The adaptation of Corynebacterium glutamicum to acetate as a carbon and energy source involves transcriptional regulation of the pta-ack operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the aceA and aceB genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as cis-regulatory elements for expression of the pta-ack operon and the aceA and aceB genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the pta-ack operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the pta-ack and the aceA/aceB promoter/operator regions. Directed deletion of the ramB gene in the genome of C. glutamicum resulted in mutant strain RG1. Whereas the wild type of C. glutamicum showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of C. glutamicum.

In Corynebacterium glutamicum, the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective ptaack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum, showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG 4-6 T/C or AC 4-5 A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum.Corynebacterium glutamicum is a nonpathogenic, aerobic grampositive soil bacterium that is widely used for the large-scale production of amino acids such as L-glutamate and L-lysine (21,24,28,30,35). In addition, the organism has gained increasing interest as a suitable model organism for the Corynebacterineae, a suborder of the actinomycetes which also includes the medically important genus Mycobacterium (51).C. glutamicum is able to grow on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy, and among these substrates are glucose and acetate (31, 34). Based on biochemical, genetic, and regulatory studies; on quantitative determination of metabolic fluxes during utilization of acetate and/or glucose; and on genome-wide comparative expression analyses, there is considerable knowledge of enzymes and genes involved in acetate metabolism of C. glutamicum (reviewed in references 9 and 17). The utilization of acetate involves its uptake and subsequent activation to acetyl coenzyme A (CoA) and, when acetate is the sole carbon substrate, also requires the operation of the glyoxylate cycle as anaplerotic pathway. The key enzymes of acetate activation, acetate kinase (AK) and phosphotransacetylase (PTA), and those of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS)...

No abstract

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the L-valine biosynthetic genes ilvBNCE, all strains produced L-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 ⌬pqo ⌬ppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) L-valine with an overall yield (Y P/S ) of 0.36 mol per mol of glucose and a volumetric productivity (Q P ) of 13.6 mM per h [1.6 g/(liter ؋ h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the L-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a Y P/S of 0.24 mol per mol of glucose and a Q P of 6.9 mM per h [0.8 g/(liter ؋ h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum L-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

The transcriptional regulator Cg1486 of Corynebacterium glutamicum ATCC 13032 is a member of the IclR protein family and belongs to the conserved set of regulatory proteins in corynebacteria. A defined deletion in the cg1486 gene, now designated ltbR (leucine and tryptophan biosynthesis regulator), led to the mutant strain C. glutamicum IB1486. According to whole-genome expression analysis by DNA microarray hybridizations, transcription of the leuB and leuCD genes encoding enzymes of the leucine biosynthesis pathway was enhanced in C. glutamicum IB1486 compared with the wild-type strain. Moreover, the genes of the trpEGDCFBA operon involved in tryptophan biosynthesis of C. glutamicum showed an enhanced expression in the cg1486 mutant strain. Bioinformatics pattern searches in the upstream regions of the differentially expressed genes revealed the common 12-bp motif CA(T/C)ATAGTG(A/G)GA that is located downstream of the ؊10 region of the mapped promoter sequences. DNA band shift assays with a streptavidin-tagged LtbR protein demonstrated the specific binding of the purified protein to 40-mers containing the 12-bp motif localized in front of leuB, leuC, and trpE, thereby confirming the direct regulatory role of LtbR in the expression of the leucine and tryptophan biosynthesis pathway genes of C. glutamicum. Genes homologous with ltbR were detected upstream of the leuCD genes in almost all sequenced genomes of bacteria belonging to the taxonomic class Actinobacteria. The ltbR-like genes of Corynebacterium diphtheriae, Corynebacterium jeikeium, Mycobacterium bovis, and Bifidobacterium longum were cloned and shown to complement the deregulation of leuB, leuCD, and trpE gene expression in C. glutamicum IB1486.