Construction and application of engineering strain for biosynthesizing resveratrol glucoside by taking resveratrol as substrate

A technology of resveratrol and engineering strains, which is applied in the construction field of biosynthetic resveratrol glycoside engineering strains, can solve the problems of high yield, high profit, limited plant resources and product purity in difficult enterprises, and achieve low cost, The effect of high synthetic yield and simple operation

Pending Publication Date: 2022-04-15
河北维达康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, resveratrol glycosides are mainly produced and prepared by plant extraction. However, plant extraction is limited by plant resources and product purity, making it difficult for enterprises to achieve the goal of high yield and high profit.

Method used

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  • Construction and application of engineering strain for biosynthesizing resveratrol glucoside by taking resveratrol as substrate
  • Construction and application of engineering strain for biosynthesizing resveratrol glucoside by taking resveratrol as substrate
  • Construction and application of engineering strain for biosynthesizing resveratrol glucoside by taking resveratrol as substrate

Examples

Experimental program
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Effect test

example 1

[0026] Example 1. Construction of pET28a-UGT plasmid and Escherichia coli recombinant strain

[0027] Construction method of pET28A-UGT plasmid:

[0028] (1) Using the pET28a vector as a template, PCR amplification was performed using specific primers. The pET28a vector is a commercial vector, purchased from Novagen; the primer sequences are shown in Table 1; the linearized vector pET28a-reverse amplification is obtained after the PCR product is recovered;

[0029] (2) According to NCBI ( https: / / www.ncbi.nlm.nih.gov / ) The amino acid sequences of glucosyltransferases in two organisms of Vitisvinifera and Arachis hypogaea published in the database were codon-optimized, the nucleic acid sequences were artificially synthesized, and then 4 pairs of specific primers were designed to amplify them. The primer sequences are shown in Table 1, and the amplified product is recovered to obtain a UGT positive amplified fragment. The gene sequence of Vitis vinifera UGT is shown in SEQ ...

example 2

[0038] Example 2. Whole-cell catalysis of recombinant strains

[0039] The two engineered bacteria were inoculated into 20 mL of LB liquid medium and cultured overnight at 37°C and 200 rpm. Then, the seed liquid was inoculated into 200 mL of LB liquid medium, and cultivated at 37 ° C and 200 rpm to bacterial OD. 600 = 0.6. Then, IPTG was added to the final concentration of 1 mM, 25° C., 150 rpm, and cultured for 16 h to induce the expression of the target protein. After induction, the cells were collected by centrifugation at 4,000 × g for 20 min, and then resuspended with PBS buffer to obtain the OD. 600 = 40 bacterial solution, centrifuged at 4,000 × g for 15 min, resuspended the bacterial cells with 5 mL of transformation solution, transferred to a reaction flask, and cultured at 37° C., 200 rpm for 24 h.

[0040]Transformation solution: 10g / L resveratrol, 30g / L glucose, 6g / L Na2HPO4, 0.5g / L NaCl, 3g / LKH2PO4, 1g / L NH4Cl, 246.5mg / LMgSO4 7H2O, 14.7mg / L CaCl2 2H2O , 27.8mg...

example 3

[0041] Example 3. Determination of resveratrol glycoside content

[0042] Detection of resveratrol glycosides by HPLC:

[0043] (1) Dissolve 20 μL of the catalytic product in 980 μL of methanol, and sonicate for 10 min;

[0044] (2) After the product extract was centrifuged at 12000rpm for 10min, the supernatant was collected;

[0045] (3) The supernatant is filtered in a brown liquid phase bottle with a 0.22 μm filter membrane to obtain a sample to be tested;

[0046] (4) The new product was analyzed by IC-MS-NMR using liquid-phase mass spectrometry: LC-MS analysis was performed using Agilent260HPLC system and Bruker-microtof-II electrospray mass spectrometer. Tnature C8 column (4.6mm×250mm, 5μm, waters) was used in high performance liquid phase, the injection volume was 10μL, the flow rate was 1.0mL / min, the UV detection wavelength was 306nm, and the mobile phase A was 900μL ice in the column oven at 25°C. Acetic acid and 1000mL water, mobile phase B is acetonitrile, grad...

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Abstract

The invention relates to the field of microbial synthesis, in particular to construction and application of an engineering strain for biosynthesizing resveratrol glucoside by taking resveratrol as a substrate. The genetically engineered bacterium expresses a glycosyl transferase (UDP-glucose transferase, UGT) gene and a UDP-glucose pyrophosphorylase gene, a UGT enzyme and a UDP-glucose pyrophosphorylase are produced, a beta-glucosidase gene is knocked out at the same time, and beta-glucosidase is not expressed. The method has the advantages of simple operation, no environmental pollution, high yield, high purity and low cost, and provides a basis for industrial production of resveratrol glucoside.

Description

technical field [0001] The invention relates to the field of microbial synthesis, in particular to the construction and application of an engineering strain for biosynthesizing resveratrol glycosides using resveratrol as a substrate. Background technique [0002] Resveratrol (Res) is a natural active substance belonging to polyphenolic compounds. The phenolic hydroxyl groups it carries have significant antioxidant and anti-free radical effects, which in turn make it anti-cancer and cardiovascular prevention and treatment. , liver protection, phytoestrogen regulation and other biological effects. However, resveratrol has disadvantages such as poor water solubility, poor oral absorption, and easy oxidation, which limits the application of resveratrol in medicine and food industries to a certain extent. Studies have found that resveratrol glycoside derivatives can improve its bioavailability and antitumor activity while improving its water solubility and reducing oxidation. ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P19/46C12N15/54C12N15/56C12R1/19
CPCY02A50/30
Inventor 赵云现杨志彬崔金旺胡江林
Owner 河北维达康生物科技有限公司
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