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Genetically engineered bacterium for efficiently synthesizing alpha-arbutin and construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and genetically engineered strains, applied in the field of genetic engineering, can solve the problem of insufficient α-arbutin yield, and achieve the effects of increasing protein expression and increasing yield

Active Publication Date: 2021-02-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the output of α-arbutin is not high enough, and there is still a distance from industrialization

Method used

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  • Genetically engineered bacterium for efficiently synthesizing alpha-arbutin and construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for efficiently synthesizing alpha-arbutin and construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for efficiently synthesizing alpha-arbutin and construction method and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Catalytic synthesis of α-arbutin by whole cells

[0035] The recombinant Bacillus subtilis to be fermented was activated by streaking on the LB solid medium containing the corresponding resistance, and cultivated overnight at 37°C. A single colony was transferred to LB liquid medium (20 mL) supplemented with corresponding resistance, and cultured on a shaker at 37° C. for 10 h to obtain a seed liquid. The seed solution was transferred to 30 mL TB medium supplemented with corresponding resistance at an inoculum amount of 1% (v / v), and cultured at 37° C. for 12 hours to obtain a fermentation solution. The fermentation broth was centrifuged (8,000 rpm, 10 min, 4° C.) to collect the cells, and washed twice with 20 mmol / L PB buffer (pH 7.0). The whole-cell catalytic system contains 310.9g / L sucrose, 50g / L HQ (the molar ratio of sucrose to HQ is 2:1), 20mmol / L PB, the cell addition amount OD 600 =20. The whole-cell catalytic reaction was carried out in a 50mL ai...

Embodiment 2

[0036] Example 2: Molecular docking and site-directed saturation mutation

[0037]Search for the homologous enzyme crystal model of SmSP in the SWISS MODEL online software (http: / / www.swissmodel.expasy.org / ) database, and the sucrose phosphorylase BaSP from B. adolescentis (PDB ID: 2gdv) is homologous to SmSP The sex is 43.74%, which meets the requirements of homology modeling, and the SmSP is modeled accordingly. We use PyMOL software to observe and analyze the crystal structure of SmSP, such as figure 2 As shown, the structure consists of two homologous subunits, which can be divided into four domains: A, B, B' and C. Domain B contains two short α-helices, an antiparallel β-sheet, and loop A. Domain B' consists of two α-helices, one long and one short, which contains a flexible loop structure loop B. Domain C is composed of five antiparallel β-sheets formed by the first 56 residues of SmSP. Among them, domains B and B' contain receptor binding sites. Combined with rele...

Embodiment 3

[0039] Embodiment 3: Genetic engineering bacterial strain BS-SmSP I336L build

[0040] Genomic integration was performed using the Cre-loxP system. Select the integration site ganA gene. First construct a linear fragment to be homologously recombined, and fuse the constructed spectinomycin integration frame (with loxP sequences at both ends of the linear spc gene) with the mutated P43-gtfA-mut gene expression frame by fusion PCR to form spc-gtfA -mut, the upstream and downstream homology arms (each 800bp) of the ganA gene were added to the two ends of spc-gtfA-mut by fusion PCR to obtain a linear fragment to be homologous recombination. Transform the linear fragment into a competent Bacillus subtilis, spread it on a spectinomycin plate, prepare the transformants that have been successfully integrated into the competent strain, and transform the temperature-sensitive Cre plasmid preserved in the laboratory into the competent , add IPTG to induce the expression of Cre recombi...

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Abstract

The invention discloses a genetically engineered bacterium for efficiently synthesizing alpha-arbutin and a construction method and application of the genetically engineered bacterium, and belongs tothe technical field of genetic engineering. Alpha-arbutin is efficiently produced by whole-cell catalysis of recombinant bacillus subtilis for expressing sucrose phosphorylase (SmSP), and a new thought and a new method are provided for production of alpha-arbutin. The protein mutant SmSPI336L with improved SmSP enzyme activity is screened through molecular docking and site-specific saturation mutagenesis, the protein expression quantity of SmSP is increased by increasing the copy number of SmSPI336L on a genome, and the yield of alpha-arbutin is greatly increased by optimizing the catalytic condition of a BS-3SmSPI336L strain. When the concentration of a substrate HQ is 50 g / L, the sucrose concentration is 310.9 g / L, the thallus concentration OD600 is 40, the yield of alpha-arbutin reaches115.8 g / L when a catalytic system catalyzes for 20 hours in a shake flask at 30 DEG C and 220 rpm, and the molar conversion rate of the substrate HQ is 93.6%.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for efficiently synthesizing α-arbutin, its construction method and application, and belongs to the technical field of genetic engineering. Background technique [0002] α-arbutin (4-hydroquinone-α-D-glucopyranoside) is a natural glycoside, which contains 1 glucose group and 1 phenol group, which are connected by α-1,4 glycosidic bonds. In recent years, α-arbutin has been widely used as a whitening agent in the cosmetic industry due to its good browning resistance to light radiation and inhibitory activity to tyrosinase. In addition, α-arbutin can not only reduce the deposition of skin pigment, but also has the effect of sterilization and anti-inflammation. It is a new type of non-irritating and non-allergic natural whitening active substance. Currently, the methods for synthesizing α-arbutin include chemical synthesis, enzymatic synthesis and fermentation. Among them, the stereoselectivity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12P19/44C12R1/125
CPCC12N9/1051C12Y204/01007C12N15/75C12P19/44
Inventor 刘龙吕雪芹陈坚堵国成李江华
Owner JIANGNAN UNIV
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