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Recombinant strain and application thereof

A technology of recombining bacterial strains and genes, applied in the field of microorganisms, can solve problems such as low yield and increased production costs

Active Publication Date: 2016-06-22
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The prior art often needs to add excessive biotin and surfactants such as Twee40 or Tween60 in the culture medium, which increases the production cost, and the yield is often low

Method used

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  • Recombinant strain and application thereof
  • Recombinant strain and application thereof
  • Recombinant strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0046] Preparation of Corynebacterium glutamicum Competent: Stretch and transfer Corynebacterium glutamicum species ATCC13869 to BHI plate in -80°C refrigerator, cultivate at 30°C; pick a single colony, and transfer to 5ml BHI liquid medium (3.7% Brain heart extract powder) test tube, 200rpm, 30 ℃ culture 12h; Inoculate 100ml BHIS liquid medium (3.7% brain heart extract powder, 9.1% sorbitol) according to 1% inoculum amount, 200rpm, 30 ℃ culture until OD600 reaches 1.5 Centrifuge at 4°C for 20min at 6000rpm, collect the cells, discard the supernatant; suspend in TGBuffer (1mM Tris, 10% glycerol (v / v), pH7.5), centrifuge at 4°C for 20min at 6000rpm, discard the supernatant, And repeat again; after suspending with 10% glycerol, centrifuge at 4°C, 6000rpm for 20min, discard the supernatant, and repeat again; add 1ml of 10% glycerol to suspend the cells and pack. Store the competent cells in a -70°C refrigerator or directly use them for electroshock transformation.

[0047] Elect...

Embodiment 1

[0059] The introduction of embodiment 1 yggB gene A106V point mutation

[0060] (1) Vector construction for yggBA106V point mutation

[0061] According to the DNA sequence (SEQ NO.1) of Corynebacterium glutamicum ATCC13869 yggB gene (GeneBank NO.: NCgl1221), primers were designed for PCR amplification. The primer sequences are shown in Table 1.

[0062] Table 1 Primer Sequence

[0063]

[0064] The whole genome sequence of wild-type Corynebacterium glutamicum C. glutamicumATCC13869 was extracted with Genome DNA Extraction Kit (Tiangen Company). Using the extracted genome as a template, A1 / A2, A3 / A4 primer pairs were used to PCR amplify under the action of Phusion ultra-fidelity polymerase (NewEngland BioLabs) to obtain the homology arms of the 106th amino acid upstream and downstream of the yggB gene, PCR parameters 98°C for 10s, 50°C for 20s, 72°C for 20s, a total of 30 cycles. The PCR products were recovered by tapping the gel with an agarose gel recovery kit (Tiangen ...

Embodiment 213869

[0068] Inactivation of odhA gene in 213869-yggBA106V bacterial strain

[0069] The glutamate biosynthetic pathway ( figure 1 ) shows that the conversion of α-ketoglutarate to succinyl-CoA catalyzed by α-ketoglutarate dehydrogenase complex (ODHC) in the tricarboxylic acid cycle is the main competitive pathway of glutamate synthesis. To reduce the activity of ODHC, it is necessary to inactivate the gene odhA encoding its Elo subunit. The method used is to introduce base deletion, so that frameshift mutation occurs during protein synthesis, so as to achieve the purpose of inactivation.

[0070] (1) Used for the construction of odhA base deletion vector

[0071] First, in the NCBIGenBank database, obtain the nucleotide sequence (SEQIDNo.9) of the Corynebacterium glutamicum (Corynebacterium glutamicumATCC13869) odhA gene (GeneBankNO.NCgl1084), thus the nucleotide sequence design introduces base deletions at specific positions so that The odhA gene was inactivated, and four prime...

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Abstract

The invention relates to the field of microorganisms, in particular to a recombinant strain and application thereof. Experimental results show that before modification, wild corynebacterium glutanmicum ATCC 13869 almost generates no glutamic acid, and the yield of strain MHZ-0112-1 glutamic acid is 4.7 g / L; the yield of MHZ-0112-2 glutamic acid is 18.5 g / L; the yield of strain MHZ-0112-4 glutamic acid and the yield of MHZ-0112-5 glutamic acid are 23.4 g / L and 26.4 g / L respectively. When a strong promoter Psod is independently utilized for expressing a gdh gene, the yield of glutamic acid is increased about 1.8 times; when a promoter Ptuf is independently utilized for expressing a gltA gene, the enzyme activity of CS is improved 4 times, and the yield of glutamic acid is increased to 30.8 g / L; after the two genes are expressed at the same time, the yield of glutamic acid continues to be increased to 35.5 g / L, and the conversion rate reaches about 59%.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to recombinant strains and applications thereof. Background technique [0002] Glutamic acid, whose scientific name is 2-amino-5-sulfoxypentanoic acid, is one of the 20 common amino acids that make up proteins. Glutamic acid is currently the most productive amino acid variety in the world, and the annual global demand for glutamic acid reaches 25 million tons. L-glutamic acid is the left-handed form of glutamic acid, which has the functions of increasing freshness, reducing blood ammonia, improving the central nervous system, improving children's intellectual development, dilating blood vessels, enhancing blood circulation, promoting flower spike development, etc., and can produce many Important downstream products, such as sodium L-glutamate, L-threonine, polyglutamic acid, etc., are widely used in food, medicine, artificial leather, cosmetics, agriculture and other industries. [0...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P13/14C12R1/15
CPCC07K14/34C12N9/0008C12N9/0016C12N9/1025C12N15/74C12N2800/101C12P13/14C12Y102/04002C12Y104/01002C12Y104/01003C12Y104/01004C12Y203/03003
Inventor 马雯雯胡丹程江红毛贤军刁刘洋
Owner MEIHUA BIOTECH LANGFANG CO LTD
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