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A method for producing an L-amino acid using a bacterium of the enterobacteriaceae family

A technology of Enterobacteriaceae and amino acids, applied in biochemical equipment and methods, enzymes, oxidoreductases, etc.

Active Publication Date: 2009-07-22
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, there has been no report so far of increasing the production of L-amino acids by fermentation in a medium containing ethanol using bacteria of the Enterobacteriaceae family, which have activity-enhanced resistance to aerobic inactivation. ) resistant native alcohol dehydrogenase or mutant alcohol dehydrogenase

Method used

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  • A method for producing an L-amino acid using a bacterium of the enterobacteriaceae family
  • A method for producing an L-amino acid using a bacterium of the enterobacteriaceae family
  • A method for producing an L-amino acid using a bacterium of the enterobacteriaceae family

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0167] Example 1. Preparation of Escherichia coli MG1655Δtdh, rhtA *

[0168] The L-threonine-producing E. coli strain MG1655 Δtdh, rhtA was constructed by * (pVIC40): The natural tdh gene encoding threonine dehydrogenase in Escherichia coli MG1655 (ATCC 700926) was inactivated using the cat gene, followed by the introduction of the rhtA23 mutation (rhtA * ), the mutation confers resistance to high concentrations of threonine (>40 mg / ml) and homoserine (>5 mg / ml). The resulting strain was then transformed with plasmid pVIC40 from E. coli VKPM B-3996. Plasmid pVIC40 is described in detail in US Patent No. 5,705,371.

[0169] In order to replace the native tdh gene, also known as "Red-mediated integration (Red-mediated integration)" described by Datsenko K.A. and Wanner B.L (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) " and / or "Red-driven integration (Red-driven integration)" approach, will carry the chloramphenicol resistance marker (Cm R ) was integrated into the chr...

Embodiment 2

[0175] Example 2. Construction of Escherichia coli MG1655::P L-tac adhE

[0176] by using P L-tac The natural promoter region of the adhE gene in the promoter replacement strain MG1655 was obtained from Escherichia coli MG1655::P L-tac adh.

[0177] To replace the native promoter region of the adhE gene, also called "Red-mediated integration" described by Datsenko K.A. and Wanner B.L (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) and / or "Red-Driven Integration" approach, will carry P L-tac Promoter and chloramphenicol resistance marker (Cm R ) was integrated into the chromosome of Escherichia coli MG1655 to replace the natural promoter region.

[0178] Escherichia coli MG1655P L-tac Chromosomal DNA of xylE (WO2006 / 043730) was used as template to obtain P L-tac Promoter and fragment of the cat gene. P L-tac The nucleotide sequence of the promoter is provided in the Sequence Listing (SEQ ID NO: 7). Primers P5 (SEQ ID NO: 8) and P6 (SEQ ID NO: 9) were used for PCR a...

Embodiment 3

[0184] Example 3. Construction of Escherichia coli MG1655Δtdh, rhtA * , P L-tac adhE

[0185] By introducing from strain MG1655::P L-tac adhE-P L-tac Promoter transduction into strain MG1655Δtdh, rhtA * coli MG1655Δtdh, rhtA * , P L-tac adhE.

[0186] with growth on the donor strain MG1655::P L-tac Phage P1 on adhE vir Infection strain MG1655Δtdh, rhtA * , obtained the strain MG1655Δtdh, rhtA * , P L-tac adhE. Check the growth of this strain on M9 plates containing 2% ethanol as the sole carbon source. Growth rate and strain MG1655::P L-tac adhE grew at the same rate.

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Abstract

A method for producing an L-amino acid is described, for example L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan, or L-glutamic acid, using a bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to enhance an activity of a wild-type alcohol dehydrogenase encoded by the adhE gene or a mutant alcohol dehydrogenase which is resistant to aerobic inactivation.

Description

technical field [0001] The present invention relates to the microbiology industry, in particular to a method for producing L-amino acids such as L-threonine, L-lysine, L- Histidine, L-Phenylalanine, L-Arginine, L-Tryptophan, L-Glutamic Acid, and L-Leucine. Background technique [0002] Conventionally, L-amino acids are produced industrially by fermentation methods using microbial strains obtained from natural sources or variants thereof. Microorganisms are often modified to increase the production yield of L-amino acids. [0003] Many techniques for increasing the yield of L-amino acids have been reported, including transformation of microorganisms with recombinant DNA (US Patent No. 4,278,765). Other techniques for increasing yield include increasing the activity of enzymes involved in amino acid biosynthesis and / or desensitizing feedback inhibition of the produced L-amino acid by the target enzyme (US Patent Nos. 4,346,170, 5,661,012, and 6,040,160). [0004] Further im...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12P13/04C12P13/06C12P13/08C12P13/10C12P13/14C12P13/22C12P13/24
CPCC12N9/0006C12P13/04C12P13/06C12P13/08C12P13/10C12P13/14C12P13/222C12P13/227C12P13/24C12Y101/01002
Inventor 利奥尼德·R·普蒂特辛伊里纳·B·奥尔特曼韦罗妮卡·A·科特利亚罗瓦奥尔佳·N·莫科瓦塔蒂亚纳·A·亚姆波尔斯卡亚尤里·I·科兹洛夫寺下优臼田佳弘松井和彦
Owner AJINOMOTO CO INC