# Protein Language Model‐Guided Engineering of a 2,3‐Butanediol Dehydrogenase for the Enantioselective Synthesis of Cyclic α‐Hydroxy Ketones

## TL;DR

Scientists improved a bacterial enzyme to make valuable chiral chemicals more efficiently using AI and protein engineering.

## Contribution

A novel strategy combining protein language models and mutagenesis to enhance and invert enzyme stereoselectivity.

## Key findings

- Engineered variants achieved >99% enantioselectivity for both trans- and cis-CHD.
- Activity improvements reached up to 5183.1-fold for multiple substrates.
- PASS strategy achieved 88.2% true-positive rate in designing thermostable enzyme variants.

## Abstract

(2R,3R)‐butanediol dehydrogenases (BDHs) are promising catalysts for the production of α‐hydroxy ketones, which are highly valuable compounds in the synthesis of fine chemicals and pharmaceuticals. However, (2R,3R)‐BDHs display limited stereoselectivity, thus restricting wider applications. In this study, we engineered a (2R,3R)‐BDH from Bacillus subtilis (BsBDH) to enhance and invert its stereoselectivity toward 1,2‐cyclohexanediol (1,2‐CHD) for the production of chiral 2‐hydroxycyclohexanone. The hot spots 115, 118, 293 of BsBDH were initially identified using the protein language model ESM‐1v. Subsequently, to obtain a stable scaffold to engineer stereoselectivity, we devised a strategy of position analysis and source search, achieving a true‐positive rate of 88.2% in designing thermostable single variants. Furthermore, iterative saturation mutagenesis was applied to the hot spots of the thermostable variant 6M2, and obtained a trans‐CHD preference variant LTF (ee > 99%) and a cis‐CHD preference variant 10M (ee > 99%). Several high‐activity variants were also obtained, including 6M2/F115C/L118F and 6M2/F115L/L118M, which demonstrated the activity improvements toward 25 substrates, with the highest enhancement reaching 5183.1‐fold. Additionally, molecular dynamics (MD) simulations and the incorporation of non‐canonical amino acids (ncAAs) were utilized to elucidate the mechanisms underlying the variants. The engineered BsBDH variants exhibit promising potential for the biocatalytic production of α‐hydroxyketones.

A (2R,3R)‐butanediol dehydrogenase from Bacillus subtilis (BsBDH) is engineered for the enantioselective synthesis of 2‐hydroxycyclohexanone. A PASS computational design strategy is proposed to enhance the thermostability of BsBDH. Moreover, ESM‐1v combined with ISM is utilized for enhancing and inverting its stereoselectivity. The engineered BsBDH variants demonstrate promising potential for the biocatalytic production of α‐hydroxyketones in the future.