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.

Lycopene β-cyclase (EC 5.5.1.19) is one of the key enzymes in the biosynthesis of β-carotene and derived carotenoids. It catalyzes isomerase reactions to form β-carotene from lycopene by β-cyclization of both of its ψ-ends. Lycopene β-cyclases are widespread in nature. We systematically analyzed the phylogeny of lycopene β-cyclases from all kingdoms of life and predicted their transmembrane structures. To this end, a collection of previously characterized lycopene β-cyclase polypeptide sequences served as bait sequences to identify their closest homologues in a range of bacteria, archaea, fungi, algae, and plant species. Furthermore, a DeepTMHMM scan was applied to search for the presence of transmembrane domains. A phylogenetic tree suggests at least five distinct clades, and the DeepTMHMM scan revealed that lycopene β-cyclases are a group of structurally different proteins: membrane-bound and cytosolic enzymes. Representative lycopene β-cyclases were screened in the lycopene-overproducing Corynebacterium glutamicum strain for β-carotene and astaxanthin production. This systematic screening facilitates the identification of new enzymes for carotenoid production. Higher astaxanthin production and less reduction of total carotenoids were achieved with the cytosolic lycopene β-cyclase CrtL from Synechococcus elongatus and the membrane-bound heterodimeric lycopene β-cyclase CrtYcd from Brevibacterium linens.

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