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However, (2\n                    <jats:italic>R<\/jats:italic>\n                    ,3\n                    <jats:italic>R<\/jats:italic>\n                    )\u2010BDHs display limited stereoselectivity, thus restricting wider applications. In this study, we engineered a (2\n                    <jats:italic>R<\/jats:italic>\n                    ,3\n                    <jats:italic>R<\/jats:italic>\n                    )\u2010BDH from\n                    <jats:italic>Bacillus subtilis<\/jats:italic>\n                    (\n                    <jats:italic>Bs<\/jats:italic>\n                    BDH) to enhance and invert its stereoselectivity toward 1,2\u2010cyclohexanediol (1,2\u2010CHD) for the production of chiral 2\u2010hydroxycyclohexanone. The hot spots 115, 118, 293 of\n                    <jats:italic>Bs<\/jats:italic>\n                    BDH were initially identified using the protein language model ESM\u20101v. Subsequently, to obtain a stable scaffold to engineer stereoselectivity, we devised a strategy of position analysis and source search, achieving a true\u2010positive 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\n                    <jats:italic>trans<\/jats:italic>\n                    \u2010CHD preference variant LTF (\n                    <jats:italic>ee<\/jats:italic>\n                    &gt; 99%) and a\n                    <jats:italic>cis<\/jats:italic>\n                    \u2010CHD preference variant 10M\u00a0(\n                    <jats:italic>ee<\/jats:italic>\n                    &gt; 99%). Several high\u2010activity 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\u2010fold. Additionally, molecular dynamics (MD) simulations and the incorporation of non\u2010canonical amino acids (ncAAs) were utilized to elucidate the mechanisms underlying the variants. The engineered\n                    <jats:italic>Bs<\/jats:italic>\n                    BDH variants exhibit promising potential for the biocatalytic production of\n                    <jats:italic>\u03b1<\/jats:italic>\n                    \u2010hydroxyketones.\n                  <\/jats:p>","DOI":"10.1002\/advs.202509314","type":"journal-article","created":{"date-parts":[[2026,1,25]],"date-time":"2026-01-25T08:47:54Z","timestamp":1769330874000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Protein Language Model\u2010Guided Engineering of a 2,3\u2010Butanediol Dehydrogenase for the Enantioselective Synthesis of Cyclic\n                    <i>\u03b1<\/i>\n                    \u2010Hydroxy Ketones"],"prefix":"10.1002","volume":"13","author":[{"given":"Haote","family":"Ding","sequence":"first","affiliation":[{"name":"Institute of Bioengineering College of Chemical and Biological Engineering Zhejiang University  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