Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing

• Published in: Metabolic engineering Vol. 31; pp. 13 - 21
• Main Authors: Li, Yifan, Lin, Zhenquan, Huang, Can, Zhang, Yan, Wang, Zhiwen, Tang, Ya-jie, Chen, Tao, Zhao, Xueming
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.09.2015
• Subjects: base pair mismatch; batch fermentation; beta Carotene - biosynthesis; beta-carotene; biochemical pathways; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Combinatorial metabolic engineering; CRISPR-Cas Systems; CRISPR/Cas9; DNA Mismatch Repair; DNA repair; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; genetic variation; genome; Genome editing; Genome, Bacterial; Metabolic Engineering; RNA Editing - genetics; β-Carotene; CRISPR; ORF; TRMR; CRISPR/Cas9; G3P; gRNA; Combinatorial metabolic engineering; β-Carotene; MAGE; DSB; MMR; MEP; PEP; Genome editing; PAM; ssDNA; dsDNA
• ISSN: 1096-7176; 1096-7184; 1096-7184
• DOI: 10.1016/j.ymben.2015.06.006

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 2.0g/L β-carotene in fed-batch fermentation. •Generated near 100% editing efficiency using dsDNA as editing template.•One cycle of genomic editing required only two days.•Strains with functional MMR system yielded increased editing efficiency.•Combinatorially optimized MEP pathway and central metabolic pathways.•Best strain produced 2.0g/L β-carotene using glucose as the sole carbon source.