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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

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.
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metabolites-05-00211-f004: 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.

Mentions: In order to increase the supply of 2-oxoglutarate as precursor for putrescine biosynthesis, two targets were chosen: glyceraldehyde 3-phosphate dehydrogenase in glycolysis and pyruvate carboxylase for anaplerosis of the TCA cycle. Since glyceraldehyde 3-phosphate dehydrogenase has been shown to be limiting glycolytic flux at least under oxygen-deprivation conditions [25], overexpression of its gene gapA in PUT21 was tested. As compared to PUT21 carrying the empty vector, PUT21 overexpressing gapA produced 39% more putrescine (44.0 ± 2.0 mM as compared to 29.5 ± 1.0 mM, Figure 4).


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495370&req=5

metabolites-05-00211-f004: 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.
Mentions: In order to increase the supply of 2-oxoglutarate as precursor for putrescine biosynthesis, two targets were chosen: glyceraldehyde 3-phosphate dehydrogenase in glycolysis and pyruvate carboxylase for anaplerosis of the TCA cycle. Since glyceraldehyde 3-phosphate dehydrogenase has been shown to be limiting glycolytic flux at least under oxygen-deprivation conditions [25], overexpression of its gene gapA in PUT21 was tested. As compared to PUT21 carrying the empty vector, PUT21 overexpressing gapA produced 39% more putrescine (44.0 ± 2.0 mM as compared to 29.5 ± 1.0 mM, Figure 4).

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