Protein engineering of multi-enzyme virus-like particle nanoreactors for enhanced chiral alcohol synthesis

In the past decade, virus-like particles (VLPs) that can encapsulate single or multiple enzymes have been studied extensively as typical nanoreactors for biocatalysis in vitro, yet their catalytic efficiencies are usually inadequate for real applications. These biocatalytic nanoreactors should be engineered like their free-enzyme counterparts to improve their catalytic performance for potential applications. Herein we engineer biocatalytic VLPs for the enhanced synthesis of chiral alcohols. Different methods including directed evolution were applied to the entire bacteriophage P22 VLPs (except the coat protein), which encapsulated a carbonyl reductase from Scheffersomyces stipitis (SsCR) and a glucose dehydrogenase from Bacillus megaterium (BmGDH) in their capsids. The best variant, namely M5, showed an enhanced turnover frequency (TOF, min^-1) up to 15-fold toward the majority of tested aromatic prochiral ketones, and gave up to 99% enantiomeric excess in the synthesis of chiral alcohol pharmaceutical intermediates. A comparison with the mutations of the free-enzyme counterparts showed that the same amino acid mutations led to different changes in the catalytic efficiencies of free and confined enzymes. Finally, the engineered M5 nanoreactor showed improved efficiency in the scale-up synthesis of chiral alcohols. The conversions of three substrates catalyzed by M5 were all higher than those catalyzed by the wild-type nanoreactor, demonstrating that enzyme-encapsulating VLPs can evolve to enhance their catalytic performance for potential applications.