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Genetically engineered bacterium for realizing high yield of L-isoleucine as well as construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and isoleucine, which is applied in the field of genetically engineered bacteria with high yield of L-isoleucine, can solve the problems of high production cost, difficult production, expensive raw materials, etc., and achieve an increase in acid production rate and a reduction in production Effect of cost, ability to improve

Active Publication Date: 2017-05-24
WUHAN GRAND HOYO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since L-isoleucine has 2 asymmetric carbon atoms, it is difficult to produce by chemical synthesis and the cost is very high
The production of L-isoleucine by the biotransformation method uses α-aminobutyric acid and α-oxobutyric acid as precursor materials for biosynthesis, but due to expensive raw materials, there is also the problem of high production costs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The acquisition of embodiment 1 mutant strain H5

[0040] 1.1 Materials and reagents

[0041] Corynebacterium glutamicum ATCC13032, a widely used standard strain, was purchased from Shanghai Fuxiang Biotechnology Co., Ltd.;

[0042] Diethyl sulfate (EDS) was purchased from Wuhan Shenshi Chemical; peptone, yeast, corn steep liquor, etc. were purchased from Biosharp; alanine, sulfaguanidine, S-(2-aminoethyl)-L-cysteine Hydrochloride, α-aminobutyric acid, DL-α-amino-β-hydroxyvaleric acid, etc. were all purchased from Sigma Company; other chemical reagents were analytically pure from Sinopharm.

[0043] 1.2 Preparation of type strains

[0044] Corynebacterium glutamicum ATCC13032 was used as the starting strain, activated on LBG solid medium, and cultured at 31°C for 12h;

[0045] Inoculate a ring of bacteria from the slant into 30ml LBG medium, culture at 31°C and 200rpm for 12h; take 1ml of the bacteria solution, centrifuge at 12000rpm for 2min, discard the supernatant...

Embodiment 2

[0051] Example 2 Construction of Genetically Engineered Bacteria C.glutamicumH5ΔargG

[0052] 2.1 Experimental materials and reagents

[0053] Plasmid pk18mobsacB and Escherichia coli DH5α were purchased from commercial channels;

[0054] DNA polymerase (KOD DNA Polymerase) was purchased from toyobo Toyobo (Shanghai) Biotechnology Co., Ltd.; restriction enzymes (EcoRI, SalI, NsiI, NotI), DNAmarker, plasmid mini-extraction kit, DNA gel recovery and purification kit , were purchased from Takara Bao Bioengineering (Dalian) Co., Ltd.; Gibson cloning and recombination kits were purchased from NEB Beijing Company;

[0055] 2.2 Construction of knockout vector argG-pk18mobsacB

[0056] The vector pk18mobsacB was digested with EcoRI / SalI double enzymes, and the reaction system was: 1ug of plasmid, 5ul of 10×buffer, 1ul of BamHI, 1ul of EcoRI, replenished with water to 50ul, and digested at 37°C for 5h. 1% agarose gel electrophoresis detection and DNA gel recovery and purification ki...

Embodiment 3

[0069] Example 3 Construction of Genetically Engineered Bacteria C.glutamicumH5ΔargGΔalaT

[0070] 3.1 Construction of knockout vector alaT-pk18mobsacB

[0071] The vector pk18mobsacB was digested with EcoRI / SalI double enzymes, and the reaction system was: 1ug of plasmid, 5ul of 10×buffer, 1ul of BamHI, 1ul of EcoRI, replenished with water to 50ul, and digested at 37°C for 5h. 1% agarose gel electrophoresis detection and DNA gel recovery and purification kit to recover 5.6kb nucleotide fragments;

[0072] The strain Corynebacterium glutamicum H5 preserved in glycerol was inoculated into 1-4ml of LBG liquid medium, cultured with shaking at 31°C and 200rpm for 16 hours; 1-4ml of the bacterial solution was centrifuged at 10,000rpm for 2 minutes, and the supernatant was discarded; Root Genome Extraction Kit to extract genomic DNA.

[0073] Using the Corynebacterium glutamicum H5 genome as a template, design primers for the upstream and downstream fragments alaT-L (shown in SEQ ...

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Abstract

The invention discloses a corynebacterium glutamicum genetically engineered bacterium for realizing high yield of L-isoleucine. The bacterium is obtained by knocking out argG genes of encoded argininosuccinate synthase and alaT genes of encoded aminotransferase from a corynebacterium glutamicum strain Corynebacterium glutamicum H5 and inserting an operon of ilvC genes of encoded acetohydroxy acid isomeroreductase into loci where the alaT genes are knocked out, wherein the corynebacterium glutamicum strain Corynebacterium glutamicum H5 is collected in China Center for Type Culture Collection (CCTCC) and has a collection number of CCTCC NO:M2016609. The invention further discloses a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium provided by the invention is used for fermenting to produce L-isoleucine, and the yield and glucose acid conversion ratio can be improved.

Description

technical field [0001] The invention relates to a genetically engineered bacterium capable of high-yielding L-isoleucine, and its construction method and application belong to the field of biotechnology. Background technique [0002] L-isoleucine, also known as L-isoleucine, chemically named L-α-amino-β-methylvaleric acid, belongs to the aspartic acid family of non-polar, water-transporting amino acids. L-isoleucine, as one of the eight essential amino acids for the human body, is the raw material for the synthesis of human hormones and enzymes. It has the effect of promoting protein synthesis and inhibiting decomposition. Adults need to ingest 20mg / kg (body weight) from the outside world every day. L-Isoleucine. Therefore, L-isoleucine has wide application and commercial value in the fields of food, medicine, feed and sports health care. [0003] Since L-isoleucine has two asymmetric carbon atoms, it is difficult to produce by chemical synthesis and the cost is very high....

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/06C12R1/15
Inventor 苏海霞邢盼盼王炯梅雪臣宋盟军万坤刘爱福
Owner WUHAN GRAND HOYO
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