Advances in in vivo continuous evolution technologies for strain development and enzyme engineering

Evolutionary engineering is a key strategy for obtaining industrial strains with high stress tolerance and productivity, as well as improving enzyme performance. However, low natural mutation rates, small in vitro mutation libraries, and lengthy phenotypic screening cycles constrain the further development of this approach. In vivo continuous evolution technologies address these challenges by implementing mutagenesis within living microbial cells, elevating mutation rates and enabling the rapid evolution of strains or genes. This review systematically examines three in vivo continuous evolution platforms based on error-prone DNA replication, DNA base modification, and DNA recombination. For each mutational mechanism, the underlying design principles, representative applications, and strategies for optimizing mutational spectra and selection regimes are discussed. By comparing their molecular mechanisms, advantages, and limitations, recent advances in the field are summarized and future directions for evolutionary engineering are outlined. Collectively, this review provides a coherent framework for understanding in vivo microbial continuous evolution technologies and offers guidance for strain development and enzyme engineering.