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在Zymomonas mobilis中开发一个可反选择的系统,用于基于CRISPR的快速有效的基因组工程

Yanli Zheng¹, Hongmei Fu¹, Jue Chen^1,2

¹College of Life Science and Technology, Wuhan Polytechnic University, Wuhan, 430023, P. R. China.

Microbial cell factories

|October 14, 2023

概括

一种新的反选择标记物clmPheS能够在Zymomonas mobilis中有效地治愈等离子体. 这加速了利用这个工业细菌进行生物燃料和化学生产的基因组工程.

科学领域:

微生物学微生物学
合成生物学合成生物学
生物技术是生物技术.

背景情况:

齐莫莫纳斯莫比利斯是生物炼油厂和合成生物学的关键工业细菌.
基于CRISPR的基因组编辑加速了Z. mobilis的工程,以实现可持续的化学,蛋白质和生物燃料生产.
高效的等离子体固化对于这些编辑技术至关重要,但对Z. mobilis.存在有限的反选择标记.

研究的目的:

开发一种新型的对抗选择标记物,用于Z. mobilis.中高效的等离子体治愈.
提高Z. mobilis的基因组工程效率,用于工业应用.

主要方法:

构建了一个Z. mobilis ZM4 pheS基因的条件致命突变 (clmPheS).
这种clmPheS突变允许纳入有毒的p-chloro-phenylalanine (4-CP) 模拟物,抑制生长.
使用强烈的P_gap促进剂来表达对4-CP.敏感性的clmPheS.

主要成果:

在Z. mobilis中,表达clmPheS的ZM4细胞对0.2mM 4-CP.具有很高的敏感性.
通过clmPheS辅助的对选择,可在突变发生后快速治愈等离子体.
减少多重染色体编辑的时间,每两轮至少减少9天;启用无痕等离子体修饰.

结论:

开发了一种策略,将CRISPR编辑与Z. mobilis.的新型反选择标记物结合起来.
实现了快速,高效的多轮多重染色体编辑和无痕的等离子体修饰.
为Z. mobilis提供了一个改进的基因组工程工具包,以及其他非模型生物的参考.

关键词:

在CRISPR-Cas基因组工程中,多回合多重基因组编辑.原生质粒编辑原生质粒编辑没有痕的突变发生.齐莫莫纳斯的移动性 clmPheS是对抗选择标记物的.

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