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基于CRISPR-Cas9的标准化代基因组编辑方法用于大肠杆菌

Huan Fang^1,2,3,4, Jianghua Zhao^1,2, Xinfang Zhao^1,5

¹Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.

ACS synthetic biology

|January 20, 2024

概括

这项研究引入了对大肠杆菌的标准化基因组编辑系统,简化了对复杂生物合成途径的多个基因的插入. 该系统实现了高效率,允许生产辅酶B12,提高了产量.

科学领域:

合成生物学合成生物学
代谢工程是代谢工程.
分子生物学分子生物学

背景情况:

将复杂的生物合成途径引入宿主基因组对于生产有价值的化合物至关重要.
现有的多基因插入方法,如CRISPR-Cas9,通常是低效和劳动密集的.

研究的目的:

开发一种标准化,代的基因组编辑系统,以便在大肠杆菌中高效地插入多基因.
促进用于化学生产的微生物细胞工厂的建设.

主要方法:

使用CRISPR-Cas9和MetClo组件,基于金门标准的模块化DNA组件.
开发了一个工具包,用于组装sgRNA/CRISPR阵列和捐赠者DNA.
优化了多重基因组插入效率,使用强大的促进器来促进tracrRNA表达.

主要成果:

在单个位点实现高达100%的基因插入效率.
证明了多重基因组插入效率为7.3%,具有优化的tracrRNA表达.
成功地将5-10基因 (5.38 Kb) 集成到E. coli中,用于辅酶B12生物合成.
产生了14个无抗生素,无等离子体的大肠杆菌生产者.
开发了一种产生1.49毫克L-1辅酶B12的重组菌株,这是大肠杆菌生产的创纪录.

结论:

开发的基因组编辑系统对于整合复杂的生物合成途径来说是用户友好和多功能.
这个工具箱加速了工业化工生产的路径工程和优化.
该系统可以在没有抗生素或等离子体选择标记的情况下创建高效的微生物细胞工厂.

关键词:

克里斯普尔-Cas9是什么意思埃舍里希亚大肠杆菌 (Escherichia coli) 是一个大肠杆菌.基因组工程是基因组工程.多基因插入插入.路径优化路径优化

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