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A kind of recombinant Corynebacterium glutamicum expressing vgb gene and application thereof

A Corynebacterium glutamicum and gene technology, applied in the field of genetic engineering, can solve the problems of reduced 4-HIL production, technical difficulties in 4-HIL production, high separation cost, etc., and achieve the effect of increasing 4-HIL production

Active Publication Date: 2019-05-10
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

Since 1973, people have continuously tried and improved the 4-HIL separation and extraction method of fenugreek seeds. Although the production of 4-HIL has been realized by this method at present, there are limitations in materials, complicated processes, high separation costs, Problems such as low yield and low purity, the total yield is less than 1%, which makes the output of 4-HIL very low and the price is extremely expensive
The chemical-enzyme synthesis method requires eight or six steps of chemical-enzymatic reactions to synthesize 4-HIL. Not only is the process complicated and the steps are cumbersome, but also the separation cost is high, the yield is low and the purity is low. go on
In 2007, researchers discovered the two-step enzymatic synthesis of 4-HIL, but due to the low yield of 4-HIL and high content of by-products, the relevant research has not been further studied.
However, when using Corynebacterium glutamicum recombinantly expressing isoleucine dioxygenase to produce 4-HIL, although L-isoleucine is the direct substrate of 4-HIL, in L-isoleucine After the acid reaches a certain concentration, increasing the supply of L-isoleucine will lead to a decrease in the production of 4-HIL, thus making the production of 4-HIL unpredictable and causing technical difficulties in the production of 4-HIL

Method used

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  • A kind of recombinant Corynebacterium glutamicum expressing vgb gene and application thereof
  • A kind of recombinant Corynebacterium glutamicum expressing vgb gene and application thereof
  • A kind of recombinant Corynebacterium glutamicum expressing vgb gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Acquisition of vector pJYW-4-ido

[0029] Using the genomic DNA of Bacillus thuringiensis YBT-1520 as a template, primers were designed according to the isoleucine dioxygenase gene ido (GenBank accession no.CP004858.1, locus_tag YBT1520_06070) sequence published by GenBank, and ido-F / ido- R is the primer PCR amplification isoleucine dioxygenase gene ido, and the primers are designed as follows:

[0030] ido-F: 5'-CCGGGGCCCAGAAGGAGGTATAGGATGAAAATGAGTGGCTTTAGCATAGAAGAAAAGG-3'; ido-R: 5'-CGAGGTTAACGTTATTTTGTCTCCTTATAAGAAAATGTT-3'.

[0031] The enzymes used to amplify the target fragments are all high-fidelity PrimerStar DNA polymerases, and the reaction system is 50 μL: 5×PSBuffer 10 μL, dNTP 4 μL, template 1 μL, upstream and downstream primers 0.5 μL, enzyme 0.25 μL, ddH 2 O make up. PCR amplification reaction conditions are: 1. Pre-denaturation at 95°C for 10 minutes; 2. Denaturation at 95°C for 30s; annealing at 55°C for 30s; 1min); the number of cycles is...

Embodiment 2

[0032] Embodiment 2: the acquisition of vector pJYW-4-ido-vgb and recombinant Corynebacterium glutamicum SN01 / pJYW-4-ido-vgb

[0033] Then, using the Genomic DNA of Vitiligo hyaline as a template, design primers according to the sequence of the Vitiligo hyaline hemoglobin gene vgb (GenBank accession no.AF292694.1) published by GenBank, and use vgb-F / vgb-R as primers to amplify the gene vgb by PCR , the primers were designed as follows:

[0034] vgb-F: 5'-ATAGGATCCAGAAGGAGATATACGATGTTAGACCAGCAAACCAT-3';

[0035] vgb-R: 5'-CCTGTCGACTTATTCAACCGCTTG-3'.

[0036] Using the plasmid pJYW-4-ido as the carrier, repeat the above steps of amplification, digestion, purification, ligation and transformation screening, the fragment vgb and the plasmid were both digested with BamH I and Sal I and purified, then ligated into pJYW- On 4-ido, get pJYW-4-ido-vgb. Figure 4 is the amplified vgb gene map, Figure 5 Shown is the verification map of recombinant plasmid pJYW-4-ido-vgb digestion. ...

Embodiment 3

[0038] Embodiment 3: 4-HIL fermentation of recombinant Corynebacterium glutamicum SN01 / pJYW-4-ido-vgb

[0039] The successful recombinant Corynebacterium glutamicum SN01 / pJYW-4-ido and SN01 / pJYW-4-ido-vgb were compared by shaking flask fermentation.

[0040] The seed medium is: glucose 25g / L, (NH 4 ) 2 SO 4 0.5g / L, urea 1.25g / L, corn steep liquor 40g / L, KH 2 PO 4 1g / L, MgSO 4 0.5g / L, pH 7.0. The seed cultivation temperature was 30° C., and the seeds were cultivated for 18 hours using a rotary shaker at 150 rpm.

[0041] The fermentation medium is: glucose 140g / L, (NH 4 ) 2 SO 4 20g / L, corn syrup 10g / L, KH 2 PO 4 1g / L, MgSO 4 0.5g / L, FeSO 4 0.5g / L, CaCO 3 20g / L, pH 7.2. During the fermentation process, the fermentation culture temperature was 30°C, and the rotary shaker was used to cultivate at 200 rpm for 144 hours. Samples were taken every 12 hours to measure the concentration of bacteria, residual sugar, 4-HIL and other amino acid production.

[0042] Figur...

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Abstract

The invention discloses recombinant corynebacterium glutamicum expressing a vgb gene and an application of the recombinant corynebacterium glutamicum, and belongs to the technical field of genetic engineering. An isoleucine dioxygenase gene ido from bacillus thuringiensis and a hemoglobin gene vgb from vitreoscilla are co-expressed to construct an expression vector pJYW-4-ido-vgb of the recombinant corynebacterium glutamicum; the expression vector is electrically transferred into corynebacterium glutamicum ssp.lactofermentum SN01 for producing L-isoleucine, and the recombinant corynebacterium glutamicum SN01 / pJYW-4-ido-vgb is obtained. The recombinant corynebacterium glutamicum can relieve inhibition of a substrate of IDO by L-isoleucine in the production process of 4-HIL (4-hydroxyisoleucine) with L-isoleucine taken as the substrate, so that the fermentation yield of 4-HIL is increased by 50.0%.

Description

technical field [0001] The invention relates to a recombinant corynebacterium glutamicum expressing vgb gene and an application thereof, belonging to the technical field of genetic engineering. Background technique [0002] According to the World Health Organization, diabetes, cancer, cardiovascular disease and respiratory disease are the four major chronic non-communicable diseases. Since 1980, the number of adults with diabetes has quadrupled to 422 million. Current research shows that fenugreek seeds can be used for clinical treatment of type 2 diabetes, and the substance that plays a key role in fenugreek seeds is (2S,3R,4S)-4-hydroxyisoleucine (4-HIL). 4-HIL is a non-protein amino acid, which has physiological functions such as promoting insulin secretion, reducing the resistance of peripheral tissues such as liver, muscle, and fat to insulin, and regulating dyslipidemia. Therefore, 4-HIL has good applications in the treatment of type 2 diabetes prospect. However, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/77C12N15/53C12N15/31C12P13/04C12R1/15
CPCC07K14/805C12N9/0069C12N15/77C12N2800/101C12P13/04
Inventor 史锋卢正珂方惠敏李永富
Owner JIANGNAN UNIV
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