Studying a Bottleneck of Multimodular Polyketide Synthase Processing: the Polyketide Structure-Dependent Performance of Ketoreductase Domains.

Ketoreductases (KRs) are canonical domains of type I polyketide synthases (PKSs). They stereoselectively reduce ACP-bound ß-ketothioester intermediates and are responsible for a large part of the stereocenters in reduced polyketides. Albeit essential for the understanding and engineering of PKS, the specific effects of altering the polyketide part of KR precursors on their performance has rarely been studied. We present investigations on the substrate-dependent performance of six isolated KR domains using a library of structurally diverse surrogates for PKS thioester intermediates. A pronounced correlation between the polyketide structure and the KR performance was observed with activity and stereoselectivity diminishing with growing deviation from the natural KR precursor structure. The extent of this decrease and the profile of arising side products was characteristic for the individual KRs. Our results reinforce the importance of structure-KR performance relationships and suggest extended studies with isolated domains and whole PKS modules.

Cross-linking of a polyketide synthase domain leads to a recyclable biocatalyst for chiral oxygen heterocycle synthesis.

The potential of polyketide synthase (PKS) domains for chemoenzymatic synthesis can often not be tapped due to their low stability and activity in vitro. In this proof-of-principle study, the immobilisation of the heterocycle-forming PKS domain AmbDH3 as a cross-linked enzyme aggregate (CLEA) is described. The AmbDH3-CLEA showed good activity recovery, stability and recyclability. Repetitive reactions on the semi-preparative scale were performed with high conversion and isolated yield. Similar to that observed for the free enzyme, the aggregate retained substrate tolerance and the ability for kinetic resolution. This first example of a successful enzymatic PKS domain immobilisation demonstrates that cross-linking can in principle be applied to this type of enzyme to increase its applicability for chemoenzymatic synthesis.

Step-Economic Synthesis of Biomimetic ß-Ketopolyene Thioesters and Demonstration of Their Usefulness in Enzymatic Biosynthesis Studies.

Studies on the biosynthetic processing of polyene thioester intermediates are complicated by limited access to appropriate substrate surrogates. We present a step-economic synthetic access to biomimetic ß-ketopolyene thioesters that is based on an Ir-catalyzed reductive Horner-Wadsworth-Emmons olefination. New ß-ketotriene and pentaenethioates of pantetheine and N-acetylcysteamine were exemplarily synthesized via short and concise routes. The usefulness of these compounds was demonstrated in an in vitro assay with the ketoreductase domain MycKRB from mycolactone biosynthesis.