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

(2 R ,3 R )-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, (2 R ,3 R )-BDHs display limited stereoselectivity, thus restricting wider applications. In this study, we engineered a (2 R ,3 R )-BDH from Bacillus subtilis ( Bs BDH) 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 Bs BDH 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 Bs BDH variants exhibit promising potential for the biocatalytic production of α -hydroxyketones.