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Method for producing L-threonine using bacteria belonging to the genus escherichia

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

Active Publication Date: 2007-07-11
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0021] The use of bacteria belonging to the genus Escherichia and having enhanced expression of genes encoding enzymes of the glycolytic pathway such as glk, pgi, pfkA, tpiA, gapA, pgk, eno, and pykA

Method used

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  • Method for producing L-threonine using bacteria belonging to the genus escherichia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1: Cloning of the glk gene from Escherichia coli, and the effect of enhanced expression of the glk gene on the production of L-threonine.

[0105] The glk gene was obtained by PCR using chromosomal DNA of Escherichia coli strain MG 1655 (VKPM B-6195) as a template and primers P1 (SEQ ID NO: 17) and P2 (SEQ ID NO: 18). Strain MG1655 is available from the American Type Culture Collection (ATCC700926). Primer P1 contains a recognition site for BamHI restriction enzyme introduced into its 5'-end. Primer P2 contains a recognition site for the SacI restriction enzyme introduced into its 5'-end. The resulting DNA fragment (968bp) containing the glk gene was treated with BamHI and SacI restriction enzymes and cloned into plasmid pMW119, which was previously modified to use the promoter P of lambda phage R replace promoter P lac , and then treated with the same restriction enzymes. Thus constructed in the promoter P R Plasmid pMW-P containing the glk gene under cont...

Embodiment 2

[0124] Example 2: Cloning of pfkA gene from Escherichia coli, and the effect of enhanced expression of pfkA gene on L-threonine production.

[0125] The pfkA gene was obtained by PCR using chromosomal DNA of E. coli strain MG 1655 (VKPM B-6195) as a template and primers P3 (SEQ ID NO: 19) and P4 (SEQ ID NO: 20). Primer P3 contains a recognition site for BamHI restriction enzyme introduced into its 5'-end. Primer P4 contains a recognition site for SacI restriction enzyme introduced into its 5'-end. The resulting DNA fragment (987 bp) containing the pfkA gene was cloned directly into vector pCR 2.1 (Invitrogen) by ligation overnight at +4°C. Then the BamHI-SacI DNA fragment containing the pfkA gene was cloned again into the plasmid pMW119, which was previously modified to use the promoter P of lambda phage R replace promoter P lac , and then treated with BamHI and SacI restriction enzymes. Thus constructed in the promoter P R Plasmid pMW-P containing the pfkA gene under con...

Embodiment 3

[0146] Example 3: Cloning of the fbaA gene from Escherichia coli, and the effect of enhanced expression of the fbaA gene on the production of L-threonine.

[0147] The fbaA gene was obtained by PCR using chromosomal DNA of Escherichia coli strain MG 1655 (VKPM B-6195) as a template and primers P5 (SEQ ID NO: 21 ) and P6 (SEQ ID NO: 22). Primer P5 contains a recognition site for BamHI restriction enzyme introduced into its 5'-end. Primer P6 contains a recognition site for the SacI restriction enzyme introduced into its 5'-end. The resulting DNA fragment (1155bp) containing the fbaA gene was treated with BamHI and SacI restriction enzymes and cloned into plasmid pMW119, which was previously modified to use the promoter P of lambda phage R replace promoter P lac , and then treated with the same restriction enzymes. Thus constructed in the promoter P R Plasmid pMW-P containing the fbaA gene under control R -fbaA. Unregulated, high-level expression of the fbaA gene can be ach...

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Abstract

The invention represents a method for preparing amino acid L-threonine by using bacterium belonging to Escherichia genus. This bacterium shows ability for production of L-threonine and modified by so manner that expression of gene chosen from the group glk, pgi, pfkA, tpiA, gapA, pgk, eno and pykA encoding glycogenolysis enzyme is enhanced.

Description

technical field [0001] The present invention relates to biotechnology, in particular to methods for producing L-amino acids by fermentation, and more particularly to genes derived from Escherichia coli bacteria. The gene is used to improve L-amino acid productivity, for example, L-threonine productivity. Background technique [0002] Conventionally, L-amino acids have been industrially produced by fermentation methods using strains of microorganisms obtained from natural sources or mutants thereof, especially mutants modified to enhance L-amino acid production ability. [0003] For example, enhancement of L-amino acid production capacity has been achieved by transforming microorganisms with recombinant DNA to amplify biosynthetic genes (see, eg, US Patent No. 4,278,765). These techniques are based on increasing the activity of enzymes involved in amino acid synthesis and / or desensitizing the target enzymes to feedback inhibition of produced L-amino acids or their by-product...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/08C12N1/21C12R1/19
Inventor 瓦勒里·Z·阿克维迪安埃卡特里纳·A·萨弗拉索瓦纳塔利亚·N·萨姆索诺瓦维拉迪米尔·Y·厄米谢夫艾林纳·B·奥尔特曼利奥尼德·R·普蒂辛
Owner AJINOMOTO CO INC
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