Method for L-threonine production

A threonine, microorganism technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as gene expression of threonine operon that are difficult to predict

Inactive Publication Date: 2003-11-19
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this approach replaces the chromosomal gene with an inducible promoter-replacement gene, and it is difficult to predict a significant increase in the expression of the threonine operon gene

Method used

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  • Method for L-threonine production
  • Method for L-threonine production
  • Method for L-threonine production

Examples

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Embodiment 1

[0022] A method for cloning the phosphoenolpyruvate carboxylase (ppc) gene is described in figure 1middle. The ppc gene was obtained from a threonine producing strain-TF4076. Chromosomal DNA was isolated, digested with restriction enzyme Sal I, and electrophoresed to selectively separate 4-5 Kb DNA fragments. Using the isolated DNA fragment as a template, the ppc gene was amplified using primer 1 (5'-aggaattcttccgcagcatttgacgtcac-3') and primer 2 (5'-aggaagcttttagccggtattacgcatacc-3'). The amplified product was digested with EcoRI and HindIII, and then electrophoresed again to finally isolate a 2.8 Kb ppc gene fragment. Cloned with a 7.6Kb pBRINT-TsGm (a pBRINT-Ts vector) from the National University of Mexico (Sylvie Le Beatriz et al., 1998, pBRINT-Ts: a family of plasmids with temperature-sensitive replicons, for designed for chromosomal integration into the lacZ gene of E. coli, Genes, 223, pp213-219). pBRINT-TsGm was double digested with the same restriction enzymes Ec...

Embodiment 2

[0023] The recombinant plasmid vector pAT94 (Korean Patent Application No. 92-24732) constructed by cloning chromosomal DNA of TF4076 was used for the threonine operon, and the recombinant plasmid pGmPPC in Example 1 was used for the ppc gene. pBRINT-TsGm (a pBRINT-Ts vector) from the National University of Mexico was used as a chromosomal DNA integration vector (Sylvie Le Beatriz et al., 1998, pBRINT-Ts: a family of plasmids with temperature-sensitive replicons for chromosomal integration into the lacZ gene of Escherichia coli., Gene., pp213-219). figure 2 The construction method of the recombinant plasmid is described. pAT94 was double digested with restriction enzymes HindIII and BamHI. The 6.4 kbp threonine operon DNA fragment was isolated from the double digest by electrophoresis. pGmPPC was double digested with HindIII and EcoRI to isolate the 2.8 kilobase pair ppc gene fragment. The pBRINT-TsGm plasmid vector was digested with EcoRI and BamHI, and the completely dig...

Embodiment 3

[0024] Transform TF4076 (a threonine-producing strain) with the recombinant plasmid pGmTN-PPC isolated from Escherichia coli strain DH5α, in LB solid medium containing 5 mg / L gentamicin (yeast extract 5 g / 10 grams / liter of tryptone for bacteria, 10 grams / liter of sodium chloride; 1.7% of agar for bacterial culture; pH7.0), cultivated at 30 degrees Celsius for 60 hours. Each single colony was inoculated into 0.5 ml of LB and incubated at 30°C for 4 hours. An aliquot of the culture was transferred to 10 mL of LB and incubated at 30°C for 6 hours and then at 37°C overnight. 10 of the culture -3 -10 -6 Dilutions were inoculated into LB solid medium containing 5 mg / L gentamicin. At this time, 12 microliters of IPTG (0.1 M) and 60 microliters of X-gal (2%) were also inoculated on the LB solid medium. Cultivate at 44 degrees centigrade for 24 hours, and screen for recombinant strains that are sensitive to carbenicillin and appear as white colonies, which cannot grow on LB solid ...

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Abstract

A method for producing L-threonine using a microorganism is provided. In the method, additional one or more copies of each of the phosphoenolpyruvate carboxylase (ppc) gene and the threonine operon are integrated into a particular site of the chromosomal DNA of a microorganism, while its inherent ppc gene and threonine operon remain. Accordingly, two or more ppc genes and threonine operons are included in the chromosomal DNA of the microorganism to thereby enhance the expression of the ppc gene encoding an enzyme to convert phosphoenolpyruvate to a threonine biosynthesis precursor, oxaloacetete, and the genes encoding enzymes involved in the synthetic pathway of threonine from oxaloacetate, including thrA (aspartokinasel-homoserine dehydrogenase), thrB (homoserine kinase), and thrC (threonine synthase), thereby markedly increasing L-threonine productivity.

Description

field of invention [0001] The present invention relates to the production of L-threonine with the participation of microorganisms. More specifically, the present invention relates to a high-yield method for producing L-threonine in which one or more additional copies of the phosphoenolpyruvate carboxylase (ppc) gene and threonine The operon is inserted into a special site of the microbial chromosomal DNA, while retaining the ppc gene and threonine operon inherent in the microorganism to improve the expression of the ppc gene and some genes, wherein the ppc gene encodes the ability to convert phosphoenolpyruvate into Enzymes of oxaloacetate, a precursor of threonine biosynthesis; certain genes described encode enzymes involved in the biosynthetic pathway from oxaloacetate to threonine, such as aspartokinase-homoserine dehydrogenation enzyme (thrA), homoserine kinase (thrB), and threonine synthase (thrC). Background technique [0002] As an essential amino acid, L-threonine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/20C12N1/21C12P13/08C12R1/19
CPCC12N1/20C12P13/08
Inventor 鲁甲秀金永哲朴宰镛金大哲李珍镐玉承汉
Owner CJ CHEILJEDANG CORP
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