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3. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum

Author

Peters-Wendisch, P. G., Schiel, B., Volker F. Wendisch, Katsoulidis, E., Möckel, B., Sahm, H., and Eikmanns, B. J.

Subjects
Threonine, growth, Lysine, Molecular Sequence Data, Restriction Mapping, amino-acids, Glutamic Acid, Gene Expression Regulation, Bacterial, Corynebacterium, nucleotide-sequence, Gene Expression Regulation, Enzymologic, Phosphoenolpyruvate Carboxylase, Recombinant Proteins, Kinetics, Species Specificity, central metabolism, expression, Escherichia coli, bacteria, carbon-flux, biosynthesis, gene, Plasmids, Pyruvate Carboxylase
Abstract

Corynebacterium glutamicum possesses both phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx) as anaplerotic enzymes for growth on carbohydrates. To analyze the significance of PCx for the amino acid production by this organism, the wild-type pyc gene, encoding PCx, was used for the construction of defined pyc-inactive and pyc-overexpressing strains and the glutamate, lysine and threonine production capabilities of these recombinant strains of C. glutamicum were tested in comparison to the respective host strains. No PCx activity was observed in the pyc-inactive mutants whereas the pyc-overexpressing strains showed eight-to elevenfold higher specific PCx activity when compared to the host strains. In a detergent-dependent glutamate production assay, the pyc-overexpressing strain showed more than sevenfold higher, the PCx-deficient strain about twofold lower glutamate production than the wild-type, Overexpression of the pyc gene and thus increasing the PCx activity in a lysine-producing strain of C. glutamicum resulted in approximately 50% higher lysine accumulation in the culture supernatant whereas inactivation of the pyc gene led to a decrease by 60%, In a threonine-producing strain of C. glutamicum, the overexpression of the pyc gene led to an only 10 to 20% increase in threonine production, however, to a more than 150% increase in the production of the threonine precursor homoserine. These results identify the anaplerotic PCx reaction as a major bottleneck for amino acid production by C. glutamicum and show that the enzyme is an important target for the molecular breeding of hyperproducing strains.

5. Riboswitch (T-box)-mediated control of tRNA-dependent amidation in Clostridium acetobutylicum rationalizes gene and pathway redundancy for asparagine and asparaginyl-trnaasn synthesis.

Author

Saad NY, Schiel B, Brayé M, Heap JT, Minton NP, Dürre P, and Becker HD

Subjects
Asparagine genetics, Aspartate-Ammonia Ligase genetics, Bacterial Proteins genetics, Clostridium acetobutylicum genetics, RNA, Bacterial genetics, RNA, Transfer, Amino Acyl genetics, Asparagine biosynthesis, Aspartate-Ammonia Ligase biosynthesis, Bacterial Proteins biosynthesis, Clostridium acetobutylicum metabolism, RNA, Bacterial metabolism, RNA, Transfer, Amino Acyl biosynthesis, Riboswitch physiology
Abstract

Analysis of the Gram-positive Clostridium acetobutylicum genome reveals an inexplicable level of redundancy for the genes putatively involved in asparagine (Asn) and Asn-tRNA(Asn) synthesis. Besides a duplicated set of gatCAB tRNA-dependent amidotransferase genes, there is a triplication of aspartyl-tRNA synthetase genes and a duplication of asparagine synthetase B genes. This genomic landscape leads to the suspicion of the incoherent simultaneous use of the direct and indirect pathways of Asn and Asn-tRNA(Asn) formation. Through a combination of biochemical and genetic approaches, we show that C. acetobutylicum forms Asn and Asn-tRNA(Asn) by tRNA-dependent amidation. We demonstrate that an entire transamidation pathway composed of aspartyl-tRNA synthetase and one set of GatCAB genes is organized as an operon under the control of a tRNA(Asn)-dependent T-box riboswitch. Finally, our results suggest that this exceptional gene redundancy might be interconnected to control tRNA-dependent Asn synthesis, which in turn might be involved in controlling the metabolic switch from acidogenesis to solventogenesis in C. acetobutylicum.

Published
2012
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6. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum.

Author

Peters-Wendisch PG, Schiel B, Wendisch VF, Katsoulidis E, Möckel B, Sahm H, and Eikmanns BJ

Subjects
Corynebacterium genetics, Corynebacterium growth & development, Escherichia coli, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Kinetics, Molecular Sequence Data, Phosphoenolpyruvate Carboxylase metabolism, Plasmids, Pyruvate Carboxylase genetics, Recombinant Proteins metabolism, Restriction Mapping, Species Specificity, Threonine biosynthesis, Corynebacterium metabolism, Glutamic Acid biosynthesis, Lysine biosynthesis, Pyruvate Carboxylase metabolism
Abstract

Corynebacterium glutamicum possesses both phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx) as anaplerotic enzymes for growth on carbohydrates. To analyze the significance of PCx for the amino acid production by this organism, the wild-type pyc gene, encoding PCx, was used for the construction of defined pyc-inactive and pyc-overexpressing strains and the glutamate, lysine and threonine production capabilities of these recombinant strains of C. glutamicum were tested in comparison to the respective host strains. No PCx activity was observed in the pyc-inactive mutants whereas the pyc-overexpressing strains showed eight-to elevenfold higher specific PCx activity when compared to the host strains. In a detergent-dependent glutamate production assay, the pyc-overexpressing strain showed more than sevenfold higher, the PCx-deficient strain about twofold lower glutamate production than the wild-type. Overexpression of the pyc gene and thus increasing the PCx activity in a lysine-producing strain of C. glutamicum resulted in approximately 50% higher lysine accumulation in the culture supernatant whereas inactivation of the pyc gene led to a decrease by 60%. In a threonine-producing strain of C. glutamicum, the overexpression of the pyc gene led to an only 10 to 20% increase in threonine production, however, to a more than 150% increase in the production of the threonine precursor homoserine. These results identify the anaplerotic PCx reaction as a major bottleneck for amino acid production by C. glutamicum and show that the enzyme is an important target for the molecular breeding of hyperproducing strains.

Published
2001

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