Cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in Corynebacterium glutamicum.
Transketolase is a key enzyme of the non-oxidative pentose phosphate pathway. The effect of its overexpression on aromatic amino acid production was investigated in Corynebacterium glutamicum, a typical amino-acid-producing organism. For this purpose, the transketolase gene of the organism was cloned on the basis of its ability to complement a C. glutamicum transketolase mutant with pleiotropically shikimic-acid-requiring, ribose- and gluconic-acid-negative phenotype. The gene was shown by deletion mapping and complementation analysis to be located in a 3.2-kb XhoI-SalI fragment of the genome. Amplification of the gene by use of low-, middle-, and high-copy-number vectors in a C. glutamicum strain resulted in overexpression of transketolase activities as well as a protein of approximately 83kDa in proportion to the copy numbers. Introduction of the plasmids into a tryptophan and lysine co-producer resulted in copy-dependent increases in tryptophan production along with concomitant decreases in lysine production. Furthermore, the presence of the gene in high copy numbers enabled tyrosine, phenylalanine and tryptophan producers to accumulate 5%-20% more aromatic amino acids. These results indicate that overexpressed transketolase activity operates to redirect the glycolytic intermediates toward the nonoxidative pentose phosphate pathway in vivo, thereby increasing the intracellular level of erythrose 4-phosphate, a precursor of aromatic biosynthesis, in the aromatic-amino-acid-producing C. glutamicum strains.
Ikeda
M
M
Technical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Yamaguchi, Japan.
Okamoto
K
K
Katsumata
R
R
D000596:Amino Acids / Q000096:biosynthesis*; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D018628:Gene Dosage; D005798:Genes, Bacterial; D015183:Restriction Mapping; D014174:Transketolase / Q000235:genetics* / Q000378:metabolism*
D000596:Amino Acids; D014174:Transketolase