Multi-faceted Set-up of a Diverse Ketoreductase Library Enables the Synthesis of Pharmaceutically-relevant Secondary Alcohols

Enzymes are valuable tools to introduce chirality into small molecules. Especially, ketoreductase (KRED)-catalyzed transformations of ketones to yield chiral secondary alcohols have become an established biocatalytic process step in the pharmaceutical and fine chemical industry. Development time, however, remains a critical factor in chemical process development and thus, the competitiveness of a biocatalytic reaction step is often governed by the availability of off-the-shelf enzyme libraries. To expand the biocatalytic toolbox with additional ketoreductases, we established a multi-faceted screening procedure to capture KRED diversity from different sources, such as literature, available genome data, and uncharacterized microbial strains. Overall, we built a library consisting of 51 KRED enzymes, 29 of which have never been described in literature before. Notably, 18 of the newly described enzymes exhibited anti-Prelog preference complementing the majority of ketoreductases which generally follow Prelog's rule. Analysis of the library's catalytic activity toward a chemically diverse ketone substrate set of pharmaceutical interest further highlighted the broad substrate scope and the complementing enantio-preference of the individual KREDs. Using the generated sequence-function data of the included short chain dehydrogenases in a bioinformatic analysis led to the identification of possible sequence determinants of the stereospecificity exhibited by these enzymes.

Automated summary

This study presents a multi-faceted screening procedure to capture ketoreductase (KRED) diversity from different sources. By establishing a library containing 51 KRED enzymes, it was found that 29 of them had never been described before, with 18 exhibiting anti-Prelog preference. Additionally, analysis of the library's catalytic activity towards a diverse ketone substrate set revealed broad substrate scope and complementing enantio-preference of the individual KREDs.