Corynebacterium glutamicum CrtR and Its Orthologs in Actinobacteria: Conserved Function and Application as Genetically Encoded Biosensor for Detection of Geranylgeranyl Pyrophosphate

Abstract

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.

Toward improved terpenoids biosynthesis: strategies to enhance the capabilities of cell factories

“…6 A). In a recent related study, the carotenoid biosynthesis regulator, crtR , in C. glutamicum was engineered to measure the intracellular GGPP concentration during growth (Henke et al 2020 ). These sensors could be employed as a high-throughput to screen for isoprenoid-producing strains.…”

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being

“…Naturally, C. glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as a yellow pigment and, correspondingly, carotenoid biosynthetic genes of the non-mevalonate pathway, involving isopentenyl pyrophosphate as a central intermediate [104]. Native carotenoid biosynthesis is controlled by the GGPP-responsive transcriptional repressor CrtR [108] and is increased via CrtR upon exposure to light [109].…”

From Aquaculture to Aquaculture: Production of the Fish Feed Additive Astaxanthin by Corynebacterium glutamicum Using Aquaculture Sidestream

“…First, the carotenoid biosynthesis pathway was terminated at lycopene by deletion of crtY e Y f Eb [ 49 , 58 ], and the precursor supply was improved [ 58 , 59 ]. Furthermore, the regulatory mechanism of the carotenoid biosynthesis by its precursor molecule geranylgeranyl pyrophosphate (GGPP) responsive transcriptional repressor CrtR was elucidated [ 57 , 60 ] and deletion of its gene was shown to improve astaxanthin production [ 46 ]. Conversion of β-carotene to astaxanthin was improved by a fusion protein of the heterologous β-carotene hydroxylase and β-carotene ketolase from Fulvimarina pelagi [ 46 ].…”