Journal
MICROORGANISMS
Volume 9, Issue 4, Pages -Publisher
MDPI
DOI: 10.3390/microorganisms9040670
Keywords
CRISPR interference; carotenoids; CRISPRi; library; metabolic engineering; terpenoids; Corynebacterium glutamicum
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Funding
- European Regional Development Fund (ERDF)
- Ministry of Economic Affairs, Innovation, Digitalization and Energy of the State of North Rhine-Westphalia [EFRE-0400184, EFRE-0300095/1703FI04]
- Deutsche Forschungsgemeinschaft
- Bielefeld University
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This study constructed a library for the repression of 74 genes of C. glutamicum using CRISPRi technology, targeting important genes involved in metabolic pathways. The results showed that CRISPRi-mediated gene repression can increase carotenoid pigment biosynthesis, while deletion of certain genes can significantly enhance pigment production.
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.
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