Accessible biocatalyst development by rapid <i>in vitro</i> semi-rational engineering (RISE) of enzymes.

ABSTRACT: 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 inver

SUBMITTER: Telek A 

PROVIDER: S-EPMC12804161 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature