Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions

Abstract

A transcriptional profiling of the metabolism of Corynebacterium glutamicum under oxygen deprivation conditions is reported. It was observed that the glucose consumption rate per cell when C. glutamicum cells were incubated under oxygen deprivation conditions was higher than that achieved by cells incubated under aerobic growth conditions. Furthermore, DNA microarray and quantitative RT-PCR analyses revealed that the genes of several key enzymes of the glycolytic and organic acid production pathways, including gapA, pgk, tpi, ppc, ldhA and mdh, were significantly upregulated under oxygen deprivation conditions. The corresponding enzymic activities consistently correlated with the regulation patterns of the genetic expression observed at the transcriptional level. Studies of lacZ fusions with the gapA, ldhA and mdh genes indicated not only that these genes are strongly induced at the onset of the stationary phase under aerobic growth conditions, but also that high expression levels are maintained under oxygen deprivation conditions. These results indicate that the genetic expression of several key metabolic enzymes in C. glutamicum cells incubated under oxygen deprivation conditions is chiefly regulated at the transcriptional level. The physiological consequence of the observed increased transcription under oxygen deprivation conditions is an increased rate of carbon source consumption, which is accompanied by a concomitant increase in organic acid production.

Involvement of Regulatory Interactions among Global Regulators GlxR, SugR, and RamA in Expression of ramA in Corynebacterium glutamicum

“…Mutations in the GlxR binding site were introduced by overlapping PCR using primers PramAmutFW and PramAmutRV, as described below. The fragments amplified were phosphorylated and cloned upstream of the lacZ gene in pCRA741 (48). The direction and sequence of the inserted fragment were confirmed by DNA sequencing.…”

Identification of a second β-glucoside phosphoenolpyruvate : carbohydrate phosphotransferase system in Corynebacterium glutamicum R

“…The promoter region of bglF or bglF2 was amplified by PCR using primers EcoRV-bglF1-400-F and EcoRV-bglF1-9-R for the bglF promoter and EcoRV-bglF2-400-F and EcoRV-bglF2-9-R for the bglF2 promoter. The amplified fragment was digested with EcoRV and cloned into the DraI site of the pCRA741 reporter plasmid, which is described elsewhere (Inui et al, 2007). Construction of mutants deleted for both RAT and the transcriptional terminator region of the bglF-or bglF2-lacZ fusion genes (PbglFDRAT-lacZ or PbglF2DRAT-lacZ) was conducted as follows.…”

“…C. glutamicum R was grown aerobically at 33 uC with shaking at 200 r.p.m. For the analysis of sugar concentration, C. glutamicum R was grown in 100 ml A medium (Inui et al, 2007) supplemented with 1 % sugar, or in BT minimal medium (Inui et al, 2007) supplemented with 0.2 % sugar. For the analysis of bgl expression, C. glutamicum R was grown in 10 ml A medium supplemented with 1 % (w/v) carbon sources.…”

Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum

“…For genetic manipulation, Escherichia coli strains were grown at 37 uC in LB medium (Sambrook et al, 1989). C. glutamicum was cultivated at 33 uC in 100 ml rich medium (A medium supplemented with 4 % glucose; Inui et al, 2007) except for the site-directed mutagenesis of CrrI and CgrI, where 10 ml rich medium was used. When appropriate, E. coli and C. glutamicum media were supplemented with 50 mg kanamycin ml 21 .…”