Comparative analysis on Corynebacterium glutamicum transcriptome in response to the change of dissolved oxygen in bioreactor
ABSTRACT: Purpose: dissolved oxygen (DO) level is an important factor that could significantly influence microorganisms’ growth, maintenance, metabolism and product yield. The goals of this study are to do comparative analysis on Corynebacterium glutamicum transcriptome in response to the change of dissolved oxygen in bioreactor, find the critical pathways and genes. Overall design: Method: three batches of fermentation were conducted with different DO level separately (50%, 30%, 0%), and sampled on 20h of fermentation, the transcriptome of Corynebacterium glutamicum generated using Illumina hiseq 2000.
Project description:Purpose:dissolved oxygen (DO) level is an important factor that could significantly influence microorganisms’ growth, maintenance, metabolism and product yield.The goals of this study are to comparative analysis on Corynebacterium glutamicum transcriptome in response to expression of eGFP under the change of dissolved oxygen in bioreactor,find the critical pathways and genes. Overall design: Method:three batches of fermentation were conducted with different DO level separately (50%, 30%, 0%),and sampled on 20h of fermentation,the transcriptome of Corynebacterium glutamicum generated using Illumina hiseq 2500.
Project description:To investigate the adaptation of Corynebacterium glutamicum to altering oxygen availabilities, we conceived a triple-phase fermentation process that describes a gradual reduction of dissolved oxygen depicting a shift from aerobiosis via microaerobiosis to anaerobiosis. The distinct process phases were clearly bordered by the bacteria’s physiologic response such as reduced growth rate, biomass substrate yield and altered yield of fermentation products. During the process, sequential samples were drawn at six points and analyzed via RNA-sequencing of Illumina TruSeq Stranded mRNA libaries sequenced paired end on an Illumina MiSeq system using 75 nt read length. We found transcriptional alterations of almost 50 % (1421 genes) of the entire protein coding genes and observed an upregulation of fermentative pathways, a rearrangement of respiration and mitigation of the basic cellular mechanisms such as transcription, translation and replication as a transient response related to the installed oxygen dependent process phases.
Project description:The exploration of scale-down models to imitate the influence of large scale bioreactor inhomogeneities on cellular metabolism is a topic with increasing relevance. While gradients of substrates, pH, or dissolved oxygen are often investigated, oscillating CO2/HCO3- levels, a typical scenario in large industrial bioreactors, is rarely addressed. Hereby, we investigate the metabolic and transcriptional response in Corynebacterium glutamicum wild type as well as the impact on L-lysine production in a model strain exposed to pCO2 gradients of (75-315) mbar. A novel three-compartment cascade bioreactor system was developed and characterized that offers high flexibility for installing gradients and residence times to mimic industrial-relevant conditions, and provides the potential of accurate carbon balancing. The phenomenological analysis of cascade fermentations imposed to the pCO2 gradients at industry-relevant residence times of about 3.55 min did not significantly impair the process performance, with growth and product formation being similar to control conditions. However, transcriptional analysis disclosed up to 66 differentially expressed genes already after 3.55min under stimulus exposure, with the overall change in gene expression directly correlateable to the pCO2 gradient intensity and the residence time of the cells.
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. Overall design: Corynebacterium glutamicum chassis C1 in comparison to the prophage free strain MB001
Project description:Metabolically engineered Corynebacterium glutamicum strains were constructed for the enhanced production of L-arginine, and their gene expression profiles were investigated Gene expression profiles of two C. glutamicum strains AR2 and AR6 were examined for the 3043 genes twice.
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. Overall design: Two conditions tested: 200 mM of NaCl vs 200 mM GABA supplemented in the culture medium. Control experiment done with the addition of 200 mM NaCl 2 technical replicates
Project description:Profiles of two major acyl-modifications, lysine acetylation and succinylation, under L-glutamate-producting and non-producing conditions in Corynebacterium glutamicum, which is industrially utilized for amino acid fermentation, was analyzed. During glutamate overproduction induced by Tween 40, global lysine acetylation was decreased, while lysine succinylation was increased. A label-free semi-quantitative proteomic analysis identified 591 acetylated proteins with 1,509 unique acetylation sites and 297 succinylated proteins with 790 unique succinylation sites. Lysine acetylation and succinylation targeted most enzymes in the central carbon metabolic pathways that are directly related to glutamate production, including the 2-oxoglutarate dehydrogenase complex (ODHC), a key enzyme for glutamate overproduction.