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

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.

Figure 1

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 (Y_P/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.

Figure 1

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 (Y_P/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.

Figure 2

Comparison of theoretical and experimental putrescine yields. The putrescine flux response was analyzed by flux balance analysis with different biomass (the split ratio between Embden-Meyerhof-Parnas pathway (EMP) and pentose phosphate pathway (PPP) pathway was 6:4) when MalE was inactive (solid line) and active (dashed line). Circles: PUT3-27 [4], open squares: NA2-8.

Figure 3

2-Oxoglutarate dehydrogenase as a target to increase production of putrescine production. Concentrations of putrescine (black bar) and N-acetylputrescine (grey bar) in supernatants and 2-oxoglutarate dehydrogenase activities in crude extracts (white bar) of different strains are given as means and standard errors of three independent cultivations. Cells were grown in CGXII medium with 20 g·L⁻¹ glucose and 1 mM IPTG.

Figure 4

Effect of pEKEx3-based overexpression of gapA and pyc in PUT21 on the production of putrescine (black bar) and N-acetylputrescine byproduct (grey bar). Cells were grown in CGXII medium with 20 g·L⁻¹ glucose and 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). Means and standard error of three independent cultivations are shown.

Figure 5

Putrescine production (black bar) and N-acetylputrescine byproduct (grey bar) in PUT21-derived strains carrying proB with different translational start codons. Cells were grown in CGXII medium with 20 g·L⁻¹ glucose and plasmid encoded genes were induced with 1 mM IPTG. Means and standard error of three independent experiments are shown.

Figure 6

Effect of deletion and overexpression of engineering target genes in C. glutamicum strain NA2 on the production of putrescine (black bars) and N-acetylputrescine (grey bars). Genetic changes introduced to the chromosome of C. glutamicum NA2 and to plasmid pVWEx1-speC-argF₂₁ are highlighted in bold. Genes for feedback-resistant N-acetylglutamate kinase (argB^A49V/M54V), pyruvate carboxylase (pyc) and glyceraldehyde 3-phosphate dehydrogenase (gapA) were added to plasmid pVWEX1-speC-argF₂₁, the translational start codon exchange of the γ-glutamate kinase gene proB from ATG to TTG was introduced in the chromosome, while the spermi(di)ne N-acetyltransferase gene snaA and the regulatory gene cgmR were deleted. Cells were grown in CGXII medium with 20 g·L^-1 glucose and 1 mM IPTG. Means and standard errors of three independent cultivations are shown.