Gene engineering bacterium for producing beta-alanine and its preparation and application

A technology of genetically engineered bacteria and alanine, applied in the field of preparation of β-alanine, can solve the problems such as the difficulty of screening strains with high activity of enzyme production

Inactive Publication Date: 2008-07-02
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at overcoming the difficulty of screening strains with high enzyme-producing activity in nature, and provides a genetically engineered bacterium with clear breeding goals, high efficiency, and strong ability to synthesize β-alanine, and the genetically engineered bacterium of the engineered bacterium. Preparation method and its application in preparation of β-alanine

Method used

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  • Gene engineering bacterium for producing beta-alanine and its preparation and application
  • Gene engineering bacterium for producing beta-alanine and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Construction of high expression vector

[0037] step:

[0038] (1) According to the sequence design primer (forward primer: AAC GGATCC TATGATTCGCACGATGCTG, the underlined sequence is restriction enzyme BamH I cutting site; reverse primer: CCA AAGCTT AGCAACCTGTACCGGAATC, the underlined sequence is the cutting point of restriction enzyme Hind III), using PCR technology to amplify the sequence of the coding region; PCR reaction conditions: denaturation at 95°C for 3min; followed by 35 cycles, the parameters are 94°C, 1min; 60°C, 1min and 72°C, 45s; finally extend at 72°C for 5min.

[0039] (2) After the PCR product was recovered by gel, it was cloned into the corresponding site of pET28b(+) with the restriction enzyme BamH I / Hind III to form the high expression vector of panD gene pET28b(+)-panD. For the plasmid construction map, see figure 1 .

[0040] (3) Take 100 μl of Escherichia coli DH5α competent cells, put them on ice, gently suspend the cells e...

Embodiment 2

[0041] Embodiment 2: the acquisition of engineering bacteria

[0042] step:

[0043] (1) Take 100 μl Escherichia coli BL21(DE3) competent cells, put them on ice, and gently suspend the cells evenly after thawing completely.

[0044] (2) Take 5 μl of the plasmid pET28b(+)-panD obtained in Example 1, add it to the competent cells and mix gently. Place on ice for 30min.

[0045] (3) Heat shock in a water bath at 42°C for 90 seconds, and place on ice for 15-20 minutes.

[0046] (4) Add 400 μl of LB medium, culture at 37° C., shake at 200-250 r / min for 1 hour.

[0047] (5) Centrifuge at 4000 r / min for 5 min at room temperature, suck off 400 μl of the supernatant with a pipette tip, and suspend the cells with the remaining medium.

[0048] (6) Spread the transformed bacteria on LB (containing 30 μg / mL kanamycin) plates.

[0049] (7) Place the plate in the forward direction at 37°C for 1 hour to absorb excess liquid, and then culture it upside down overnight. After the strains g...

Embodiment 3

[0050] Embodiment 3: comparative experiment of β-alanine synthesis ability

[0051] The genetically engineered bacteria obtained in Example 2 were cultured on LB medium for 16-24 hours, then inserted into LB liquid medium containing 30 μg / mL of kanamycin, and cultured overnight at 37° C. and 200 r / min on a shaking table. Then transfer to the same LB liquid medium with 1% inoculum size, fill 250ml with 30ml LB liquid medium, culture at 37°C, 200r / min shaker until OD 600 When it is about 0.4 to 1.0, add inducer (IPTG final concentration is 0.4mmol / L or add lactose to final concentration is 8g / L), 30℃, 200r / min shaker culture for 12~20h, then put in 37℃, Cultivate on a shaker at 200r / min for 8h. Centrifuge at 4500r / min for 10min at 4°C, collect the cells, wash twice with 10mL sterile water, and finally add 5mL of 0.2mol / L L-asp (adjust the pH to 7.0 with NaOH), transform at 37°C and 200r / min 2d. The transformation solution was centrifuged at 4500r / min for 10min to obtain the s...

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Abstract

The invention provides genetic engineering bacteria for producing beta-alanine. The genetic engineering bacteria are obtained by the following method of amplifying L-aspartic acid alpha-decarboxylase gene panD of a donor containing panD gene by PCY, cloning the panD to a plasmid capable of expressing a foreign gene at high efficiency to obtain a vector for abundant expression of the panD gene, and transforming the vector into a receiver to obtain the genetic engineering bacteria for producing beta-alanine. The genetic engineering bacteria and preparation and application thereof have the main advantages that (1) the engineering bacteria of beta-alanine can be synthesized by the preparation of Escherichia coli; (2) the engineering bacteria has high ability in synthesizing beta-alanine, and the primary study determines that the synthesis ability is up to 2.94 g/l and that the beta-alanine is unlikely be determined by an identical method using a starting strain; and (3) the Escherichia coli is improved by using genetic engineering technology to achieve definite breeding target and high efficiency.

Description

(1) Technical field [0001] The invention relates to a genetically engineered bacterium producing beta-alanine, a preparation method thereof, and an application thereof in preparing beta-alanine. (2) Background technology [0002] β-alanine, also known as β-alanine. In 1972, it was discovered by Ross and Monroe in the degradation products of uracil. It is a non-protein amino acid and the only β-type amino acid in nature. The main physiological activity of β-alanine is to synthesize pantothenic acid and coenzyme A. Modern medical research has found that in the nervous system of mammals, it can act as a neurotransmitter in the brain; activate ion channels; and treat liver damage caused by tissue hypoxia. [0003] In the field of fine chemical industry, β-alanine is used to synthesize polymerized β-alanine, electroplating buffer, dye, etc. In the pharmaceutical industry, β-alanine can be used as an intermediate of many drugs, such as calcium pantothenate, which is a vitamin ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/06C12R1/19
Inventor 裘娟萍高丽娟
Owner ZHEJIANG UNIV OF TECH
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