Accessible biocatalyst development by rapid in vitro semi-rational engineering (RISE) of enzymes
| Autoři | |
|---|---|
| Rok publikování | 2026 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | iScience |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | https://www.sciencedirect.com/science/article/pii/S2589004225025180 |
| Doi | https://doi.org/10.1016/j.isci.2025.114257 |
| Klíčová slova | SITE-DIRECTED MUTAGENESIS; MOLECULAR-DYNAMICS |
| Přiložené soubory | |
| Popis | 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 forACE2 inhibitor drugs. RISE bridges the gap between inefficient wild-type enzymes and expensive directed evolution, promoting biocatalysis implementation in early chemical development. |
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