Rational engineering of Corynebacterium glutamicum for the production of the dicarboxylic acid glutarate
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ABSTRACT:
ORGANISM(S): Corynebacterium glutamicum Corynebacterium glutamicum ATCC 13032
PROVIDER: GSE117175 | GEO | 2018/11/20
REPOSITORIES: GEO
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Project description:5-aminovalerate (5AVA), L-lysine derived compound, represents a potential building block for the production of the bio-plastic nylon-5. Escherichia coli has been engineered for the production of 5AVA, but Corynebacterium glutamicum has never been engineered for the production of 5AVA, but, a lot of work was done in the last decades to optimize the production of the precursor L-lysine and more recently cadaverine. 5AVA added to the growth medium hardly affected growth rate of C. glutamicum, since, a half-inhibitory concentration of 1.1 M 5AVA was determined. While in E. coli, 5AVA production was engineered by using the DavBA pathway from Pseudomonas putida, here a pathway based on the route described in P. aeruginosa was established. C. glutamicum wild type was converted into a 5AVA producing strain by heterologous expression of L-lysine decarboxylase (LdcC), putrescine transaminase (PatA) and γ-aminobutyraldehyde dehydrogenase (PatD) genes from E. coli. 5AVA production was improved by using a strain previously engineered for high L-lysine production, by de-repressing phosphoenolpyruvate phosphotransferase system (PTS) and glycolysis and by avoiding formation of the by-product L-lactate. 5AVA accumulation by this strain was increased to 44.9 mM, representing a yield of 202 mmol mol-1 glucose, which is about three times higher than the highest yield achieved in E. coli for the production of 5AVA from glucose fermentation.
2016-06-17 | GSE83413 | GEO
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.
2015-10-22 | GSE74258 | GEO
Project description:Ciprofloxacin, an inhibitor of bacterial gyrase and topoisomerase IV, was shown to inhibit growth of C. glutamicum with concomitant excretion of L-glutamate. C. glutamicum strains overproducing L-lysine, L-arginine, L-ornithine, and putrescine, respectively, produced L-glutamate instead of the desired amino acid when exposed to ciprofloxacin. Even in the absence of the putative L-glutamate exporter gene yggB, ciprofloxacin effectively triggered L-glutamate production. When C. glutamicum wild type was cultivated under nitrogen-limiting conditions, 2-oxoglutarate rather than L-glutamate was produced as consequence of exposure to ciprofloxacin. Transcriptome analysis revealed that ciprofloxacin increased RNA levels of genes involved in DNA synthesis, repair and modification. Enzyme assays showed that 2-oxoglutarate dehydrogenase activity was decreased due to ciprofloxacin addition. Here, it was shown for the first time that production of L-glutamate by C. glutamicum may be triggered by an inhibitor of DNA synthesis and L-glutamate titers of up to 37 ± 1 mM and a substrate specific L-glutamate yield of 0.13 g/g were reached.
2016-01-26 | GSE77189 | GEO
Project description:Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of L-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of L-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory formby changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L-1 in fed-batch cultivation at maximum volumetric productivities up to 1.34 g L-11 h1-1, the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N-acetyl-glucosamine that do not have competing uses in the food or feed industries was established.
2016-05-21 | GSE81695 | GEO
Project description:Corynebacterium glutamicum as a powerful host for the production of 5-aminovalerate from glucose fermentation
| PRJNA325936 | ENA
Project description:The Gram-positive soil bacterium Corynebacterium glutamicum is widely used in industrial fermentative processes for the production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose is taken up into the C. glutamicum cell by the phosphotransferase system PTS which can be replaced and/or enhanced by a permease and a glucokinase. Heterologous expression of the gene for the high-affinity glucose permease from Streptomyces coelicolor and of the Bacillus subitilis glucokinase gene fully compensated for the absence of the PTS in ï??hpr strains and strains grew as fast with glucose as C. glutamicum wild type. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and the glucokinase from Bacillus subtilis were overproduced using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid, a 40% higher L-lysine titer and a 30% higher volumetric productivity as compared to GRLys1(pEKEx3) resulted. The non-proteinogenic amino acid pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the L-lysine producing strain, the L-Lysine dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were expressed as synthetic operon. This enabled C. glutamicum to L-PA with a yield of 0.49 ± 0.03 gg-1 and a volumetric productivity of 0.04 ± 0.00 gL-1h-1.To the best of our knowledge, this is the first fermentative process for the production of L-PA. Two conditions tested, 200 mM NaCl Vs 200 mM pipecolic supplemented in the culture medium, control experiments done with the addition of 200mM of NaCl. Four technical replicates.
2016-03-30 | E-GEOD-79690 | biostudies-arrayexpress
Project description:The Gram-positive soil bacterium Corynebacterium glutamicum is widely used in industrial fermentative processes for the production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose is taken up into the C. glutamicum cell by the phosphotransferase system PTS which can be replaced and/or enhanced by a permease and a glucokinase. Heterologous expression of the gene for the high-affinity glucose permease from Streptomyces coelicolor and of the Bacillus subitilis glucokinase gene fully compensated for the absence of the PTS in hpr strains and strains grew as fast with glucose as C. glutamicum wild type. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and the glucokinase from Bacillus subtilis were overproduced using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid, a 40% higher L-lysine titer and a 30% higher volumetric productivity as compared to GRLys1(pEKEx3) resulted. The non-proteinogenic amino acid pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the L-lysine producing strain, the L-Lysine dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were expressed as synthetic operon. This enabled C. glutamicum to L-PA with a yield of 0.49 ± 0.03 gg-1 and a volumetric productivity of 0.04 ± 0.00 gL-1h-1.To the best of our knowledge, this is the first fermentative process for the production of L-PA.
2016-03-30 | GSE79690 | GEO
Project description:Previous findings have demonstrated that the NADH/NAD+ ratio has a strong impact on the glycolytic flux in C. glutamicum under anaerobic conditions in the absence of external electron acceptors. During an attempt to rewire anaerobic metabolism to achieve high yield formation of ethanol, we inactivated the malate dehydrogenase and lactate dehydrogenase in a C. glutamicum strain expressing pyruvate decarboxylase and alcohol dehydrogenase, to eliminate formation of the by-products succinate and lactate, respectively. This modification increased the yield of ethanol but had a negative effect on glycolysis, which we found to correlate with an elevated NADH/NAD+ ratio. The pyruvate dehydrogenase (PDH) of C. glutamicum is active under anaerobic conditions, and can potentially exacerbate the negative effect on glycolysis, due to NADH formation. To reduce PDH activity under anaerobic conditions, we decided to replace the gene encoding the E3 subunit of PDH with its Escherichia coli counterpart, as E. coli PDH has been reported to be functional under aerobic conditions only. The resultant strain JS133 produced far less acetate with a further increased ethanol production, however, the glycolytic flux was still low. After observing differences in glycolytic flux for JS133 on glucose and fructose, we speculated that the pentose phosphate pathway (PPP) might be involved in the reduced flux on glucose. To prove this, we deleted the zwf gene, encoding glucose-6-phosphate dehydrogenase, which is the entry point into PPP, and immediately observed a stimulating effect on glycolysis. Subsequent characterization revealed a direct correlation between the intracellular NADH/NAD+ and NADPH/NADP+ ratios under anoxic conditions. Based on these findings we managed to re-channel the metabolism of C. glutamicum successfully towards either to ethanol or D-lactate with 92% and 98% of the theoretical yield respectively, which is the highest yields for D-lactate production thus far reported in the literature.
2020-10-02 | GSE143977 | GEO
Project description:Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO2 + H2 as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H2) and the other steel mill off-gas (2% H2). Results were characterised using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimisation of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens.
2019-02-03 | GSE115837 | GEO
Project description:Cell proliferation is achieved by numerous enzyme reactions. Temperature governs the activity of each enzyme, which overall determines the optimal growth temperature. Synthesizing useful chemicals and fuels utilizes only part of the metabolic pathways, especially the central metabolic pathways such as glycolysis and TCA cycle to metabolize glucose. However, the optimal temperature for the activity of the central metabolic pathways is inconclusive whether it is correlated with the optimal temperature for cell proliferation. Here, we found that Corynebacterium glutamicum wild type increased the metabolic activity to consume glucose under anaerobic (oxygen deprived) conditions at 42.5ºC, in which the cell hardly grows under aerobic conditions. Glucose consumption rate was increased by 24% at 42.5ºC compared to that at the optimal growth temperature of 30.0ºC. Transcriptional analysis showed that gapX gene encoding glycelaldehydre-3-phosphate dehydrogenase, glucokinase gene, and a gene involved in glucose uptake were upregulated at 42.5ºC. Production of fermentative lactate was increased by 69% than that at 30.0ºC, whereas succinate production was decreased by 13%. In addition to several glycolytic enzymes, the activity of pyruvate kinase was increased with increasing the temperature, whereas the activity of phosphoenolpyruvate carboxylase in anapleotic pathway leading to succinate synthesis was decreased. However, a metabolically engineered succinate over-producing strain, in which lactate production was shut off and pyruvate carboxylase encoding gene in another anapleotic pathway was overexpressed, produced 34% higher succinate at 42.5ºC than that at 30.0ºC with increased glucose consumption. This study provides the evidence that the optimal reaction temperature for production of fermentative products can be set beyond upper limit of growth temperature in C. glutamicum, and hence it could be applicable for producing various chemicals and fuels in C. glutamicum under anaerobic conditions and non-growing cell reactions in other microorganisms.
2020-06-23 | GSE123063 | GEO