Advances in Strategies for In Vivo Directed Evolution of Targeted Functional Genes

Enzymes are indispensable in fields such as biotechnology, medicine, and industrial manufacturing due to their high catalytic specificity and efficiency under mild conditions. However, their natural versions often suffer from limitations, including low activity toward non-natural substrates, poor stability under extreme conditions, and narrow substrate spectra. Directed evolution, a key protein engineering strategy that optimizes protein function via genetic diversity introduction and directed selection, has become the primary solution to these limitations. Among its mature methodological systems, in vivo evolution platforms (advanced by synthetic biology) are particularly efficient, as they integrate in-cell mutation, translation, selection, and replication into an automated process, significantly improving experimental efficiency. This review will focus on two core strategies that enhance these platforms: in vivo targeted gene hypermutation and heterologous polymerase-mediated targeted hypermutation. These techniques enable the rapid optimization of enzymes to acquire novel functions, as well as the comprehensive engineering of microbial strains to enhance their performance and stress tolerance. Analyzing these strategies provides a robust technical framework for enzyme engineering and promises to drive future innovations across multiple fields.