Limits...
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 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 (argBA49V/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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4495370&req=5

metabolites-05-00211-f006: 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 (argBA49V/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.

Mentions: Strain improvement was based on C. glutamicum PUT21, which produced 29.2 ± 2.8 mM putrescine from 20 g·L−1 glucose (Table 2). Strain NA2 carried the chromosomal changes odhATTG and odhIT15A and produced 38.1 ± 0.2 mM (Table 2). Overexpression of gapA in NA2 yielded strain NA3, which showed increased glyceraldehyde 3-phosphate dehydrogenase activity (160 ± 10 as compared 130 ± 10 mU/mg) and produced 41.9 ± 1.1 mM putrescine (Table 2, Figure 6). Additional overexpression of pyc did not increase putrescine production by strain NA4 (Figure 6). However, NA5, which showed increased feedback-resistant N-acetyl glutamate kinase activity due to additional overexpression of argBA49V/M54V [36], produced more putrescine, namely 48.3 ± 3.3 mM (Figure 6). Overexpression of argBA49V/M54V improved putrescine overproduction although arginine accumulation was avoided in all putrescine producing strains described in this study due to leaky expression of argF. Thus, it is possible that overexpression of genes for various other feedback-alleviated N-acetylglutamate kinase variants [37,38] may increase putrescine production further. Although all N-acetylglutamate kinase variants have been selected for loss of feedback inhibition by arginine, it is conceivable that N-acetylglutamate kinase is inhibited by putrescine, ornithine or other arginine biosynthesis intermediates and that some of the described variants may have lost this putative feedback inhibition as well.


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

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

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 (argBA49V/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.
© Copyright Policy
Related In: Results  -  Collection

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

metabolites-05-00211-f006: 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 (argBA49V/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.
Mentions: Strain improvement was based on C. glutamicum PUT21, which produced 29.2 ± 2.8 mM putrescine from 20 g·L−1 glucose (Table 2). Strain NA2 carried the chromosomal changes odhATTG and odhIT15A and produced 38.1 ± 0.2 mM (Table 2). Overexpression of gapA in NA2 yielded strain NA3, which showed increased glyceraldehyde 3-phosphate dehydrogenase activity (160 ± 10 as compared 130 ± 10 mU/mg) and produced 41.9 ± 1.1 mM putrescine (Table 2, Figure 6). Additional overexpression of pyc did not increase putrescine production by strain NA4 (Figure 6). However, NA5, which showed increased feedback-resistant N-acetyl glutamate kinase activity due to additional overexpression of argBA49V/M54V [36], produced more putrescine, namely 48.3 ± 3.3 mM (Figure 6). Overexpression of argBA49V/M54V improved putrescine overproduction although arginine accumulation was avoided in all putrescine producing strains described in this study due to leaky expression of argF. Thus, it is possible that overexpression of genes for various other feedback-alleviated N-acetylglutamate kinase variants [37,38] may increase putrescine production further. Although all N-acetylglutamate kinase variants have been selected for loss of feedback inhibition by arginine, it is conceivable that N-acetylglutamate kinase is inhibited by putrescine, ornithine or other arginine biosynthesis intermediates and that some of the described variants may have lost this putative feedback inhibition as well.

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