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Fermentative production of the diamine putrescine: system metabolic engineering of corynebacterium glutamicum.

Nguyen AQ, Schneider J, Reddy GK, Wendisch VF - Metabolites (2015)

Bottom Line: Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering.Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%.Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant.

View Article: PubMed Central - PubMed

Affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany. anguyen@cebitec.uni-bielefeld.de.

ABSTRACT
Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g-1·h-1 and a yield on glucose of 0.26 g·g-1.

No MeSH data available.


Related in: MedlinePlus

Metabolic flux distribution in C. glutamicum (A) and the relative flux through glucose 6-phosphate dehydrogenase Zwf and malate enzyme (MalE) (B) as a function of putrescine production. (A) Objective function was biomass flux, except for 100% putrescine flux. The metabolic flux was distributions were calculated in C. glutamicum without (in black) and with (in red) putrescine secretion to obtain yield coefficient (YP/S) of 25, 50, 75, 94%, respectively, relative to the glucose uptake rate. All fluxes are given in percent and are normalized to glucose uptake. Values are sorted by increasing putrescine flux. Solid line: Zwf flux, dotted line: MalE flux. For abbreviations: 1,3PG: 1,3-Bisphosphogylceric acid, 2OXO: 2-Oxoglutaric acid , 2PG: 2-Phosphoglyceric acid, 3PG: 3-Phosphoglyceric acid, AC-CoA: Acetyl-CoA, CIT: Citric acid, DHAP: Dihydroxyacetonephosphate, F6P: Fructose-6-phosphate, G6P: Glucose-6-phosphate, GA3P: Glyceraldyehyde-3-phosphate, GLC: Glucose, GLC-LAC: 6-Phosphogluconolactone, GLC6P: 6-Phosphogluconic acid, GLU: l-Glutamic acid, GLY: Glycerol, GLY3P: Glycerol-3-phosphate, ICI: Isocitric acid, l-RIB5P: l-Ribulose-5-phosphate, MAL: Malic acid, NAC-GLU: N-Acteylglutamic acid, OAA: Oxalacetic acid, ORN: l-Ornithine, PEP: Phosphoenolpyruvic acid, PUT: Putrescine, PYR: Pyruvic acid, RIB: LRibulose, RIB5P: Ribulose-5-phosphate, RIBO5P: Ribose-5-phosphate, S7P: Sedoheptulose-7-phosphate, E4P: Erythrose-4-phosphate, SUC: Succinic acid. Arrows from intermediates marked in grey boxes perpendicular to the metabolic reactions indicate flux into biomass.
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metabolites-05-00211-f001: Metabolic flux distribution in C. glutamicum (A) and the relative flux through glucose 6-phosphate dehydrogenase Zwf and malate enzyme (MalE) (B) as a function of putrescine production. (A) Objective function was biomass flux, except for 100% putrescine flux. The metabolic flux was distributions were calculated in C. glutamicum without (in black) and with (in red) putrescine secretion to obtain yield coefficient (YP/S) of 25, 50, 75, 94%, respectively, relative to the glucose uptake rate. All fluxes are given in percent and are normalized to glucose uptake. Values are sorted by increasing putrescine flux. Solid line: Zwf flux, dotted line: MalE flux. For abbreviations: 1,3PG: 1,3-Bisphosphogylceric acid, 2OXO: 2-Oxoglutaric acid , 2PG: 2-Phosphoglyceric acid, 3PG: 3-Phosphoglyceric acid, AC-CoA: Acetyl-CoA, CIT: Citric acid, DHAP: Dihydroxyacetonephosphate, F6P: Fructose-6-phosphate, G6P: Glucose-6-phosphate, GA3P: Glyceraldyehyde-3-phosphate, GLC: Glucose, GLC-LAC: 6-Phosphogluconolactone, GLC6P: 6-Phosphogluconic acid, GLU: l-Glutamic acid, GLY: Glycerol, GLY3P: Glycerol-3-phosphate, ICI: Isocitric acid, l-RIB5P: l-Ribulose-5-phosphate, MAL: Malic acid, NAC-GLU: N-Acteylglutamic acid, OAA: Oxalacetic acid, ORN: l-Ornithine, PEP: Phosphoenolpyruvic acid, PUT: Putrescine, PYR: Pyruvic acid, RIB: LRibulose, RIB5P: Ribulose-5-phosphate, RIBO5P: Ribose-5-phosphate, S7P: Sedoheptulose-7-phosphate, E4P: Erythrose-4-phosphate, SUC: Succinic acid. Arrows from intermediates marked in grey boxes perpendicular to the metabolic reactions indicate flux into biomass.

Mentions: The next step was to investigate the in silico flux distributions associated with different putrescine production rates (Figure 1). As shown in Figure 1A, the split ratio of carbon flux at the glucose-6-phosphate node (Pgi/Zwf) without putrescine production was 72% to 21%. This ratio differed slightly from the flux measured in the wild type and the flux calculated in simulation experiments by Shinfuku and colleagues, who determined ratios of 59% to 41% and 60% to 40%, respectively [12]. When putrescine secretion had been increased stepwise up to 94%, a flux redistribution was observed. The flux through the pentose phosphate pathway (PPP) increased up to 71% at 50% putrescine production with respect to the glucose uptake, indicating an increased NADPH demand for putrescine production (Figure 1B). Interestingly, if putrescine production increased even further, this did not lead to an increased flux through the PPP. Rather a decrease to 35% was observed at 94% putrescine flux. This decrease was compensated by an active malate enzyme (MalE) at a putrescine flux above 50% (Figure 1B). MalE in combination with pyruvate carboxylase (Pyc) and malate dehydrogenase (Mdh) constitutes a transhydrogenase cycle [13], might supply NADPH for the reduction of glutamate to putrescine.


Fermentative production of the diamine putrescine: system metabolic engineering of corynebacterium glutamicum.

Nguyen AQ, Schneider J, Reddy GK, Wendisch VF - Metabolites (2015)

Metabolic flux distribution in C. glutamicum (A) and the relative flux through glucose 6-phosphate dehydrogenase Zwf and malate enzyme (MalE) (B) as a function of putrescine production. (A) Objective function was biomass flux, except for 100% putrescine flux. The metabolic flux was distributions were calculated in C. glutamicum without (in black) and with (in red) putrescine secretion to obtain yield coefficient (YP/S) of 25, 50, 75, 94%, respectively, relative to the glucose uptake rate. All fluxes are given in percent and are normalized to glucose uptake. Values are sorted by increasing putrescine flux. Solid line: Zwf flux, dotted line: MalE flux. For abbreviations: 1,3PG: 1,3-Bisphosphogylceric acid, 2OXO: 2-Oxoglutaric acid , 2PG: 2-Phosphoglyceric acid, 3PG: 3-Phosphoglyceric acid, AC-CoA: Acetyl-CoA, CIT: Citric acid, DHAP: Dihydroxyacetonephosphate, F6P: Fructose-6-phosphate, G6P: Glucose-6-phosphate, GA3P: Glyceraldyehyde-3-phosphate, GLC: Glucose, GLC-LAC: 6-Phosphogluconolactone, GLC6P: 6-Phosphogluconic acid, GLU: l-Glutamic acid, GLY: Glycerol, GLY3P: Glycerol-3-phosphate, ICI: Isocitric acid, l-RIB5P: l-Ribulose-5-phosphate, MAL: Malic acid, NAC-GLU: N-Acteylglutamic acid, OAA: Oxalacetic acid, ORN: l-Ornithine, PEP: Phosphoenolpyruvic acid, PUT: Putrescine, PYR: Pyruvic acid, RIB: LRibulose, RIB5P: Ribulose-5-phosphate, RIBO5P: Ribose-5-phosphate, S7P: Sedoheptulose-7-phosphate, E4P: Erythrose-4-phosphate, SUC: Succinic acid. Arrows from intermediates marked in grey boxes perpendicular to the metabolic reactions indicate flux into biomass.
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Related In: Results  -  Collection

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metabolites-05-00211-f001: Metabolic flux distribution in C. glutamicum (A) and the relative flux through glucose 6-phosphate dehydrogenase Zwf and malate enzyme (MalE) (B) as a function of putrescine production. (A) Objective function was biomass flux, except for 100% putrescine flux. The metabolic flux was distributions were calculated in C. glutamicum without (in black) and with (in red) putrescine secretion to obtain yield coefficient (YP/S) of 25, 50, 75, 94%, respectively, relative to the glucose uptake rate. All fluxes are given in percent and are normalized to glucose uptake. Values are sorted by increasing putrescine flux. Solid line: Zwf flux, dotted line: MalE flux. For abbreviations: 1,3PG: 1,3-Bisphosphogylceric acid, 2OXO: 2-Oxoglutaric acid , 2PG: 2-Phosphoglyceric acid, 3PG: 3-Phosphoglyceric acid, AC-CoA: Acetyl-CoA, CIT: Citric acid, DHAP: Dihydroxyacetonephosphate, F6P: Fructose-6-phosphate, G6P: Glucose-6-phosphate, GA3P: Glyceraldyehyde-3-phosphate, GLC: Glucose, GLC-LAC: 6-Phosphogluconolactone, GLC6P: 6-Phosphogluconic acid, GLU: l-Glutamic acid, GLY: Glycerol, GLY3P: Glycerol-3-phosphate, ICI: Isocitric acid, l-RIB5P: l-Ribulose-5-phosphate, MAL: Malic acid, NAC-GLU: N-Acteylglutamic acid, OAA: Oxalacetic acid, ORN: l-Ornithine, PEP: Phosphoenolpyruvic acid, PUT: Putrescine, PYR: Pyruvic acid, RIB: LRibulose, RIB5P: Ribulose-5-phosphate, RIBO5P: Ribose-5-phosphate, S7P: Sedoheptulose-7-phosphate, E4P: Erythrose-4-phosphate, SUC: Succinic acid. Arrows from intermediates marked in grey boxes perpendicular to the metabolic reactions indicate flux into biomass.
Mentions: The next step was to investigate the in silico flux distributions associated with different putrescine production rates (Figure 1). As shown in Figure 1A, the split ratio of carbon flux at the glucose-6-phosphate node (Pgi/Zwf) without putrescine production was 72% to 21%. This ratio differed slightly from the flux measured in the wild type and the flux calculated in simulation experiments by Shinfuku and colleagues, who determined ratios of 59% to 41% and 60% to 40%, respectively [12]. When putrescine secretion had been increased stepwise up to 94%, a flux redistribution was observed. The flux through the pentose phosphate pathway (PPP) increased up to 71% at 50% putrescine production with respect to the glucose uptake, indicating an increased NADPH demand for putrescine production (Figure 1B). Interestingly, if putrescine production increased even further, this did not lead to an increased flux through the PPP. Rather a decrease to 35% was observed at 94% putrescine flux. This decrease was compensated by an active malate enzyme (MalE) at a putrescine flux above 50% (Figure 1B). MalE in combination with pyruvate carboxylase (Pyc) and malate dehydrogenase (Mdh) constitutes a transhydrogenase cycle [13], might supply NADPH for the reduction of glutamate to putrescine.

Bottom Line: Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering.Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%.Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant.

View Article: PubMed Central - PubMed

Affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany. anguyen@cebitec.uni-bielefeld.de.

ABSTRACT
Corynebacterium glutamicum shows great potential for the production of the glutamate-derived diamine putrescine, a monomeric compound of polyamides. A genome-scale stoichiometric model of a C. glutamicum strain with reduced ornithine transcarbamoylase activity, derepressed arginine biosynthesis, and an anabolic plasmid-addiction system for heterologous expression of E. coli ornithine decarboxylase gene speC was investigated by flux balance analysis with respect to its putrescine production potential. Based on these simulations, enhancing glycolysis and anaplerosis by plasmid-borne overexpression of the genes for glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase as well as reducing 2-oxoglutarate dehydrogenase activity were chosen as targets for metabolic engineering. Changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and changing threonine 15 of OdhI to alanine reduced 2-oxoglutarate dehydrogenase activity about five fold and improved putrescine titers by 28%. Additional engineering steps improved further putrescine production with the largest contributions from preventing the formation of the by-product N-acetylputrescine by deletion of spermi(di)ne N-acetyltransferase gene snaA and from overexpression of the gene for a feedback-resistant N-acetylglutamate kinase variant. The resulting C. glutamicum strain NA6 obtained by systems metabolic engineering accumulated two fold more putrescine than the base strain, i.e., 58.1 ± 0.2 mM, and showed a specific productivity of 0.045 g·g-1·h-1 and a yield on glucose of 0.26 g·g-1.

No MeSH data available.


Related in: MedlinePlus