CRISPRi-Library-Guided Target Identification for Engineering Carotenoid Production by Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum is a prominent production host for various value-added compounds in white biotechnology. Gene repression by dCas9/clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) allows for the identification of target genes for metabolic engineering. In this study, a CRISPRi-based library for the repression of 74 genes of C. glutamicum was constructed. The chosen genes included genes encoding enzymes of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, regulatory genes, as well as genes of the methylerythritol phosphate and carotenoid biosynthesis pathways. As expected, CRISPRi-mediated repression of the carotenogenesis repressor gene crtR resulted in increased pigmentation and cellular content of the native carotenoid pigment decaprenoxanthin. CRISPRi screening identified 14 genes that affected decaprenoxanthin biosynthesis when repressed. Carotenoid biosynthesis was significantly decreased upon CRISPRi-mediated repression of 11 of these genes, while repression of 3 genes was beneficial for decaprenoxanthin production. Largely, but not in all cases, deletion of selected genes identified in the CRISPRi screen confirmed the pigmentation phenotypes obtained by CRISPRi. Notably, deletion of pgi as well as of gapA improved decaprenoxanthin levels 43-fold and 9-fold, respectively. The scope of the designed library to identify metabolic engineering targets, transfer of gene repression to stable gene deletion, and limitations of the approach were discussed.

Recent advances in the metabolic pathways and microbial production of coenzyme Q

“…In addition, combining the Entner–Doudoroff pathway with the MEP pathway increased isoprene titers in E. coli three-fold (Liu et al 2013 ). With the advent of CRISPRi-mediated repression, fast screening of many target genes among different pathways allows to find suitable candidates to direct flux towards IPP and DMAPP in shorter time as it has been shown for E. coli (Tian et al 2019 ) and C. glutamicum (Göttl et al 2021 ). Lastly, cofactor economy plays an important role as some enzymes of the MEP and MVA pathways as well as of the CoQ 10 synthesis are dependent on NAD(P)H, ATP or SAM.…”

Rational Engineering of Non-Ubiquinone Containing Corynebacterium glutamicum for Enhanced Coenzyme Q10 Production

“…In order to improve precursor supply for CoQ 10 production, overexpression of the MEP pathway genes dxs and idi is a common way to increase flux towards IPP and DMAPP and has been shown to increase patchoulol production in an engineered C. glutamicum strain [ 29 ]. Other strategies aim at the supply and distribution of the molecules of the MEP pathway entry point, glyceraldehyde 3-phosphate and pyruvate [ 54 ], e.g., by the modification of central carbon metabolism [ 55 ], CRISPRi-mediated repression [ 56 ], and increase in the NAD(P)H pool [ 57 ]. A different kind of approach is membrane engineering that involves the expression of proteins with membrane-bending properties and the overall increase in membrane synthesis to expand the membrane surface area and storage capacity for CoQ 10 .…”

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

“…Large-scale CRISPRi screens and selections have been developed to investigate genotype-phenotype relationships through gRNA fitness ( Figure 1D ). These assays can use small, targeted libraries, such as essential genes or genes in a metabolic pathway ( Shields et al, 2020 ; Göttl et al, 2021 ), or large genome-wide libraries targeting nearly all genes in the bacterial genome ( Lee et al, 2019 ; Jiang et al, 2020 ). Additionally, CRISPRi libraries can be constructed in two major forms—pooled libraries, where cells containing different gRNA are mixed during library construction ( Bosch et al, 2021 ; Rahman et al, 2021 ), a strategy known as multiplexing, or arrayed libraries where different gRNA designs are constructed individually in different clonal populations, typically arrayed in microtiter plates ( Liu et al, 2017 ; Göttl et al, 2021 ).…”

“…These assays can use small, targeted libraries, such as essential genes or genes in a metabolic pathway ( Shields et al, 2020 ; Göttl et al, 2021 ), or large genome-wide libraries targeting nearly all genes in the bacterial genome ( Lee et al, 2019 ; Jiang et al, 2020 ). Additionally, CRISPRi libraries can be constructed in two major forms—pooled libraries, where cells containing different gRNA are mixed during library construction ( Bosch et al, 2021 ; Rahman et al, 2021 ), a strategy known as multiplexing, or arrayed libraries where different gRNA designs are constructed individually in different clonal populations, typically arrayed in microtiter plates ( Liu et al, 2017 ; Göttl et al, 2021 ). Pooled competitive selections are more common due to the ease of DNA construction and analysis of large, genome-scale gRNA libraries with >10,000 designs by next-generation sequencing ( Lee et al, 2019 ; Bosch et al, 2021 ).…”