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Herein, we report the directed evolution of a tandem\u2010fused 4\u2010OT enzyme that achieves efficient activation of ketone substrates for enantioselective aldol reactions with substituted benzaldehydes. The engineered biocatalyst yields a range of chiral \u03b2\u2010hydroxy ketone products with up to 99% yield and moderate enantioselectivity, while enabling access to both target enantiomers. This work expands the substrate promiscuity and reaction scope of 4\u2010OT, laying a foundation for rational design of next\u2010generation 4\u2010OT variants for advanced organic synthesis and industrial biocatalysis.<\/jats:p>","DOI":"10.1002\/cbic.70438","type":"journal-article","created":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T10:57:56Z","timestamp":1782298676000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Unlocking Ketone Activation for Enantioselective Aldol Reactions by 4\u2010Oxalocrotonate Tautomerase"],"prefix":"10.1002","volume":"27","author":[{"given":"Yi","family":"Fu","sequence":"first","affiliation":[{"name":"School of Life Sciences and Health Engineering Jiangnan University  Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuhui","family":"Sheng","sequence":"additional","affiliation":[{"name":"School of Life Sciences and Health Engineering Jiangnan 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