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Engineering bacterium for producing alpha-arbutin, construction method and application thereof

A construction method and technology of arbutin, applied in the field of bioengineering, can solve the problems of toxicity, unfavorable industrial production of α-arbutin, and high cost

Active Publication Date: 2021-04-13
ANHUI HUAHENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hydroquinone added in the existing method of α-arbutin is not only toxic, but also has high cost, which is not conducive to the industrialized production of α-arbutin

Method used

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  • Engineering bacterium for producing alpha-arbutin, construction method and application thereof
  • Engineering bacterium for producing alpha-arbutin, construction method and application thereof
  • Engineering bacterium for producing alpha-arbutin, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Recombinant plasmid pZE - pchB - salABCD - GD - xgtA

[0037] The recombinant plasmid pZE-pchB-salABCD-GD-xgtA provided in this example is mainly obtained by linking the genes xgtA, pchB, salABCD, and GD to the E. coli expression vector pZE12-luc.

[0038] The method for constructing the above-mentioned recombinant plasmid provided in this example specifically includes the following steps. Screening of bacterial, fungal or protein engineered enzymes encoding α-glucosidase (XgtA), isochoristate pyruvate lyase (PchB), anthranilate 5-hydroxylase (SalABCD) and gentisate decarboxylase (GD) gene. PCR amplification of target genes encoding α-glucosidase (XgtA), isochorisate pyruvate lyase (PchB), anthranilate 5-hydroxylase (SalABCD) and gentisate decarboxylase (GD) After the target fragment is obtained, the target fragment and the vector are digested with an appropriate enzyme, and the digested fragment is recovered, and then inserted into the expression plasmid pZE12-luc (P...

Embodiment 2

[0040] Engineering bacteria producing α-arbutin: Recombinant Escherichia coli BW1, BW2, BW3

[0041] The engineering bacteria producing α-arbutin provided by the present invention have no special requirements on the type of host strain used to construct the expression plasmid. In the embodiment of the present invention, strain BW25113 is used as the initial host for constructing the plasmid.

[0042] First, pick fresh BW25113 colonies and inoculate them into 4 mL of LB medium, incubate at 37°C for 8–12 h, then inoculate 1 mL into 100 mL of LB medium, and incubate at 37°C until OD 600 When the growth reaches 0.6, centrifuge at 6000 rpm for 10 min at 4 °C to collect the bacteria, wash with 10 mL of 10% pre-cooled glycerol, centrifuge at 6000 rpm for 10 min, repeat the glycerol washing step, and drain the remaining glycerol as much as possible after centrifugation. Finally, an appropriate amount of 10% glycerol was added to resuspend the cells to obtain competent cells. Take 90 ...

Embodiment 3

[0045] Application of recombinant Escherichia coli: production of α-arbutin through fermentation and cultivation of recombinant Escherichia coli BW1, BW2, and BW3 respectively

[0046] Pick fresh engineering single colonies of recombinant Escherichia coli BW1, BW2, and BW3 from the plate and inoculate them into 4 mL LB test tubes containing the corresponding antibiotics. After culturing at 37°C for 8 h, transfer them to a shaker containing 50 mL medium containing the corresponding antibiotics. The fermentation culture was carried out in the bottle, the inoculum size was 2%, the fermentation temperature was 30°C or 37°C, and the rotation speed was 200 rpm. Wherein, the medium: 2 g∙L‾ 1 MOPS, 20 g∙L‾ 1 Maltose, 10 g∙L‾ 1 Glycerin, 5 g∙L‾ 1 Yeast powder, 6 g∙L‾ 1 NaHPO 4 , 0.5 g∙L‾ 1 NaCl, 3 g∙L‾ 1 K H 2 PO 4 , 2 g∙L‾ 1 NH 4 Cl, 246.5 mg∙L‾ 1 MgSO 4 , 14.7 mg∙L‾ 1 CaCl 2 , and add corresponding antibiotics according to the actual situation.

[0047] The induct...

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Abstract

The invention provides an engineering bacterium for producing alpha-arbutin. The engineering bacterium comprises genes for co-expressing and encoding alpha-arbutin synthetase, PchB, SalABCD, and GD in a host, wherein the alpha-arbutin synthetase is one of or any combination of HGE, XgtA, and DGAS. The engineering bacterium further comprises genes for co-expressing AroL, ppsA, tktA, aroG<fbr>, and EntC from escherichia coli, so that the yield of the alpha-arbutin is increased. The invention also provides a construction method and application of the engineering bacterium for producing the alpha-arbutin. By utilizing the engineering bacterium for producing the alpha-arbutin, the alpha-arbutin is produced by using carbon sources such as maltose and / or glycerol, so that the denovo synthesis of the alpha-arbutin is realized.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to an engineering bacterium for producing α-arbutin and its construction method and application. Background technique [0002] Arbutin, also known as arbutin, is white needle crystal or powder. Extracted from the leaves of bearberry, it can prevent the production of melanin by inhibiting the activity of tyrosinase in the body. Accelerates the decomposition and excretion of melanin, thereby reducing skin pigmentation, removing spots and freckles, and does not produce toxic, irritating, sensitizing and other side effects on melanocytes, and also has bactericidal and anti-inflammatory effects. It is the safest and most effective whitening raw material popular today. It is mainly used in the preparation of high-grade cosmetics. It can be formulated into skin cream, freckle cream, high-grade pearl cream, etc. It can not only beautify skin, but also reduce inflammation and anti-ir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P19/44C12P19/18
CPCC12N9/1085C12N9/1205C12N9/1022C12N9/90C12Y207/01071C12Y207/0104C12Y202/01001C12Y205/01054C12Y504/04002C12N15/70C12P19/44C12P19/18
Inventor 袁其朋申晓林陈昕程瑶王佳孙新晓
Owner ANHUI HUAHENG BIOTECH
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