Advances and applications of microbial genome evolution engineering

Abstract:

The construction of microbial cell factories frequently encounters bottlenecks due to the complexity of metabolic pathways, insufficient enzyme functionality, and poor environmental tolerance. Directed evolution, as a pivotal technology for overcoming these engineering constraints, can significantly enhance the titer, productivity, and overall robustness of target-producing strains. Its development originated from traditional random mutagenesis, a method that generates genetic diversity through the imposition of artificial growth pressures and is currently in widespread use. However, owing to the uncontrollable direction of mutagenesis and the low frequency of beneficial mutations, this approach suffers from low screening efficiency and is both time-consuming and labor-intensive. To accelerate the evolutionary rate, global accelerated evolution strategies based on tools such as DNA repair deficiencies and cytidine deaminases have emerged, achieving orders-of-magnitude increases in genome-wide mutation rates. Targeted continuous evolution strategies centered on CRISPR/Cas and T7 RNA polymerase systems enable the precise introduction of mutations within predefined genomic regions, thereby greatly improving the enrichment efficiency of beneficial mutations while minimizing non-productive mutations. This review will focus on three core strategies—traditional random evolution, global accelerated evolution, and targeted continuous evolution—systematically elucidating their developmental trajectories, applications in the construction of microbial cell factories, and future directions. Collectively, this review aims to provide methodological guidance for overcoming the engineering bottlenecks in the construction of microbial cell factories.