Cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in Corynebacterium glutamicum |
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Authors: | M Ikeda K Okamoto R Katsumata |
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Institution: | (1) Technical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Hofu, Yamaguchi 747-8522, Japan Fax: +81-835-22-2466, JP;(2) Laboratory of Animal Microbiology, Faculty of Agriculture, Tohoku University, Aobaku, Sendai 981-0914, Japan, JP |
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Abstract: | Transketolase is a key enzyme of the nonoxidative 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.
Received: 27 July 1998 / Received last revision: 12 October 1998 / Accepted: 24 October 1998 |
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