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Method for producing L-arginine or L-lysine by fermentation

A technology of arginine and lysine, applied in the field of L-arginine and L-lysine

Active Publication Date: 2004-12-01
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The technical effect of this patented method involves improving production efficiency for lysines from microorganisms like Corynobacters that are able to convert argininosuccinate into citrullonium ion (LCA) which has potential applications in various industries such as food industry.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving the efficiency with which certain types of organics like lactic acid produced during industrial processes such as brewing and manufacturing. Specifically, there may exist various ways to modify their properties without affecting other components involved in them' activities.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0188] Example 1: Construction of adenylyltransferase (GlnE) deficient bacterial strain

[0189] (1) Preparation of the plasmid used to remove GlnE

[0190] The glnE sequence of Brevibacterium flavum ATCC 14067 has long been known (see EP1229121 A2). Based on the reported nucleotide sequence, primers shown in SEQ ID NOS: 5 and 6 were synthesized, and the internal sequence of glnE was amplified by using the chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 strain as a template by PCR.

[0191] The chromosomal DNA of the Brevibacterium lactofermentum ATCC 13869 strain was prepared using a bacterial genomic DNA purification kit (Advanced Genetic Technologies Corp.). PCR was cycled 30 times with denaturation at 94°C for 30 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 2 minutes each, using Pyrobest DNA polymerase (Takara Shuzo).

[0192] The PCR product was purified by a conventional method, and blunt-ended using a Blunting kit (Takara Shuzo). T...

Embodiment 2

[0204] Example 2: Evaluation of GS adenylation site modified strains

[0205] (1) Construction of a plasmid modified at the GlnA adenylation site

[0206] The adenylation site of the glnA gene product (GlnA) of coryneform bacteria has long been known (see FEMS Microbiology Letters, 303-310, (173) 1999). A strain modified at the GlnA adenylation site is obtained by replacing the glnA gene on the chromosome with the glnA gene modified at the GlnA adenylation site. The specific process will be described below.

[0207] First, using the PCR method, using the chromosomal DNA of the Brevibacterium lactofermentum ATCC 13869 strain as a template, and using the synthesized DNA shown in SEQ ID NOS: 7 and 8 as primers, the N-terminal side amplification product of the glnA gene was obtained. In addition, in order to obtain the amplified product of the C-terminal side of the glnA gene, PCR used the chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 strain as a template, and the ...

Embodiment 3

[0216] Example 3: Obtaining and evaluating AmtR-deficient strains

[0217] (1) Preparation of a plasmid for removing AmtR

[0218] A plasmid for removal of the AmtR gene product (AmtR) from coryneform bacteria was prepared as follows.

[0219] First, the chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 was extracted and used as a template to perform PCR together with the synthetic DNA shown in SEQ ID NOS:13 and 14. The resulting DNA fragment was blunt-ended and inserted into the HincII site of pHSG299 (Taka Shuzo). The resulting plasmid was named pΔAmtRT.

[0220] The above-mentioned pΔAmtR does not contain a sequence that enables it to replicate autonomously in coryneform bacteria cells, so when coryneform bacteria are transformed with this plasmid, the strain in which the plasmid is incorporated into the chromosome due to homologous recombination behaves as a transformant, although the frequency of its occurrence extremely low.

[0221] (2) Introduce pΔAmtR in...

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Abstract

L-Arginine or L-lysine is produced by culturing a coryneform bacterium having an L-arginine- or L-lysine-producing ability and modified so that glutamine synthetase activity is enhanced, e.g., a coryneform bacterium which is modified so that activity regulation of glutamine synthetase by adenylylation is eliminated, in a medium to produce and accumulate L-arginine or L-lysine in the medium and collecting the L-arginine or L-lysine from the medium.

Description

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Claims

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Application Information

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Owner AJINOMOTO CO INC
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