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Method for biological production of hydroxytyrosol

A technology for producing hydroxytyrosol and hydroxytyrosol, applied in the field of biomedicine, can solve the problems of unsuitability for industrial production, toxic chemical reagents, low yield and the like, and achieves the effects of reducing production costs, solving source problems, and being beneficial to industrial production.

Inactive Publication Date: 2018-02-23
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, hydroxytyrosol is mainly obtained through chemical degradation and synthesis, but the yield of chemical degradation is low, while the reaction process of chemical synthesis is complicated, the yield is low, and the catalyst is expensive, the chemical reagents are toxic, and the environment is seriously polluted, so it is not suitable for industrialization. Production

Method used

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  • Method for biological production of hydroxytyrosol
  • Method for biological production of hydroxytyrosol
  • Method for biological production of hydroxytyrosol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Design of keto-decarboxylase gene kdc and 4-hydroxyphenylacetic acid-3-monooxygenase gene hpaBC

[0034] Preferred Pichia pastoris GS115 keto decarboxylase gene, use JCAT online codon optimization software (http: / / www.jcat.de) in conjunction with OPTIMIZER online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER / ) , optimize the codon preference of Escherichia coli, design the full-length kdc gene, and add the trc promoter before the gene.

[0035] Keto decarboxylase gene kdc with trc promoter, its nucleotide sequence is shown in SEQ ID No.1.

[0036] The 4-hydroxyphenylacetic acid-3-monooxygenase gene of preferred Escherichia coli uses JCAT online codon optimization software (http: / / www.jcat.de) in conjunction with OPTIMIZER online codon optimization tool (http: / / genomes.de). urv.es / OPTIMIZER / ), optimize the codon preference of Escherichia coli, design the full-length hpaBC gene, and add the tac promoter in front of the gene.

[0037] The nucleotide ...

Embodiment 2

[0038] Embodiment 2 Construction of recombinant expression vector pKB

[0039] The expression vector backbone pBPE004 was fully chemically synthesized, and its nucleotide sequence is shown in SEQ ID No.3.

[0040] The nucleotide sequence shown in SEQ ID No.1 and the nucleotide sequence shown in SEQ ID No.2 were connected to the nucleotide sequence shown in SEQ ID No.3 by enzyme-cut ligation, and positive clones were screened to obtain the recombinant vector pKB ;

[0041]The nucleotides shown in SEQ ID No.1 obtained by chemical total synthesis are respectively added with EcoRI and HindIII restriction sites at both ends of the fragment by PCR method, and FastDigest endonucleases EcoRI and HindIII are used for digestion. The reaction system It is: 5 μL 10*FD buffer, 2.5 μL EcoRI, 2.5 μL PstI, 30 μL nucleotides shown in SEQ ID No.1 with enzyme cutting sites and 10 μL ultrapure water. The reaction conditions are: 37°C, 2h. Using a PCR purification kit, add 250 μL of Bingding Bu...

Embodiment 3

[0043] Embodiment 3 Recombinant expression vector pKB is transformed into Escherichia coli chassis strain SyBE-002447

[0044] The detailed construction steps of recombinant vector pKB transformed into Escherichia coli SyBE-002447 are as follows:

[0045] 1. Inoculate 100 μL of activated Escherichia coli strain SyBE-002447 in 10 ml of LB medium, cultivate to OD at 37°C, 220 rpm 600 When the temperature is 0.8-1.0, transfer to a 10ml centrifuge tube, centrifuge at 4500rpm / min for 5min in a pre-cooled 4°C centrifuge, remove the supernatant, and collect the bacteria;

[0046] 2. Wash the cells with 5ml of pre-cooled sterilized 10% glycerin, centrifuge at 4500 rpm / min for 5 min in a pre-cooled 4°C centrifuge, remove the supernatant, collect the cells, and repeat washing three times;

[0047] 3. Pour off the supernatant as much as possible, add 100 μL of 10% glycerol to resuspend the bacteria, and make SyBE-002447 competent cells;

[0048] 4. Add 2.5 μL of the pKB plasmid constru...

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Abstract

The invention discloses a method for biological production of hydroxytyrosol. The method comprises the following steps: performing complete chemical synthesis of a ketone group decarboxylase gene kdcwith a trc promoter and a 4-hydroxyphenylacetic acid-3-monooxygenase gene hpaBC with a tac promoter, performing complete chemical synthesis of a carrier skeleton pBPE004, connecting the kdc and the hpaBC onto pBPE004 to obtain a recombinant vector pKB by using an enzyme digestion connection method, shifting the pKB into a base tray strain SyBE-002447 to obtain a SyBE-KB1 strain; integrating the kdc gene and the hpaBC gene onto a SyBE-002447 chromosome to obtain an SyBE-KB2 strain. Glucose is used as a precursor, and the SyBE-KB1 and SyBE-KB2 strains are fermented to produce the hydroxytyrosol.According to the method, engineering escherichia coli is used for producing the hydroxytyrosol, the source problem of the hydroxytyrosol can be solved, and the cost can be reduced.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a recombinant bacterium producing hydroxytyrosol, a construction method and a biological production method of hydroxytyrosol. Background technique [0002] Hydroxytyrosol (HT) is a natural polyphenolic compound with a chemical name of 3,4-dihydroxyphenethyl alcohol, which has strong antioxidant activity and mainly exists in the fruit and leaves of olives in the form of esters. In the process, oleuropein was hydrolyzed to obtain HT monomer. Studies have shown that hydroxytyrosol has various biological and pharmacological activities such as anticancer, antibacterial, and anti-inflammatory, and has attracted extensive attention from researchers at home and abroad. [0003] At present, hydroxytyrosol is mainly obtained through chemical degradation and synthesis, but the yield of chemical degradation is low, while the reaction process of chemical synthesis is complicated, the yield...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P7/22C12R1/19
CPCC12N9/0073C12N9/88C12P7/22C12Y114/13002
Inventor 赵广荣马雅婷
Owner TIANJIN UNIV
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