{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T11:50:04Z","timestamp":1788436204981,"version":"build-2803163510"},"reference-count":45,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2021,6,26]],"date-time":"2021-06-26T00:00:00Z","timestamp":1624665600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biotechnology Journal"],"published-print":{"date-parts":[[2021,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>Aldo\u2010keto reductases\u2010catalyzed transformations of ketones to chiral alcohols have become an established biocatalytic process step in the pharmaceutical industry. Previously, we have discovered an aldo\u2010keto reductase (AKR) from <jats:italic>Kluyveromyces marxianus<\/jats:italic> that is active to the aliphatic <jats:italic>tert<\/jats:italic>\u2010butyl 6\u2010substituted (5<jats:italic>R<\/jats:italic>\/<jats:italic>S<\/jats:italic>)\u2010hydroxy\u20103\u2010oxohexanoates, but it is inactive to aromatic ketones. In order to acquire an excellent <jats:italic>Km<\/jats:italic>AKRmutant for ensuring the simultaneous improvement of activity\u2010thermostability toward <jats:italic>tert<\/jats:italic>\u2010butyl 6\u2010cyano\u2010(5<jats:italic>R<\/jats:italic>)\u2010hydroxy\u20103\u2010oxohexanoate ((5<jats:italic>R<\/jats:italic>)\u2010<jats:bold>1<\/jats:bold>) and broadening the universal application prospects toward more substrates covering both aliphatic and aromatic ketones, a fluorescence\u2010based high\u2010throughput (HT) screening technique was established.<\/jats:p><\/jats:sec><jats:sec><jats:title>Main Methods and Major Results<\/jats:title><jats:p>The directed evolution of <jats:italic>Km<\/jats:italic>AKR variant M5 (<jats:italic>Km<\/jats:italic>AKR\u2010W297H\/Y296W\/K29H\/Y28A\/T63M) produced the \u201cbest\u201d variant M5\u2010Q213A\/T23V. It exhibited enhanced activity\u2010thermostability toward (5<jats:italic>R<\/jats:italic>)\u2010<jats:bold>1<\/jats:bold>, improved activity toward all 18 test substrates and strict <jats:italic>R<\/jats:italic>\u2010stereoselectivity toward 10 substrates in comparison to M5. The enhancement of enzymatic activity and the extension of substrate scope covering aromatic ketones are proposed to be largely attributed to pushing the binding pocket of M5\u2010Q213A\/T23V to the enzyme surface, decreasing the steric hindrance at the entrance and enhancing the flexibility of loops surrounding the active center. In addition, combined with 0.94\u00a0g dry cell weight (DCW) L<jats:sup>\u22121<\/jats:sup> glucose dehydrogenase from <jats:italic>Exiguobacterium sibiricum<\/jats:italic> (<jats:italic>Es<\/jats:italic>GDH) for NADPH regeneration, 2.81\u00a0g DCW L<jats:sup>\u22121<\/jats:sup> M5\u2010Q213A\/T23V completely converted (5<jats:italic>R<\/jats:italic>)\u2010<jats:bold>1<\/jats:bold> of up to 450\u00a0g L<jats:sup>\u22121<\/jats:sup> at 120\u00a0g g<jats:sup>\u20131<\/jats:sup> substrates\/catalysts (S\/C), yielding the corresponding optically pure <jats:italic>tert<\/jats:italic>\u2010butyl 6\u2010cyano\u2010(3<jats:italic>R<\/jats:italic>,5<jats:italic>R<\/jats:italic>)\u2010dihydroxyhexanoate ((3<jats:italic>R<\/jats:italic>,5<jats:italic>R<\/jats:italic>)\u2010<jats:bold>2<\/jats:bold>,\u00a0&gt;\u00a099.5% <jats:italic>d.e<\/jats:italic>.<jats:sub>p<\/jats:sub>) with a space\u2010time yield (STY) of 1.08\u00a0kg L<jats:sup>\u22121<\/jats:sup> day<jats:sup>\u22121<\/jats:sup>.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>A fluorescence\u2010based HT screening system was developed to tailor <jats:italic>Km<\/jats:italic>AKR's activity, thermostability and substrate scope. The \u201cbest\u201d variant M5\u2010Q213A\/T23V holds great potential application for the synthesis of aliphatic\/aromatic <jats:italic>R<\/jats:italic>\u2010configuration alcohols.<\/jats:p><\/jats:sec>","DOI":"10.1002\/biot.202100130","type":"journal-article","created":{"date-parts":[[2021,6,14]],"date-time":"2021-06-14T11:02:00Z","timestamp":1623668520000},"source":"Crossref","is-referenced-by-count":11,"title":["Fluorescence\u2010based screening for engineered aldo\u2010keto reductase <i>Km<\/i>AKR with improved catalytic performance and extended substrate scope"],"prefix":"10.1002","volume":"16","author":[{"given":"Shuai","family":"Qiu","sequence":"first","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province College of Biotechnology and Bioengineering Zhejiang University of Technology  Hangzhou P. R. 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