title: Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system.
abstract: BACKGROUND
  Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid.
  
  
  RESULTS
  By knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defense (Cascade), we constructed a native CRISPRi system in E. coli. Downregulation of the target gene from 6 to 82% was demonstrated using green fluorescent protein. Regulation of the citrate synthase gene (gltA) in the TCA cycle affected host metabolism. The effect of metabolic flux regulation was demonstrated by the poly-3-hydroxbutyrate (PHB) accumulation in vivo.
  
  
  CONCLUSIONS
  By regulating native gltA in E. coli using an engineered endogenous type I-E CRISPR system, we redirected metabolic flux from the central metabolic pathway to the PHB synthesis pathway. This study demonstrated that the endogenous type I-E CRISPR-Cas system is an easy and effective method for regulating internal metabolic pathways, which is useful for product synthesis.
authors.affiliation: State Key Laboratory of Microbial Technology, Jinan, 250100, China.
authors.forename: Yizhao
authors.initials: Y
authors.lastname: Chang
authors.affiliation: State Key Laboratory of Microbial Technology, Jinan, 250100, China.
authors.forename: Tianyuan
authors.initials: T
authors.lastname: Su
authors.affiliation: State Key Laboratory of Microbial Technology, Jinan, 250100, China. qiqingsheng@sdu.edu.cn.
authors.forename: Qingsheng
authors.initials: Q
authors.lastname: Qi
authors.affiliation: State Key Laboratory of Microbial Technology, Jinan, 250100, China. liangquanfeng@sdu.edu.cn.
authors.forename: Quanfeng
authors.initials: Q
authors.lastname: Liang
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002950:Citrate (si)-Synthase / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000172:deficiency / Q000235:genetics; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D005818:Genetic Engineering; D006885:Hydroxybutyrates / Q000378:metabolism; D011091:Polyesters / Q000378:metabolism; D000094122:Polyhydroxybutyrates
chemical_list: D064130:CRISPR-Associated Proteins; D002950:Citrate (si)-Synthase; D004265:DNA Helicases; D029968:Escherichia coli Proteins; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C574631:ygcB protein, E coli
keywords: Escherichia coli; Poly-3-hydroxbutyrate; TCA cycle; Type I-E CRISPR-Cas system