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In this study, a novel SDR from\n                    <jats:italic>Bacillus subtilis<\/jats:italic>\n                    (\n                    <jats:italic>Bs<\/jats:italic>\n                    SDR) was identified and engineered for the asymmetric synthesis of (\n                    <jats:italic>S<\/jats:italic>\n                    )\u22121\u2010(2,6\u2010dichloro\u20103\u2010fluorophenyl)ethanol ((\n                    <jats:italic>S<\/jats:italic>\n                    )\u2010CFL). By integrating in silico molecular docking with FuncLib based design, a focused mutant library of\n                    <jats:italic>Bs<\/jats:italic>\n                    SDR targeting key hotpots was constructed. Four of the constructed 31 variants exhibited markedly enhanced activity. Subsequent combinatorial mutagenesis generated a \u201cbest\u201d variant\n                    <jats:italic>Bs<\/jats:italic>\n                    SDR\n                    <jats:sub>M2<\/jats:sub>\n                    (I178L\/T179L), which displayed a 107.4\u2010fold increase in catalytic activity, along with excellent enantioselectivity (&gt;\u200999.9%\n                    <jats:italic>e.e<\/jats:italic>\n                    .) and broad substrate scope. Notably, unlike most SDRs that are strictly dependent on NAD(H) or NADP(H), the\n                    <jats:italic>Bs<\/jats:italic>\n                    SDR also possesses a remarkable capacity to utilize the noncanonical cofactor NMNH. Moreover, in addition to robust enantioselective reductive activity toward CFA, the\n                    <jats:italic>Bs<\/jats:italic>\n                    SDR also exhibits glucose\u2010oxidizing activity, driving cofactors regeneration. In the presence of NMNH,\n                    <jats:italic>Bs<\/jats:italic>\n                    SDR\n                    <jats:sub>M2<\/jats:sub>\n                    displayed a 33.3\u2010fold improvement in catalytic efficiency (\n                    <jats:italic>k<\/jats:italic>\n                    <jats:sub>cat<\/jats:sub>\n                    \/\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>M<\/jats:sub>\n                    ) toward substrate 1\u2010(2,6\u2010dichloro\u20103\u2010fluorophenyl)ethanone (CFA) compared to the wild\u2010type (WT), along with an extended half\u2010life (\n                    <jats:italic>t<\/jats:italic>\n                    <jats:sub>1\/2<\/jats:sub>\n                    ) of 52.1\u2009h at 45\u00b0C. Using\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    expressing\n                    <jats:italic>Bs<\/jats:italic>\n                    SDR\n                    <jats:sub>M2<\/jats:sub>\n                    as the only biocatalyst and glucose as the cosubstrate, 100\u2009g\/L CFA was reduced within 6\u2009h in 92.1% conversion and &gt;\u200999.9%\n                    <jats:italic>e.e<\/jats:italic>\n                    ., achieving a space\u2010time yield (STY) of 368.2\u2009g\/(L\u00b7d). This work highlights the potential of\n                    <jats:italic>Bs<\/jats:italic>\n                    SDR as a cost\u2010effective and sustainable biocatalyst for the efficient synthesis of (\n                    <jats:italic>S<\/jats:italic>\n                    )\u2010CFL and other high\u2010value chiral alcohols.\n                  <\/jats:p>","DOI":"10.1002\/bit.70239","type":"journal-article","created":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T06:38:01Z","timestamp":1778740681000},"page":"2062-2079","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering a Novel Cofactor\u2010Versatile Short\u2010Chain Dehydrogenase for Efficient Synthesis of Chiral Alcohols"],"prefix":"10.1002","volume":"123","author":[{"given":"Li\u2010Li","family":"Yao","sequence":"first","affiliation":[{"name":"College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou Zhejiang 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