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Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides., (© 2023 Wiley-VCH Verlag GmbH.)

Thioesterases (TEs) are fundamentally important enzymes present in all bacteria and eukaryotes, where they have conserved functions in fatty acid biosynthesis and secondary metabolism. This work provides the first structural insights into a functionally distinct group of TEs that perform diverse acylations in polyketide and peptide biosynthesis (TE B s). Structural analysis of the oocydin (OocS) TE B domain facilitated identification and engineering of the active site to modulate acyl-group acceptance. In this way, we achieved higher reactivity using a structure-based approach, building a foundation for biocatalytic development of TE B -mediated O-acylation, a modification known to improve the bioactivity of oocydin-type polyketides. Lastly, the promiscuity of the OocS TE B motivated us to investigate, and ultimately provide evidence for, the production of longer chain branched oocydins in the native host Serratia plymuthica 4Rx13. This work frames the OocS TE B and homologs as invaluable synthetic biology tools for polyketide drug development., (© 2022 Wiley-VCH GmbH.)

Bacterial multimodular polyketide synthases (PKSs) are large enzymatic assembly lines that synthesize many bioactive natural products of therapeutic relevance. While PKS catalysis is mostly based on fatty acid biosynthetic principles, polyketides can be further diversified by post-PKS enzymes. Here, we characterized a remarkably versatile trans-acyltransferase (trans-AT) PKS from Serratia that builds structurally complex macrolides via more than ten functionally distinct PKS modules. In the oocydin PKS, we identified a new oxygenation module that α-hydroxylates polyketide intermediates, a halogenating module catalyzing backbone γ-chlorination, and modular O-acetylation by a thioesterase-like domain. These results from a single biosynthetic assembly line highlight the expansive biochemical repertoire of trans-AT PKSs and provide diverse modular tools for engineered biosynthesis from a close relative of E. coli., (© 2022 Wiley-VCH GmbH.)