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Herein, we applied a computer\u2010assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, <jats:italic>Ls<\/jats:italic>CR<jats:sub>M4<\/jats:sub> (N101D\/A117G\/F147L\/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1<jats:italic>S<\/jats:italic>)\u20102\u2010chloro\u20101\u2010(3,4\u2010difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I\u2010Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant <jats:italic>Ls<\/jats:italic>CR<jats:sub>M4<\/jats:sub>\u2010V99L\/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4\u00b0C increase in melting temperature compared to <jats:italic>Ls<\/jats:italic>CR<jats:sub>M4<\/jats:sub>. Our computer\u2010assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.<\/jats:p>","DOI":"10.1002\/bit.28662","type":"journal-article","created":{"date-parts":[[2024,1,24]],"date-time":"2024-01-24T05:59:48Z","timestamp":1706075988000},"page":"1532-1542","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Computer\u2010directed rational design enhanced the thermostability of carbonyl reductase <i>Ls<\/i>CR for the synthesis of ticagrelor precursor"],"prefix":"10.1002","volume":"121","author":[{"given":"Shen\u2010Yuan","family":"Xu","sequence":"first","affiliation":[{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province Zhejiang University of Technology Hangzhou China"},{"name":"Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education Zhejiang University of Technology Hangzhou Zhejiang China"},{"name":"The 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