Cloning, expression and molecular evolution of Komagataella phaffii ketoreductase with enhanced catalytic activity

The synthesis of optically active alcohols is vital for the pharmaceutical and fine chemical industries, yet traditional chemical methods generally lack efficiency and stereoselectivity. Although ketoreductases are a more environmentally friendly option, many of them need to have their catalytic performance improved to satisfy industrial demands. This study focused on engineering Komagataella phaffii ketoreductase (KpKR), the first characterized ketoreductase from this yeast, by applying an innovative combination of error-prone PCR and ribosome display for rapid directed evolution. Through the use of kinetic analysis, molecular docking, homology modeling, and substrate specificity assays, five evolved variants (M1-M5) were identified and characterized. Compared to a range of microbial ketoreductases, such as Bacillus sp. ECU0013 and yeast-derived enzymes, the KpKR variants demonstrated efficient catalytic performance; notably, M5 showed enhanced catalytic efficiency (k_cat/k_m = 199.58 s^-1mM^-1) toward ketoesters, while M1 demonstrated remarkable activity for halogenated substrates, outperforming Bacillus enzymes by thousands-fold. Structural investigation identified key C-terminal changes that likely contributed to improved active site accessibility. Furthermore, sequence analysis confirmed KpKR as a member of the short-chain dehydrogenase/reductase (SDR) family. These results underscore the innovative utility of ribosome display in enzyme engineering and establish the evolved KpKR variants as powerful, highly efficient biocatalysts suitable for challenging pharmaceutical synthesis.