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

• Published in: International journal of molecular sciences Vol. 21; no. 15; p. 5482
• Main Authors: Henke, Nadja A, Austermeier, Sophie, Grothaus, Isabell L, Götker, Susanne, Persicke, Marcus, Peters-Wendisch, Petra, Wendisch, Volker F
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 31-07-2020; MDPI
• Subjects: Actinobacteria - genetics; Actinobacteria - metabolism; Bacterial Proteins - physiology; Binding Sites; Biosensing Techniques; biosensor; Biosensors; Biosynthesis; C. glutamicum; Carotenoids; Carotenoids - metabolism; Conserved sequence; Correlation analysis; Corynebacterium glutamicum - genetics; Corynebacterium glutamicum - metabolism; Deletion mutant; Deoxyribonucleic acid; DNA; Engineering; Flow cytometry; Fluorescence; Gene Expression Regulation, Bacterial; Genes; Genetic code; Genomes; GGPP; Homology; Intracellular; Metabolism; Metabolites; Nucleotide sequence; Pigmentation; Polyisoprenyl Phosphates - analysis; Promoter Regions, Genetic; Proteins; regulation of carotenogenesis; Transcription; Transcription Factors - physiology; GGPP; biosensor; regulation of carotenogenesis; C. glutamicum
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21155482

Carotenoid biosynthesis in
is controlled by the MarR-type regulator CrtR, which represses transcription of the promoter of the
operon (P
) and of its own gene (P
). 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
, five CrtR orthologs were characterized in more detail. EMSA assays showed that the CrtR orthologs from
,
,
,
and
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
bound to DNA regions upstream of the orthologous
genes that contained a 15 bp DNA sequence motif conserved between the tested bacteria. Moreover, the CrtR orthologs functioned in
in vivo at least partially, as they complemented the defects in the pigmentation and expression of a P
_
uv transcriptional fusion that were observed in a
deletion mutant to varying degrees. Subsequently, the utility of the P
_
uv transcriptional fusion and chromosomally encoded CrtR from
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.