Process for producing L-lysine by fermenting

A lysine, amino acid technology, applied in the field of DNA and microorganisms

Inactive Publication Date: 2008-04-30
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such a DDPS mutant enzyme has not been described in the previous literature, and although there is a report on an AKIII mutant enzyme (Boy, E., et al., J. Bacteriol., 112, 84 (1972)), there is no known The example proposed that such a mutant enzyme can improve the yield of L-lysine

Method used

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  • Process for producing L-lysine by fermenting
  • Process for producing L-lysine by fermenting
  • Process for producing L-lysine by fermenting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0155] Embodiment 1: Preparation of mutant DDPS gene

[0156] (1) Cloning of wild-type dapA gene

[0157] The nucleotide sequence of the dapA gene of E. coli has been reported (Richaud, F. et al., J. Bacteriol., 297 (1986)), and its known openable frame (ORF) includes 876 base pairs and Encodes a protein comprising 292 amino acid residues. Since it is unknown how the dapA gene is regulated, only the SD sequence and the ORF region except the promoter region were amplified and cloned by PCR method.

[0158] The total genomic DNA of E. coli K-12MC1061 strain was extracted by the same method as that of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963)). Prepare two kinds of primers with sequences shown in SEQ ID NO: 1 and NO: 2, and use the primers to complete the PCR reaction and amplify the target DNA according to the method consistent with Erlich et al. (PCR Technology, Stockton press (1989)) . The obtained DNA was inserted into a commercially available cloning vec...

Embodiment 2

[0202] Embodiment 2: Preparation of mutant AKIII gene

[0203] (1) Cloning of wild-type lysC gene

[0204] The nucleotide sequence of the E. coli AKIII gene (lysC) has been reported (Cassan, M., Parsot, C., Cohen, G.N., and Patte, J.C., J. Biol. Chem., 261, 1052 (1986)) , and its known translational frame (ORF) comprises 1347 base pairs, and encodes a protein comprising 449 amino acid residues. In this gene, there is an operon, which is repressed by L-lysine. Therefore, in order to remove this operon region, the region containing only one SD sequence and ORF was amplified and cloned by PCR.

[0205] Total genomic DNA of E. coli K-12MC1061 strain was prepared according to the method of Saito and Miura (Biochem. Biophys. Acta., 72619 (1963)). Two primers having the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 were prepared, and a PCR reaction was performed according to the method of Erlich et al. (PCR Technology, Stocktonpress (1989)) to amplify the lysC gene. The obta...

Embodiment 3

[0263] Example 3: import dapA with * Strains fermented to produce L-lysine

[0264] In order to produce L-lysine with E. coli, as pointed out in Japanese Patent Application Laid-open No. 56-18596, U.S. Patent No. 4,346, 170 and Applied Microbiology and Biotechnology, 15, 227-231 (1982), for Enhancing DDPS hosts is required for aspartokinase, which has been altered not to be inhibited by L-lysine. L-threonine-producing bacteria can be exemplified as such strains. Because of E. coli producing L-threonine, B-3996 has the highest yield among currently known strains. So the B-3996 strain was used to determine dapA * the host. The B-3996 strain extrachromosomally anchored pVIC40 as the only plasmid. See Japanese Patent Laid-open No. 3-501682 (PCT) for details. This microorganism is deposited with the Research Institute for Genetics and Industrial Microorganism Breeding under accession number RIA1867.

[0265] On the other hand, dapA present in pdapAS24 (in which the 118th...

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Abstract

A bacterium belonging to the genus Escherichia, which is transformed by introducing, into its cells, a DNA coding for a dihydrodipicolinate synthase originating from a bacterium belonging to the genus Escherichia having mutation to desensitize feedback inhibition by L-lysine and a DNA coding for an aspartokinase III originating from a bacterium belonging to the genus Escherichia having mutation to desensitize feedback inhibition by L-lysine; preferably a bacterium belonging to the genus Escherichia in which a dihydrodipicolinate reductase gene and a diaminopimelate dehydrogenase gene originating from Brevibacterium lactofermentum (or a succinyldiaminopimelate transaminase gene and a succinyldiaminopimelate deacylase gene) are further enhanced, is cultivated in an appropriate medium, L-lysine is produced and accumulated in a culture thereof, and L-lysine is collected from the culture.

Description

technical field [0001] The present invention relates to microorganism industry, in particular to a method for producing L-lysine by fermentation, as well as DNA and microorganisms used in the production method. Background technique [0002] In the prior art, when producing L-lysine by a fermentation method, in order to increase the yield, a microbial strain isolated from a natural environment or an artificially mutated strain derived from the strain was used. Many artificial mutant strains producing L-lysine are known. Most of them are S-2-aminoethylcysteine ​​(ACE) resistant mutant strains, and belong to the genus Brevibacterium, Corynebacterium, Bacillus or Escherichia. Also, various techniques for improving the yield of amino acids have been disclosed, such as transformants obtained by the method using recombinant DNA (United States Patent No. 4,278,765). [0003] Regarding those strains belonging to the genus Escherichia, for example, the use of a A method for produci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12P13/08C12R1/185C12N15/09C12N9/02C12N9/06C12N9/10C12N9/12C12N9/88C12N15/00C12N15/52C12N15/54C12N15/70C12P13/06C12R1/19
CPCC12P13/08C12N9/88C12N9/1096C12N9/12C12N9/0016C12N9/001C12Y103/01026C12Y104/01016C12N15/52C12N1/02C12P13/06
Inventor 儿岛宏之尾川由理川村和枝佐野孝之辅
Owner AJINOMOTO CO INC
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