Research Advances in Targeted Mutagenesis-mediated Microbial Evolutionary Engineering

doi:10.13560/j.cnki.biotech.bull.1985.2025-0191

Abstract

Abstract:

Natural mutations in organism's genomes confer diverse traits that facilitate evolutionary adaptation to environmental challenges. However, the slow evolutionary rates inherent to natural settings limit their practical utility. Strategies involving targeted base modifications at specific DNA sequences enable accelerated laboratory evolution, which has been widely applied in non-rational protein engineering and metabolic pathway optimization. Nevertheless, conventional mutagenesis methods remain labor-intensive, suffer from low efficiency, and lack targeting precision. In contrast, targeted mutagenesis technologies introduce mutations at precisely defined genomic loci, significantly improving efficiency and shortening evolution cycles. In recent years, targeted mutagenesis has emerged as a key area of research in microbial evolutionary engineering. These technologies enhance the optimization of microbial metabolic pathways, improve stress tolerance in chassis strains, and have found widespread applications in biocatalysis, environmental remediation,and industrial fermentation. This review summarizes recent advances in targeted mutagenesis systems developed for key microbial chassis organisms, focusing on the design principles and applications of error-prone replication systems, CRISPR-Cas-mediated mutagenesis systems, and the MutaT7 mutagenesis system. Additionally, it analyzes the strengths and limitations of current laboratory evolution systems and discusses future prospects for precision-targeted mutagenesis technologies.

Key words: directed evolution, targeted mutagenesis, gene editing, RNA polymerase, CRISPR-Cas

DIAO Chen-yang, CUI You-zhi, LI Bing-zhi. Research Advances in Targeted Mutagenesis-mediated Microbial Evolutionary Engineering[J]. Biotechnology Bulletin, 2025, 41(8): 11-21.

Figures/Tables 6

Fig. 1 Schematic diagram of DNA targeted evolution technologies

Fig. 2 Phage-assisted continuous evolution system (PACE)

Fig. 3 Research progress on mutation technology based on orthogonal DNA replication systemsA: Orthogonal DNA replication system in Saccharomyces cerevisiae (OrthoRep). B: Orthogonal DNA replication system in Bacillus thuringiensis(BacORep). C: Orthogonal DNA replication system in Escherichia coli (EcORep)

Fig. 4 Schematic diagram of the EvolvR mutation process

Fig. 5 Schematic diagram of the MutaT7 mutation process

Fig. 6 Research progress on targeted mutagenesis systems based on T7 RNA polymerase

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