Empowering Protein Engineering through Recombination of Beneficial Substitutions.

ABSTRACT

Directed evolution stands as a seminal technology for generating novel protein functionalities, a cornerstone in biocatalysis, metabolic engineering, and synthetic biology. Today, with the development of various mutagenesis methods and advanced analytical machines, the challenge of diversity generation and high-throughput screening platforms is largely solved, and one of the remaining challenges is how to empower the potential of single beneficial substitutions with recombination to achieve the epistatic effect. This review overviews experimental and computer-assisted recombination methods in protein engineering campaigns. In addition, integrated and machine learning-guided strategies were highlighted to discuss how these recombination approaches contribute to generating the screening library with better diversity, coverage, and size. A decision tree was finally summarized to guide the further selection of proper recombination strategies in practice, which was beneficial for accelerating protein engineering.