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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

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.
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metabolites-05-00211-f003: 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.

Mentions: 2-Oxogluatarate is a branch point metabolite, which can be funneled either into the TCA cycle or glutamate biosynthesis. ODHC is a key enzyme in the TCA cycle consisting of three subunits: E1o is encoded by odhA [16], E2o is encoded by aceF, and E3 is encoded by lpd [17]. Putrescine production should benefit from the reduction of ODHC activity as suggested by the flux simulation and two approaches were followed: decreasing synthesis of OdhA, a subunit of ODHC [16] and/or inhibiting ODHC by maintaining its binding with OdhI [18]. Reducing synthesis of OdhA by exchanging the translational start codon from GTG to TTG in the chromosome of strain PUT21 reduced ODHC activity from 11 ± 2 mU/mg to 7 ± 1 mU/mg (Figure 3) and increased putrescine production by 15% (Figure 3). Alternatively, maintaining inhibition of ODHC by exchanging threonine residues 14 or 15 or both of OdhI to alanine resulted in partial or complete loss of phosphorylation and inactivation of OdhI by PknG [18]. Strains PUT21odhIT14A and PUT21odhIT15A showed ODHC activities of 10 ± 2 and 7 ± 1 mU/mg, respectively (Figure 3) and produced 32 ± 3 mM and 32 ± 2 mM putrescine (Figure 3). Combining the chromosomal changes odhATTG and odhIT15A yielded strain NA2, reduced ODHC activity to 2 ± 0 mU/mg (Figure 3) and increased putrescine production by 28% (38.1 ± 0.2 mM) as compared to PUT21 (29.2 ± 2.8 mM, Figure 3). Significant concentrations (>2 mM) of glutamate or other amino acids were not detected in culture broth at the end of all cultivations.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4495370&req=5

metabolites-05-00211-f003: 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.
Mentions: 2-Oxogluatarate is a branch point metabolite, which can be funneled either into the TCA cycle or glutamate biosynthesis. ODHC is a key enzyme in the TCA cycle consisting of three subunits: E1o is encoded by odhA [16], E2o is encoded by aceF, and E3 is encoded by lpd [17]. Putrescine production should benefit from the reduction of ODHC activity as suggested by the flux simulation and two approaches were followed: decreasing synthesis of OdhA, a subunit of ODHC [16] and/or inhibiting ODHC by maintaining its binding with OdhI [18]. Reducing synthesis of OdhA by exchanging the translational start codon from GTG to TTG in the chromosome of strain PUT21 reduced ODHC activity from 11 ± 2 mU/mg to 7 ± 1 mU/mg (Figure 3) and increased putrescine production by 15% (Figure 3). Alternatively, maintaining inhibition of ODHC by exchanging threonine residues 14 or 15 or both of OdhI to alanine resulted in partial or complete loss of phosphorylation and inactivation of OdhI by PknG [18]. Strains PUT21odhIT14A and PUT21odhIT15A showed ODHC activities of 10 ± 2 and 7 ± 1 mU/mg, respectively (Figure 3) and produced 32 ± 3 mM and 32 ± 2 mM putrescine (Figure 3). Combining the chromosomal changes odhATTG and odhIT15A yielded strain NA2, reduced ODHC activity to 2 ± 0 mU/mg (Figure 3) and increased putrescine production by 28% (38.1 ± 0.2 mM) as compared to PUT21 (29.2 ± 2.8 mM, Figure 3). Significant concentrations (>2 mM) of glutamate or other amino acids were not detected in culture broth at the end of all cultivations.

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