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Process for producing L-threonine with the use of bacterium belonging to the genus escherichia

A kind of Escherichia, threonine technology, applied in biological field

Inactive Publication Date: 2007-08-08
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The use of bacteria belonging to the genus Escherichia (Escherichia) having enhanced aspartate transaminase activity has not been reported so far for the production of L-threonine

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1: Cloning the aspC gene from Escherichia coli to the pMW119 vector

[0075] The aspC gene was obtained from the chromosomal DNA of E. coli strain K-12 by PCR using the primers shown in SEQ ID NOs: 3 and 4 in the Sequence Listing. The obtained DNA fragment was treated with restriction endonuclease PvuII and EcoRI and connected to the P lac Stable low copy plasmid pMW119 (replicon pSC101 ) under promoter control. In this way, the pMW-P lac -aspC plasmid.

[0076] Likewise, placing the aspC gene in the strong P of phage lambda R promoter instead of P lac under the control of the promoter. Use chemically synthesized 5'-phosphorylated oligonucleotides shown in SEQ ID NO: 5 and 6 in the Sequence Listing to form P-containing R Promoter DNA double strand. Subsequently, the DNA duplex was ligated into pMW-P previously treated with restriction enzymes PvuII and HindII lac -aspC plasmid. This constructs the pMW-P R -aspC plasmid.

[0077] High level expression ...

Embodiment 2a

[0079] Effect of embodiment 2 aspC gene amplification on threonine production

[0080] Escherichia coli strain B-3996 (pMW-P lac -aspC) and B-3996 (pMW-P R -aspC) were grown on L-agar plates containing streptomycin (100 μg / ml) at 37°C for 18-24 hours. One ring of cells was then transferred to 50 ml of L-broth with the following composition: tryptophan - 10 g / l, yeast extract - 5 g / l, NaCl - 5 g / l. Cells were grown (50 ml, OD540-20.u.) on a shaker (240 rpm) at 37°C for 5 hours to inoculate 450 ml of fermentation medium. Batch fermentations were carried out in laboratory fermenters with a volume of 1.0 L at 37° C. under ventilation (1 / 1 vvm) at a stirring speed of 1200 rpm. The pH was automatically maintained at 6.6 with 8% ammonia.

[0081] The results are shown in Table 1.

[0082] Composition of fermentation medium (g / l):

[0083] Sucrose 100.0

[0084] NH 4 Cl 1.75

[0085] K H 2 PO 4 1.0

[0086] MgSO 4 .7H 2 O 0.8

[0087] FeSO 4 .7H 2 O 0.01

...

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PUM

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Abstract

There is disclosed a method for producing L-threonine using bacterium belonging to the genus Escherichia wherein the bacterium has L- theonine productivity and has been modified to enhance an activity of aspartate aminotransferase.

Description

technical field [0001] The present invention relates to biotechnology, in particular to a method for producing L-amino acids by fermentation and more particularly to a gene derived from Escherichia coli. This gene is useful for increasing the productivity of L-amino acids such as L-threonine. Background technique [0002] Traditionally, L-amino acids have been industrially produced by fermentation using microbial strains obtained from natural sources or mutants of the same strains specifically modified to increase the productivity of L-amino acids. [0003] In order to increase the productivity of L-amino acids, for example, amplification of biosynthetic genes by transformation of microorganisms with recombinant DNA has been used (see, eg, US Pat. No. 4,278,765). These techniques are based on enhancing the activity of enzymes involved in amino acid synthesis and / or making target enzymes insensitive to feedback inhibition by the produced L-amino acids or their by-products (s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N1/21C12N9/10C12P13/08C12R1/19
CPCC12P13/08C12N9/1096C12N1/20
Inventor V·Z·阿克维迪安E·A·萨拉索瓦A·M·卡普兰A·O·洛巴纳夫Y·I·科滋洛夫
Owner AJINOMOTO CO INC
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