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Genetic engineering strain for synthesizing alpha-arbutin as well as construction method and application of genetic engineering strain

A genetically engineered strain, arbutin technology, applied in the field of genetic engineering, can solve the problems of unfavorable industrial production and low yield

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

AI Technical Summary

Problems solved by technology

However, the existing construction of Bacillus subtilis with heterologous expression of sucrose phosphorylase has a low yield of synthetic arbutin, which is not conducive to industrial production.

Method used

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  • Genetic engineering strain for synthesizing alpha-arbutin as well as construction method and application of genetic engineering strain
  • Genetic engineering strain for synthesizing alpha-arbutin as well as construction method and application of genetic engineering strain
  • Genetic engineering strain for synthesizing alpha-arbutin as well as construction method and application of genetic engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Catalytic synthesis of α-arbutin by whole cells

[0031] 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, 20mmol / L PB, and the amount of cell addition is OD 600 =20. The whole-cell catalytic reaction was carried out in a 50mL airtight container, and placed in ...

Embodiment 2

[0032] Embodiment 2: Construction of genetic engineering strain BS-HT-SmSP

[0033] Table 1

[0034]

[0035] According to the codon preference of Bacillus subtilis, the gtfA gene derived from S. mutans UA159 was artificially synthesized, and the gene sequence is shown in SEQ ID NO.1. Using gtfA-F (BamH I) and gtfA-R (Sma I) as primers, the gtfA gene was amplified by PCR to obtain the gtfA gene with restriction sites at both ends. The E. coli-Bacillus subtilis shuttle vector pHT01-Pgrac100 was selected as the expression vector, and the gtfA gene was cloned into the restriction site BamH I and Sma I by restriction enzyme ligation to obtain the recombinant plasmid pHT01-Pgrac100-gtfA , transforming the plasmid into Bacillus subtilis B. subtilis WB600 to obtain a genetic engineering strain BS-HT-SmSP capable of catalyzing the synthesis of α-arbutin. The result is as figure 2 As shown, using the whole cell catalyzed method for synthesizing α-arbutin in Example 1, the output...

Embodiment 3

[0036] Embodiment 3: Optimization of sucrose phosphorylase (SmSP) expression vector

[0037] Table 2

[0038]

[0039] Note: The underline indicates the restriction site, and the italic letters indicate the homology arm sequence

[0040] Three recombinant expression vectors pSTOP1622-PxylA-gtfA, pMA0911.1-PHpaII-gtfA and pP43NMK were obtained by one-step cloning and ligation of gtfA gene to pSTOP1622-PxylA and pP43NMK-P43 plasmids, and restriction restriction to pMA0911.1-PHpaII plasmid -P43-gtfA. The above three plasmids were respectively transferred into B.subtilisWB600 to obtain three recombinant strains of BS-STOP-SmSP, BS-MA-SmSP and BS-NMK-SmSP, and the whole cells in Example 1 were used to catalyze the synthesis of α-ursi Glycoside method, the three recombinant strains and the BS-HT-SmSP strain were catalytically verified, and the results were as follows image 3 As shown, the yield of α-arbutin catalyzed by BS-NMK-SmSP was higher than that of the other three recomb...

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Abstract

The invention discloses a genetic engineering strain for synthesizing alpha-arbutin as well as a construction method and application of the genetic engineering strain, and belongs to the technical field of genetic engineering. The alpha-arbutin is produced through whole-cell catalysis of recombinant bacillus subtilis expressing sucrose phosphorylase (SmSP), and a new thought and method are provided for production of the alpha-arbutin. By optimizing an expression vector and promoter of the sucrose phosphorylase (SmSP), the expression quantity of the SmSP is increased, and the yield of the alpha-arbutin is increased to 41.0 g / L from 10.6 g / L of an original strain. Due to the fact that plasmid expression is not stable, an optimized gene expression cassette P43-gtfA is integrated into a B.subtilis WB600 genome, and a genetically engineered strain BSP43-SmSP is obtained. When the concentration of a substrate HQ is 50g / L, the sucrose concentration is 310.9 g / L, the thallus concentration OD600 is equal to 20, and a catalytic system catalyzes in a shake flask at 30 DEG C and 220 rpm for 20 hours, the yield of the strain alpha-arbutin is stable and is increased to 61.1 g / L compared with a contrast, and the molar conversion rate of the substrate namely hydroquinone is 49.4%.

Description

technical field [0001] The invention relates to a genetic engineering bacterial strain for 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 of the c...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N15/63C12P19/44C12R1/125
CPCC12N15/75C12N15/63C12P19/44C12N9/1051C12Y204/01007
Inventor 刘龙吕雪芹陈坚堵国成李江华
Owner JIANGNAN UNIV
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