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Beta-alanine synthetase mutant, coding gene, genetically engineered bacterium and application

A technology of genetically engineered bacteria and coding genes, applied in the field of beta alanine synthase mutants, can solve the problem of low yield and the like, and achieve the effects of increased yield, clear breeding goals and high efficiency

Active Publication Date: 2021-12-17
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although β-alanine has been synthesized, the yield is still not high, and there is still a long way to go before practical application

Method used

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  • Beta-alanine synthetase mutant, coding gene, genetically engineered bacterium and application
  • Beta-alanine synthetase mutant, coding gene, genetically engineered bacterium and application
  • Beta-alanine synthetase mutant, coding gene, genetically engineered bacterium and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Escherichia coli panD K43Y Construction of Gene Mutants

[0038] (1) Acquisition of the pET28b(+)-panD plasmid containing the Escherichia coli panD gene: from the laboratory through one-step cloning of the constructed Escherichia coli BL21-pET28b(+)-panD (construction process: the plasmid PET28b(+) PCR as a template and detection of the carrier by agarose gel electrophoresis, the panD fragment shown in SEQ ID No.3 obtained by PCR was connected to the carrier PET28b (+) by a one-step cloning method to obtain PET28b (+)-panD recombinant plasmid) inoculation In LB liquid culture medium, culture at 37°C and 200rpm on a shaking table for 12h, centrifuge at 8000rpm for 5min, collect bacterial cells, and use a plasmid extraction kit to extract plasmid pET28b(+)-panD; take 2μL of plasmid DNA and use ND500 The concentration and purity of the sample were measured by an ultra-micro ultraviolet-visible spectrophotometer; the composition of the LB liquid medium was: pept...

Embodiment 2

[0041] Example 2: Carry out pre-cultivation of the empty strain, the parent and the genetically engineered mutant strain bacteria respectively

[0042] 1. Pick single colonies from the plate and inoculate them into 5mL LB test tubes containing 50mg / L kanamycin resistance, incubate at 37°C for 6-8 hours and transfer to 50mL LB shakers containing 50mg / L kanamycin resistance. In the bottle, culture OD600=0.5-0.6, add 0.5mmol IPTG, culture on a shaker at 37°C, induce for 10 hours, and centrifuge to collect bacteria.

[0043] 2. Transfer the bacterial cells obtained in step 1 to shake flasks, and prepare β-alanine by fed-batch culture at 37° C., 180 rpm, for 36 hours. Such as figure 2 As shown, the β-alanine production of the mutant reached 5.72g / L in 24 hours, which was 1.84 times and 2.23 times higher than that of the parent and wild type under the same culture conditions.

[0044] 3. Wash and resuspend the thalline obtained in step 1 twice with PBS, and control the concentrat...

Embodiment 3

[0045] Embodiment 3: the research of the kinetic characteristic of mutant bacterial strain

[0046] In Example 1, the correct transformants, parental strains and empty strains were inserted into 5mL LB test tubes containing 50 mg / L, cultured at 37°C for 6-8 hours, and then transferred to 50 mg / L kanamycin containing 50mL LB shake flask, to OD 600 When =0.5~0.6, add 0.5mmol IPTG, culture on a shaker at 37°C, and after 10h, centrifuge at 6000rpm at 4°C for 5min to obtain three kinds of cells expressing panD. Wash the strain twice with PBS, and finally resuspend it with PBS, sonicate the cells, and centrifuge at 10000rpm at 4°C for 5 minutes to collect the supernatant, which is the crude enzyme solution, and then use the protein purification kit to purify the target protein through the NI column to obtain the final product The concentration of pure protein was measured with BCA kit. The protein concentrations of the empty strain, the parent strain (K43R) and the mutant strain (...

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Abstract

The invention relates to the field of gene engineering, in particular to a beta-alanine synthetase mutant with improved catalytic efficiency, a coding gene, a genetically engineered bacterium and application. The amino acid sequence of the beta-alanine synthetase mutant is as shown in SEQ ID No. 2. According to the invention, the influence of panD genes of different strains on the yield of beta-alanine is compared; and it is determined that the amount of beta-alanine produced by whole-cell fermentation of obtained recombinant escherichia coli reaches 5.04 g / L when mutation is carried out on the No.43 amino acid site of the panD gene and Lys is changed into Tyr, while the amount of beta-alanine produced by wild strains is 1.58 g / L, which is increased by about 3.2 times. According to the invention, a site-directed mutagenesis technology is used for carrying out directed transformation on the panD gene; the yield of the obtained mutant strain beta-alanine is obviously improved; the breeding target is clear; the efficiency is high; and the application prospect is good.

Description

(1) Technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a beta-alanine synthetase mutant with improved catalytic efficiency, coding gene, genetic engineering bacteria and application. (2) Background technology [0002] β-alanine, also known as β-alanine, is widely used in medicine, cosmetics, food, and chemical products. β-alanine is an important precursor substance, which can be derived to synthesize pantothenic acid, calcium pantothenate and carnosine. At present, the global demand for calcium D-pantothenate is huge, resulting in a shortage of products, and the market demand for β-alanine and its derivatives in other fields is also rising. At present, the industrial production of β-alanine is mainly chemical and biological. The cost of chemical synthesis is high, and a large number of toxic by-products will be produced during the production process, which is not conducive to separation and purification, and will...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/06C12R1/19
CPCC12N9/88C12N15/70C12P13/06C12Y401/01011
Inventor 孙东昌原攀红陈德刚张萍费明月胡诗龙付贝贝
Owner ZHEJIANG UNIV OF TECH
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