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Process for prodn. of L-Thr

A technology of threonine and antibiotics, applied in the field of producing L-threonine

Inactive Publication Date: 2003-10-08
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of these expression regulatory plasmids may only be applicable when the final product is a protein

Method used

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  • Process for prodn. of L-Thr
  • Process for prodn. of L-Thr
  • Process for prodn. of L-Thr

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Constructing a recombinant plasmid and using it to knock out the tdc gene

[0037] Genomic DNA of the threonine-producing strain pGmTN-PPC (KCCM-10236) was isolated using the QIAGEN Genomic-tip system. Using genomic DNA as a template, a 3.1kb tdc operon gene fragment (5295pb) containing tdc B and tdc C was amplified by polymerase chain reaction (PCR). Primers used were 5'-agg agg gga tcc ggt atg tct tct gag gcg-3' and 5'-agg agg gaa ttc atc ggc aac agg cac ag-3'. PCR was performed as follows, 30 cycles of amplification, each cycle including denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 3 minutes and 30 seconds.

[0038] The PCR product was electrophoresed on a 0.7% agarose gel, and the band of desired size was eluted. Elution band and pT7Blue cloning carrier (Novagen Co,) blunt-end ligated overnight in order to obtain recombinant plasmid pT7Blue / tdc at 16 ℃ (referring to figure 1 ). Escherichia coli DH5α...

Embodiment 2

[0040] Example 2: Screening for strains integrated with recombinant plasmids

[0041] Escherichia coli DH5α was transformed with the recombinant plasmid pT7Δtdc::loxpKan, plated on a solid medium containing 50 mg / L ampicillin and 15 mg / L kanamycin, and cultured overnight at 37°C. Pick colonies from the culture with a toothpick, inoculate into 3 ml of liquid medium containing ampicillin and kanamycin, and cultivate overnight at 200 rpm. Plasmid DNA was isolated from the cultures using a QIAGEN miniprep kit, and the size of the plasmid DNA was identified. Plasmid DNA was digested with restriction enzymes and loaded onto 0.7% agarose to confirm its orientation. The identified plasmid DNA was digested with restriction enzyme PVU II and electrophoresed on a 0.7% agarose gel to elute a DNA fragment of about 3840 kb (Δtdc::loxpKan). The threonine-producing strain pGmTN-PPC12 was transformed with the DNA fragment Δtdc::loxpKan by electroporation, and plated on a solid medium conta...

Embodiment 3

[0042] Embodiment 3: the shake flask culture comparison threonine output of the recombinant bacterial strain that obtains

[0043] The L-threonine production comparison of 30 single colonies of the recombinant strains cultured in the kanamycin-containing solid medium in Example 2 was screened in an Erlenmeyer flask (flask) using a threonine titer medium. The composition of the threonine titer medium used in each case is shown in Table 1.

[0044] components

content per liter

glucose

70g

(NH 4 ) 2 SO 4

28g

K H 2 PO 4

1.0g

MgSO 4 ·7H 2 o

0.5g

FeSO 4 ·7H 2 o

5mg

MnSO 4 ·8H 2 o

5mg

calcium carbonate

30g

L-methionine

0.15g

yeast extract

2g

PH7.0

[0045] Single colonies were grown overnight at 32°C on LB solid medium in an incubator. One full loop of each culture was inoculated into 25 ml of titer medium a...

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Abstract

A method for producing L-threonine using a microorganism is provided. In the method, the threonine dehydratase (tdc) gene existing in the genomic DNA of the microorganism is partially deactivated using a recombination technique. For a microorganism strain with enhanced activity of threonine operon-containing enzymes and the phosphoenolpyruvate carboxylase (ppc) gene, the tdc gene engaged in one of the four threonine metabolic pathways is specifically deactivated, thereby markedly increasing the yield of L-threonine.

Description

field of invention [0001] The present invention relates to the production of L-threonine associated with microorganisms. More specifically, the present invention relates to a method for producing L-threonine with a high yield using a microorganism, wherein the threonine dehydratase (tdc) gene of the genomic DNA of the microorganism is partially inactivated by recombinant technology, thereby significantly increasing the L-threonine Threonine production. Background technique [0002] L-threonine is an essential amino acid that is widely used as animal feed and food additives, as well as fluids and synthetic materials for medical or pharmaceutical purposes. L-threonine is produced by fermentation using synthetic mutants derived from wild-type Escherichia coli, Corynebacterium, Serratia, Providencia and the like. These mutants are known to include amino acid analogue- and drug-resistant mutants and their synthetic mutants causing diaminopimelate, methionine, lysine or isoleuci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/21C12N9/88C12N15/00C12N15/11C12N15/70C12P13/08C12R1/19
CPCC12N9/88C12P13/08C12N15/70
Inventor 朴宰镛李炳春金大哲李珍镐赵载容朴英薰
Owner CJ CHEILJEDANG CORP
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