Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system.
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.


CONCLUSION
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.
['Chang|Yizhao|Y|', 'Su|Tianyuan|T|', 'Qi|Qingsheng|Q|', 'Liang|Quanfeng|Q|']
D064130:CRISPR-Associated Proteins D064113:CRISPR-Cas Systems D002950:Citrate (si)-Synthase D004265:DNA Helicases D015536:Down-Regulation D004926:Escherichia coli D029968:Escherichia coli Proteins D055786:Gene Knockout Techniques D005818:Genetic Engineering D006885:Hydroxybutyrates D011091:Polyesters
Escherichia coli Type I-E CRISPR-Cas system TCA cycle Poly-3-hydroxbutyrate