Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi)

Abstract

Corynebacterium glutamicum is an important organism for the industrial production of amino acids. Metabolic pathways in this organism are usually engineered by conventional methods such as homologous recombination, which depends on rare double-crossover events. To facilitate the mapping of gene expression levels to metabolic outputs, we applied CRISPR interference (CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C. glutamicum. We then determined the effects of target repression on amino acid titers. Single-guide RNAs directing dCas9 to specific targets reduced expression of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement ratios of l-lysine and l-glutamate production comparable to levels achieved by gene deletion. This approach for C. glutamicum metabolic engineering, which only requires 3 days, indicates that CRISPRi can be used for quick and efficient metabolic pathway remodeling without the need for gene deletions or mutations and subsequent selection.

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

“…A significant level of repression of ␤-galactosidase expression via CRISPRi was reported in E. coli in which dCas9 and a lacZ-targeting gRNA were maintained in plasmids (29). In the same study, up to 300-fold repression of expression of a monomeric red fluorescent protein (mRFP) was observed, which is similar to the CRISPR-dCas9-mediated RFP repression levels achieved in Corynebacterium glutamicum (39). The differences in repression efficiency observed between ␤-galactosidase and RFP suggests that certain targets may be more susceptible to CRISPRi.…”

“…On the other hand, CRISPRi provides excellent transcriptional control and is simple to implement in many organisms. The CRISPRdCas9 system has been applied to genome-scale transcriptional repression (35) and activation (35,36) for interrogation of gene function in human cell lines as well as to genetic and metabolic engineering of Escherichia coli (37,38), Corynebacterium glutamicum (39), and mycobacteria (40).…”

Amino acids production focusing on fermentation technologies – A review

“…Moreover, the deletion of pck and pyk genes, results in an accumulation of Lglutamate through an enhanced flux of oxaloacetate in the TCA cycle, due to the absence of the transformation of oxaloacetate to phospoenolpyruvate. They demonstrated that final L-lysine and L-glutamate yields obtained with the reduced expression of pgi, pck, and pyk genes are comparable to the one obtained by gene deletion (Cleto et al, 2016). Therefore CRISPRi represents a quick and efficient solution to modify the pathway without complex gene deletions or mutations (Dominguez et al, 2016).…”

“…One of the most innovative procedures is the engineering of new enzymes, such as glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which can produce 2 moles of NADPH from 1 moles of glucose (Bommareddy et al, 2014) leading to a higher yield of the target amino acid. Moreover, innovative approaches utilizing CRISPR interference (CRISPRi) to modify quickly and efficiently the metabolic pathway without gene deletions and mutation has been recently developed (Cleto et al, 2016) (see section 3.2.7 for more details).…”

“…Furthermore, since the central genes for metabolism directly impact the production of amino acids, techniques such as riboswitch and CRISPRi enable the construction of optimized microorganisms, improving the production of amino acids and other high value chemicals (Zhou and Zeng, 2015b;Wendisch et al, 2016). CRISPRi interference technology with the deactivated Cas9 protein (dCas9) was used by Cleto et al (2016) to determine the effect of target gene repression on L-lysine and the L-glutamate production. In their study they demonstrated that the application of CRISPRi/dCas9-mediated for pathway engineering in C. glutamicum strongly enhanced L-lysine and the L-glutamate production.…”