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Carotenoid biosynthesis in Corynebacteriumglutamicum is controlled by the MarR-type regulator CrtR, which represses transcription of the promoter of the crt operon (PcrtE) and of its own gene (PcrtR). Geranylgeranyl pyrophosphate (GGPP), and to a lesser extent other isoprenoid pyrophosphates, interfere with the binding of CrtR to its target DNA in vitro, suggesting they act as inducers of carotenoid biosynthesis. CrtR homologs are encoded in the genomes of many other actinobacteria. In order to determine if and to what extent the function of CrtR, as a metabolite-dependent transcriptional repressor of carotenoid biosynthesis genes responding to GGPP, is conserved among actinobacteria, five CrtR orthologs were characterized in more detail. EMSA assays showed that the CrtR orthologs from Corynebacteriumcallunae, Acidipropionibacteriumjensenii, Paenarthrobacternicotinovorans, Micrococcusluteus and Pseudarthrobacterchlorophenolicus bound to the intergenic region between their own gene and the divergently oriented gene, and that GGPP inhibited these interactions. In turn, the CrtR protein from C. glutamicum bound to DNA regions upstream of the orthologous crtR genes that contained a 15 bp DNA sequence motif conserved between the tested bacteria. Moreover, the CrtR orthologs functioned in C. glutamicum in vivo at least partially, as they complemented the defects in the pigmentation and expression of a PcrtE_gfpuv transcriptional fusion that were observed in a crtR deletion mutant to varying degrees. Subsequently, the utility of the PcrtE_gfpuv transcriptional fusion and chromosomally encoded CrtR from C. glutamicum as genetically encoded biosensor for GGPP was studied. Combined FACS and LC-MS analysis demonstrated a correlation between the sensor fluorescent signal and the intracellular GGPP concentration, and allowed us to monitor intracellular GGPP concentrations during growth and differentiate between strains engineered to accumulate GGPP at different concentrations.

Corynebacterium glutamicum is a natural producer of the C50 carotenoid decaprenoxanthin. The crtEcg0722crtBIYEb operon comprises most of its genes for terpenoid biosynthesis. The MarR-type regulator encoded upstream and in divergent orientation of the carotenoid biosynthesis operon has not yet been characterized. This regulator, named CrtR in this study, is encoded in many actinobacterial genomes co-occurring with terpenoid biosynthesis genes. CrtR was shown to repress the crt operon of C. glutamicum since DNA microarray experiments revealed that transcript levels of crt operon genes were increased 10 to 70-fold in its absence. Transcriptional fusions of a promoter-less gfp gene with the crt operon and crtR promoters confirmed that CrtR represses its own gene and the crt operon. Gel mobility shift assays with purified His-tagged CrtR showed that CrtR binds to a region overlapping with the −10 and −35 promoter sequences of the crt operon. Isoprenoid pyrophosphates interfered with binding of CrtR to its target DNA, a so far unknown mechanism for regulation of carotenogenesis. The molecular details of protein-ligand interactions remain to be studied. Decaprenoxanthin synthesis by C. glutamicum wild type was enhanced 10 to 30-fold upon deletion of crtR and was decreased 5 to 6-fold as result of crtR overexpression. Moreover, deletion of crtR was shown as metabolic engineering strategy to improve production of native and non-native carotenoids including lycopene, β-carotene, C.p. 450 and sarcinaxanthin.