Tailoring natural enzymes to synthetic needs is often associated with high costs and long timelines, hindering the broader adoption of biocatalysis in the chemical and pharmaceutical industries. To address this, we developed the RISE (rapid in vitro semi-rational engineering) workflow that makes enzyme engineering accessible to chemistry laboratories. RISE integrates four key concepts: computational design of focused variant libraries, rapid generation of linear mutant DNA libraries via PCR, cell-free protein synthesis from linear template DNA, and iterative cycles of mutagenesis, expression, and testing to accumulate beneficial mutations. In a proof-of-concept study, we engineered a ketimine reductase from Rattus norvegicus (RnKIRED), achieving stereoselectivity inversion in one reductive amination reaction and a 400-fold activity improvement in another. These engineered variants enabled the gram-scale synthesis of key intermediates for ACE2 inhibitor drugs. RISE bridges the gap between inefficient wild-type enzymes and expensive directed evolution, promoting biocatalysis implementation in early chemical development.

Department of Applied Biotechnology and Food Science Faculty of Chemical Technology and Biotechnology Budapest University of Technology and Economics Budapest 1111 Hungary

Eötvös Loránd University Institute of Chemistry Budapest 1117 Hungary

Institute of Molecular Life Sciences Research Centre for Natural Sciences HUN REN Budapest 1117 Hungary

International Clinical Research Center St Anne's University Hospital Brno 625 00 Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Brno 625 00 Czech Republic

Servier Research Institute of Medicinal Chemistry Budapest 1037 Hungary

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