Project description:Differential gene expression analysis of C. glutamicum ATCC 13032 in presence of 2.5 mM indole compared to control conditions without indole. C. glutamicum ATCC 13032 cells were cultivated in CGXII minimal medium with 40 g per litre glucose in presence of 2.5 mM indole and harvested during exponential phase (o.d.600 4).
Project description:To identify genes which are differentially expressed in Corynebacterium glutamicum chassis C1 in comparison to the prophage free strain MB001, we performed DNA microarray analyses of C. glutamicum C1 against MB001. For this purpose RNA was isolated from cells cultivated in CGXII minimal medium with 2% glucose (w v-1) and harvested in the exponential growth phase at an OD600 of 5. Four biological replicates were performed.
Project description:Differential gene expression analysis of C. glutamicum C1 in presence of 3 mM indole-alanine dipeptide compared to control conditions without indole-alanine dipeptide. C. glutamicum C1 cells were cultivated in CGXII minimal medium with 40 g per litre glucose in presence or absence of 3 mM indole-alanine dipeptide and harvested during exponential phase (o.d.600 6).
Project description:In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also harbor a proteasome, suggesting fates for pupylated proteins other than degradation via a proteasome or degradation at all. In the present study we set out to study pupylation in the proteasome-lacking non-pathogenic model microorganism and biotechnological workhorse Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew as the control indicating that pupylation seems to be dispensable under the conditions tested. By expression of homologous Pup carrying a poly-histidine tag in C. glutamicum ATCC 13032 we purified the first pupylome of a microorganism lacking a proteasome. Multidimensional Protein Identification Technology (MudPIT) unraveled 54 proteins being pupylated in this organism. Similar to mycobacteria, the majority of pupylated proteins in C. glutamicum can be classified as enzymes of the metabolism or as involved in translation. These results help to elucidate the common target pathways of pupylation in bacteria. Sample 1: For growth in CGXII minimal media, a preculture 1 was grown in 5 ml BHI medium inoculated with a single colony from a fresh BHI agar plate and incubated at 170 rpm for 8 hours. Then 500 M-BM-5l of preculture 1 were used to inoculate preculture 2 in 100 ml shake flasks with 20 ml CGXII minimal medium containing 4 % (w/v) glucose and incubated over night (140 rpm , 30 M-BM-0C ). Subsequent main cultures (50 ml CGXII medium with 4 % glucose) were inoculated with cells from preculture 2 to an OD600 of about 0.4. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done. Sample 2-4: For growth in CGXII minimal media, a preculture 1 was grown in 5 ml BHI medium inoculated with a single colony from a fresh BHI agar plate and incubated at 170 rpm for 8 hours. Then 500 M-BM-5l of preculture 1 were used to inoculate preculture 2 in 100 ml shake flasks with 20 ml CGXII minimal medium containing 4 % (w/v) glucose and incubated over night (140 rpm , 30 M-BM-0C ). Subsequent main cultures (50 ml CGXII medium with 4 % glucose) were inoculated with cells from preculture 2 to an OD600 of about 0.4. For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done for each growth medium. Sample 5-6: For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done.
Project description:Corynebacterium glutamicum shows a great potential for the production of gamma-aminobutyric acid (GABA) from glucose fermentation via putrescine. GABA, a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods and the biodegradable plastic polyamide 4. Here, the effect of GABA in the growth of C. glutamicum was evaluated. It was estimated that the presence 1.1 M of GABA in the medium reduces the maximum growth rate of C. glutamicum to half. It was also shown that the presence of GABA in the medium negatively affects the growth of C. glutamicum in ethanol as sole carbon source. Furthermore, a new route for the production of GABA in C. glutamicum was established. GABA production from glucose fermentation via putrescine was achieved by plasmid-based overexpression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) in a putrescine production strain. The resultant strain can produce 5.3 ± 0.1 g L-1 of GABA. GABA production was improved by avoiding the formation of N-acetylputrescine and by reducing the amount of nitrogen in CGXII medium. Deletion of the genes responsible for GABA catabolism and GABA re-uptake led to an increase in the GABA production of 21% achieving a titer 8.0 ± 0.3 g L-1 and an increase in the volumetric productivity of 41% reaching a productivity of 0.31 g L-1 h-1, the highest volumetric productivity achieved so far for GABA production in C. glutamicum from glucose fermentation in flasks fermentations. The results obtained hitherto are very promising and competitive compared to the traditional pathway for the production of GABA.
Project description:To understrand the altered global gene expression levels in C. glutamicum wild type in presence of furfural, transcriptome profiling was performed. Transcriptome profiles of the wild type grown in CgXII medium without furfural and with furfural stresses (each 6.5 mM, 13 mM, and 20 mM) were compared by using the samples taken at the OD600 of 6 (for the control and experiments). Each experiment was performed with a duplicate.