靶向诱变介导的微生物进化技术研究进展

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

摘要/Abstract

摘要：

生物基因组自然突变赋予其不同性状，驱动生物进化适应生存环境。然而，生物自然条件下进化速度缓慢。通过在特定DNA序列引入碱基突变的策略可实现生物实验室内的快速进化，广泛应用于蛋白功能非理性改造和代谢路径优化。然而，传统的诱变进化方法存在着实验工作量大、突变效率低，突变窗口不精确等缺点，而靶向突变技术通过精确定位特定DNA范围引入突变，不仅能有效提高突变效率，还能显著缩短实验室进化周期。近年来，在目标序列靶向引入突变已成为微生物进化技术重要研究方向。靶向突变技术能够优化特定微生物的代谢途径、提升底盘耐受性，广泛应用于生物催化、环境保护、工业发酵等领域。本文总结了在主要模式微生物底盘中开发的靶向突变进化技术，重点介绍了基于复制易错系统、CRISPR-Cas系统和MutaT7系统的靶向突变系统的设计原理与应用，同时分析了现有实验室进化系统的优缺点，并讨论了未来靶向诱变技术发展方向。

关键词: 定向进化, 靶向突变, 基因编辑, RNA聚合酶, CRISPR-Cas

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

刁辰洋, 崔有志, 李炳志. 靶向诱变介导的微生物进化技术研究进展[J]. 生物技术通报, 2025, 41(8): 11-21.

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.

图/表 6

图1 DNA靶向进化技术示意图

Fig. 1 Schematic diagram of DNA targeted evolution technologies

图2 噬菌体辅助连续进化系统（PACE）

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

图3 基于正交DNA复制系统的突变技术研究进展A：酿酒酵母正交DNA复制系统（OrthoRep）；B：苏云金芽孢杆菌正交DNA复制系统（BacORep）；C：大肠杆菌正交DNA复制系统（EcORep）

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)

图4 EvolvR突变过程示意图

Fig. 4 Schematic diagram of the EvolvR mutation process

图5 MutaT7系统突变过程示意图

Fig. 5 Schematic diagram of the MutaT7 mutation process

图6 基于T7 RNA聚合酶的靶向诱变系统研究进展

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

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