Method for constructing stably inherited genetic engineering strain for efficient biosynthesis of beta-arbutin and application thereof

A technology of genetically engineered strains and genetically engineered bacteria, applied in the field of bioengineering, can solve the problems of low selectivity, reduced maintenance coefficient of Escherichia coli, and low yield, so as to alleviate the inhibition of biological enzyme activity and the impact of production instability , The effect of reducing production costs

Pending Publication Date: 2022-03-01
BEIJING UNIV OF CHEM TECH
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  • Abstract
  • Description
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Problems solved by technology

There are four main ways to obtain arbutin: plant extraction, chemical synthesis, enzyme conversion, and biosynthesis. The production process of plant extraction is complicated, and at the same time, it is affected by the environmental season, making its output low; due to low Catalytic efficiency and low selectivity, the method of chemical synthesis of arbutin is not the first choice; due to the toxicity of hydroquinone, the activity of the enzyme is inhibited, and the separation of the substrate and the product is difficult, resulting in a waste of energy; the biosynthesis method can be glucose as Carbon source, constructing a plasmid to transform the pathway gene into the host bacteria to synthesize arbutin from scratch, but the genetic instability of the plasmid affects the activity of the production strain, which reduces the maintenance coefficient of engineered E. coli

Method used

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  • Method for constructing stably inherited genetic engineering strain for efficient biosynthesis of beta-arbutin and application thereof
  • Method for constructing stably inherited genetic engineering strain for efficient biosynthesis of beta-arbutin and application thereof
  • Method for constructing stably inherited genetic engineering strain for efficient biosynthesis of beta-arbutin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0034] 2. Embodiment 2 constructs genetically engineered escherichia coli BW1

[0035] Insert the gene TAL, 4Cl2, phdE / B / C into the back of the gene pgi by using the crispr cas9 technology, the specific implementation method

[0036] (1) Electroporation method: Introduce the vector pCas 9 into E. coli BW, culture it on a spectinomycin plate at 30 degrees for 20 hours, select the positive clone transformants and name them BW-pCas 9, and pick the BW-pCas 9 growing on the plate Single clones were inoculated into 1.5ul / mL Spectinomycin LB liquid medium and cultured at 30°C.

[0037] (2) Construction of gene pgi site sgRNA plasmid

[0038] i. The targeting sequence used by sgRNA in this study is shown in Table 1

[0039] Table 1. Targeting sequences used by sgRNA at gene pgi site

[0040]

[0041] ii. The primer sequences used in this study are shown in Table 2

[0042] Table 2 Primer sequence list

[0043]

[0044] iii. Construction of gene pgi site sgRNA plasmid

[00...

Embodiment 3

[0058] 3. Example 3 Construction of recombinant Escherichia coli BW2

[0059] Applying crispr cas9 technology, gene 4HB1H, TGS replaces the pseudosense gene yneO on the genome, specific implementation methods

[0060] (1) Construction of gene yneO site sgRNA plasmid

[0061] i. The targeting sequence used by sgRNA in this study is shown in Table 1

[0062] Table 4. Targeting sequences used by sgRNA at gene pgi site

[0063]

[0064] ii. The primer sequences used in this study are shown in Table 5

[0065] Table 5 Primer sequence list

[0066]

[0067] iii. Construction of gene yneO site sgRNA plasmid

[0068] P9 / P2 is the primer, pTarget plasmid is the template, and the nucleotide sequence length containing sgRNA obtained by PCR is 2200kbp. After agarose gel electrophoresis, the PCR product is purified and recovered by gel recovery kit, and the PCR purified solution is chemically transformed The method was introduced into Escherichia coli DH5α competent cells, and s...

Embodiment 5

[0126] 6. The application of the genetically engineered bacterial strain BW3 of embodiment 5

[0127] With reference to Example 4, the above-mentioned genetically engineered strain BW3 was placed in the culture medium, and 1 mL was sampled every 12 hours to determine the growth status of the bacteria and the yield of the target product. The results were as follows: Figure 5 shown.

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Abstract

The invention provides a method for constructing a stably inherited genetic engineering strain for efficient biosynthesis of beta-arbutin and application of the genetic engineering strain. The method comprises the following steps: firstly, integrating genes for coding tyrosine lytic enzyme, coumaric acid coenzyme A ligase, beta-cinnamyl hydroxylase, beta-cinnamyl oxidase and beta-cinnamyl deacylase (phdC) in a host, and constructing a host strain capable of realizing high yield of p-hydroxybenzoic acid; secondly, genes for coding 4-hydroxybenzoic acid hydroxylase and glucosyltransferase are integrated in the host, and a strain capable of producing beta-arbutin is constructed. And finally, integrating a gene of a coding core pathway and a gene of a shikimic acid pathway onto a genome of the engineering bacterium, and knocking out a gene of a competitive pathway to construct a stably inherited beta-arbutin efficient biosynthesis gene engineering strain, so that the influence of unstable engineering bacterium production caused by application of plasmids is avoided; the method has an application prospect in industrial production of beta-arbutin.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the construction of high-yield beta-arbutin strains and its method and application. Background technique [0002] β-arbutin, also known as hydroquinone glucoside, 4-hydroxyphenyl-β-D-glucopyranoside, exists in the leaves of bearberry trees, lingonberry trees, pear trees, holly trees, etc., and has Inhibiting the ability of tyrosinase to prevent the formation of melanin, it has the effect of whitening and skin care, and is widely used in the medical and cosmetic industries. There are four main ways to obtain arbutin: plant extraction, chemical synthesis, enzyme conversion, and biosynthesis. The production process of plant extraction is complicated, and at the same time, it is affected by the environmental season, making its output low; due to low Catalytic efficiency and low selectivity, the method of chemical synthesis of arbutin is not the first choice; due to the toxici...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/54C12N15/53C12N15/60C12N15/52C12N1/21C12P19/44C12R1/19
CPCC12N15/70C12N15/52C12N9/0073C12N9/1092C12N9/1051C12N9/1205C12N9/1096C12N9/1085C12N9/88C12N9/93C12P19/44C12Y114/13064C12Y602/01012C12Y207/01071C12Y206/01086C12Y205/01054C12Y205/01019C12Y403/01023
Inventor 申晓林王晓蕾袁其朋王佳孙新晓
Owner BEIJING UNIV OF CHEM TECH
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