Process for the preparation of l-amino acids using strains of the enterobacteriaceae family

A technology of Enterobacteriaceae and amino acids, applied in the field of L-threonine and microorganisms, can solve the problem of undetectable

Inactive Publication Date: 2007-02-07
EVONIK OPERATIONS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Null mutations of malK result in constitutive expression of the regulon, but its overexpression results in barely detectable expression

Method used

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  • Process for the preparation of l-amino acids using strains of the enterobacteriaceae family

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Example 1: Construction of expression plasmid pTrc99AmalT

[0157] The malT gene of E. coli K12 was amplified using polymerase chain reaction (PCR) and synthetic oligonucleotides. Starting from the nucleotide sequence of the malT gene of E. coli K12 MG1655 (Accession No. AE000418, Blattner et al. (Science 277: 1453-1474 (1997)), PCR primers were synthesized (MWG Biotech, Ebersberg, Germany). The primers contain sequences of restriction enzymes, which are underlined in the nucleotide sequences shown below.Primer malT1 contains a restriction cleavage site for XabI, and primer malT2 contains a restriction cleavage site for HindIII.

[0158] malT1:

[0159] 5′-CCTCAT TCTAG ACAGTGAAGTGATTAA-3' (SEQ ID No: 1)

[0160] malT2:

[0161] 5′-GGCGCGTTATC AAGCTT AACTTACAC-3' (SEQ ID No: 2)

[0162] Chromosomal DNA of E. coli K12 MG1655 for PCR was isolated using Qiagen Genomic-tips 100 / G (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Using specific p...

Embodiment 2

[0164] Example 2: Preparation of L-threonine with bacterial strain MG442 / pTrc99AmalT

[0165] The Escherichia coli strain MG442 producing L-threonine is described in patent specification US-A-4,278,765, deposited with the Russian State Collection of Industrial Microorganisms (VKPM, Moscow, Russia) under the Budapest Treaty under the deposit number CMIM B-1628 and with the deposit number DSM 16574 is deposited at the German Collection of Microorganisms (DSMZ, Braunschweig, Germany).

[0166]Strain MG442 was transformed with the expression plasmid pTrc99AmalT and the vector pTrc99A described in Example 1, and cells carrying the plasmids were selected on LB agar with 50 μg / ml ampicillin. Successful transformation was confirmed by control cleavage with the enzymes HpaI, HindIII / XbaI and EcoRV after isolation of the plasmid DNA. In this way the strains MG442 / pTrc99AmalT and MG442 / pTrc99A were formed. Selected individual colonies were then further propagated on minimal medium with...

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Abstract

The invention relates to a process for the preparation of L-amino acids, in particular L-threonine, in which the following steps are carried out: a) fermentation of the microorganisms of the Enterobacteriaceae family which produce the desired L-amino acid and in which the malT gene or nucleotide sequences or alleles which code for it are enhanced, b) concentration of the desired L-amino acid in the medium or in the cells of the microorganisms, and c) isolation of the desired L-amino acid.

Description

field of invention [0001] The present invention relates to a method for preparing L-amino acids, especially L-threonine, using recombinant microorganisms of the family Enterobacteriaceae. The malT gene in the strains used is enhanced, especially overexpressed. The present invention also relates to these microorganisms. Background of the invention [0002] L-amino acids, especially L-threonine, are used in human medicine and in the pharmaceutical industry, in the food industry, and especially in animal nutrition. [0003] It is known that L-amino acids are produced by fermentation of Enterobacteriaceae strains, especially Escherichia coli (E.coli) and Serratia marcescens. Due to the extreme importance of L-amino acids, Attempts are ongoing to improve production methods. Improvements in production methods may involve fermentation measures such as agitation and oxygen supply, or the composition of the nutrient medium such as sugar concentration during fermentation, or the gradu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/04C12P13/08C07K14/24C07K14/245C07K14/255
CPCC07K14/245C07K14/24C12P13/08C12N1/20
Inventor 梅希特希尔德·里平托马斯·赫尔曼
Owner EVONIK OPERATIONS GMBH
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