The CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway

• Published in: Metabolic engineering Vol. 43; no. Pt A; pp. 37 - 45
• Main Authors: Zhu, Xinna, Zhao, Dongdong, Qiu, Huanna, Fan, Feiyu, Man, Shuli, Bi, Changhao, Zhang, Xueli
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.09.2017
• Subjects: biochemical pathways; chromosomes; Combinatorial metabolic engineering; CRISPR-Cas Systems; CRISPR/Cas9; CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO); Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; gene editing; genes; Genome editing; Metabolic Engineering; plasmids; Plasmids - genetics; Plasmids - metabolism; regulatory sequences; transcription (genetics); xylose; Xylose - genetics; Xylose - metabolism; CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO); Genome editing; CRISPR/Cas9; Combinatorial metabolic engineering
• ISSN: 1096-7176; 1096-7184; 1096-7184
• DOI: 10.1016/j.ymben.2017.08.003

One of the most important research subjects of metabolic engineering is the pursuit of balanced metabolic pathways, which requires the modulation of expression of many genes. However, simultaneously modulating multiple genes on the chromosome remains challenging in prokaryotic organisms, including the industrial workhorse - Escherichia coli. In this work, the CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique was developed to simultaneously modulate the expression of multiple genes on the chromosome. To implement it, two plasmids were employed to target Cas9 to regulatory sequences of pathway genes, and a donor DNA plasmid library was constructed containing a regulator pool to modulate the expression of these genes. A modularized plasmid construction strategy was used to enable the assembly of a complex donor DNA plasmid library. After genome editing using this technique, a combinatorial library was obtained with variably expressed pathway genes. As a demonstration, the CFPO technique was applied to the xylose metabolic pathway genes in E. coli to improve xylose utilization. Three transcriptional units containing a total of four genes were modulated simultaneously with 70% efficiency, and improved strains were selected from the resulting combinatorial library by growth enrichment. The best strain, HQ304, displayed a 3-fold increase of the xylose-utilization rate. Finally, the xylose-utilization pathway of HQ304 was analyzed enzymologically to determine the optimal combination of enzyme activities. •A novel CRISPR/Cas9 CFPO technique was established.•CFPO was applied to modulate expression of three genetic components on chromosome.•One of the best E. coli xylose utilization strain was obtained by CFPO technique.