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Metab Eng. 2015 Sep;31:13-21. doi: 10.1016/j.ymben.2015.06.006. Epub 2015 Jun 30.

Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing.

Author information

1
Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, People's Republic of China; School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China. Electronic address: yfl@tju.edu.cn.
2
Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, People's Republic of China; School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China. Electronic address: zhenquanlin@tju.edu.cn.
3
Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, People's Republic of China; School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China. Electronic address: 1210990526@qq.com.
4
Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, People's Republic of China; School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China. Electronic address: zhangyan12@tju.edu.cn.
5
Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, People's Republic of China; School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China. Electronic address: zww@tju.edu.cn.
6
Key Laboratory of Fermentation Engineering, Ministry of Education, Hubei University of Technology, Wuhan 430068, People's Republic of China. Electronic address: yajietang@hotmail.com.
7
Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, People's Republic of China; School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China. Electronic address: chentao@tju.edu.cn.
8
Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, People's Republic of China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, People's Republic of China; School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People's Republic of China. Electronic address: xmzhao@tju.edu.cn.

Abstract

Engineering cellular metabolism for improved production of valuable chemicals requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli. This system enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously. We also found that cells with intact mismatch repair system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential, we used our method to integrate the β-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate (MEP) pathway and central metabolic pathways for β-carotene overproduction. We collectively tested 33 genomic modifications and constructed more than 100 genetic variants for combinatorially exploring the metabolic landscape. Our best producer contained15 targeted mutations and produced 2.0 g/L β-carotene in fed-batch fermentation.

KEYWORDS:

CRISPR/Cas9; Combinatorial metabolic engineering; Genome editing; β-Carotene

PMID:
26141150
DOI:
10.1016/j.ymben.2015.06.006
[Indexed for MEDLINE]

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