In this way, genome mining can provide access to the expansive treasure trove of novel biochemistry from trans -AT PKSs. The polyketide oocydin is the product of a pathway containing numerous unusual features. A combination of in vitro biochemical characterization and genetic manipulation of the producer Serratia plymuthica provided functional evidence for new enzymology. Together, these data facilitated the discovery of novel modules that install three unprecedented on-line PKS modifications: α-hydroxylation, carbon backbone chlorination, and O -acetylation 1 . Responsible for α-hydroxylation is the flavin-dependent monooxygenase OocM, a trans -acting component of an oxygenating module. OocM homologs and similar module architectures occur in many other trans -AT PKS biosynthetic pathways, suggesting that α-hydroxylation is a widespread feature among trans -AT PKSs. These results provide a striking example of the expansive repertoire of unique enzymology in trans -AT PKSs, highlighting their potential to serve as an enzymatic toolbox to expand the chemical space accessible to synthetic biology. References 1. F. Hemmerling, R. A. Meoded, A. E. Fraley, H. A.Minas, C. L. Dieterich, M. Rust, R. Ueoka, K.Jensen, E. J. N. Helfrich, C. Bergande, M. Biedermann,N. Magnus, B. Piechulla, and J. Piel: "Modular Halogenation, α-Hydroxylation, and Acylation by a Remarkably Versatile Polyketide Synthase" Angew. Chem. Int. Ed. 2022, e202116614
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