Evaluating Ketoreductase Exchanges as a Means of Rationally Altering Polyketide Stereochemistry

Abstract

Modular polyketide synthases (PKSs) are multidomain multienzymes responsible for the biosynthesis in bacteria of a wide range of polyketide secondary metabolites of clinical value. The stereochemistry of these molecules is an attractive target for genetic engineering in attempts to produce analogues exhibiting novel therapeutic properties. The exchange of ketoreductase (KR) domains in model PKSs has been shown in several cases to predictably alter the configuration of the β-hydroxy functionalities but not of the α-methyl groups. By systematic screening of a broad panel of KR domains, we have identified two donor KRs that afford modification of α-methyl group stereochemistry. To the best of our knowledge, this provides the first direct in vivo evidence of KR-catalyzed epimerization. However, none of the introduced KRs afforded simultaneous alteration of methyl and hydroxy configurations in high yield. Therefore, swapping of whole modules might be necessary to achieve such changes in stereochemistry.

Towards Precision Engineering of Canonical Polyketide Synthase Domains: Recent Advances and Future Prospects

“…A recent RLS study focused on exchanging non-epimerizing A1-KRs with a panel of epimerizing A2 and B2 KRs confirmed the limitations of this method to introduce functional epimerase activity. Out of the sixteen epimerizing KRs swapped into the DEBS KR6 position, only two retained any activity [ 81 ]. A current hypothesis based on proteolytic analysis of modules with epimerizing and non-epimerizing KRs suggests that epimerizing modules may possess a more open architecture, which is important for proper KR functioning.…”

Polyene macrolide biosynthesis in streptomycetes and related bacteria: recent advances from genome sequencing and experimental studies

“…This motif is absent from A-type ketoreductases that form 3L-3-hydroxyacyl-ACP intermediates. Ketoreductase domains also determine methyl stereochemistry after incorporation of branched extenders (Weissman et al, 1997;Valenzano et al, 2009;Zheng et al, 2013;Annaval et al, 2015). Additional amino acid residues have been identified that allow prediction of C-2 methyl as well as C-3 alcohol stereochemistry in an extended chain (Keatinge-Clay, 2007).…”

Harnessing natural product assembly lines: structure, promiscuity, and engineering

“…Most of the focus has been directed at the reductive domains through either domain swapping or mutagenesis. Whole PKS reductive loops have been successfully swapped to alter the stereochemical outcome of a truncated product from a bimodular PKS [67,3]. Using similar bimodular PKSs for screening, site-directed mutagenesis of the amphotericin ketoreductase afforded a change in stereochemistry [155].…”