Method for biosynthesis of tyrosol in Escherichia coli and application of tyrosol

A technology of Escherichia coli and biosynthesis, applied in the field of bioengineering, can solve the problems of difficult industrialization, cumbersome process, low concentration, etc., and achieve the effect of important economic value and social benefit

Active Publication Date: 2014-10-15
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tyrosol was originally derived from olive oil, but due to its low concentration in olive oil (25mg / kg), the extraction process is difficult to achieve industrialization
Due to industrial needs, tyrosol is currently mainly synthesized by chemistry, but the process is cumbersome and pollutes the environment

Method used

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  • Method for biosynthesis of tyrosol in Escherichia coli and application of tyrosol
  • Method for biosynthesis of tyrosol in Escherichia coli and application of tyrosol
  • Method for biosynthesis of tyrosol in Escherichia coli and application of tyrosol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Knockout of Escherichia coli phenylacetaldehyde dehydrogenase encoding gene FeaB

[0028] (1) Escherichia coli MG1655, plasmid pKD46 (temperature-sensitive type, containing exo, bet and gam genes regulated by the arabinose promoter, Amp r ), pKD4 (contains Kanna resistance gene with FRT sites at both ends, Kan r ), pCP20 (temperature-sensitive type, encoding FLP recombinase capable of recognizing FRT sites, Amp r ) are kept by the laboratory.

[0029] (2) Primer design and synthesis: According to the sequence information of FeaB in GenBank and Kan gene in plasmid pKD4, primers were designed and synthesized by Shenzhen Huada Gene Technology Co., Ltd. Primers FeaB-5FPL / FeaB-3RPL are used to amplify the Kan gene to replace the coding region of the FeaB gene; primers MG1655-5FP / MG1655-3RP are primers for identification of recombinant bacteria after FeaB gene knockout, and the two primers are located on the left and right of the target sequence region sides.

...

Embodiment 2

[0037] Example 2 Amplification of yeast 4-hydroxyphenylpyruvate decarboxylase encoding gene ARO10 and expression vector construction and transformation

[0038](1) According to the gene sequence of yeast 4-hydroxyphenylpyruvate decarboxylase coding gene ARO10 provided in NCBI, primers ARO10-5FP and ARO10-3RP were designed, and the genomic DNA of Saccharomyces cerevisiae S288C was used as a template for PCR, amplified to obtain the ORF of ARO10, such as image 3 shown. The primer sequences are as follows (the restriction site is indicated by the underline):

[0039]

[0040] reaction system:

[0041]

[0042]

[0043] Reaction program: initial denaturation at 98°C for 30 sec; 30 cycles of 98°C for 8 sec, 55°C for 30 sec, and 72°C for 1 min; final extension at 72°C for 10 min.

[0044] (2) The ARO10 and plasmid pTrcHisB amplified in step (1) were digested by SacI and NcoI respectively, recovered, and the digested ARO10 was connected into pTrcHisB to obtain the ARO10 ...

Embodiment 3

[0046] The fermentation culture of embodiment 3 recombinant strains

[0047] Shake flask fermentation: the recombinant strain was cultured overnight at 37°C in 2mL LB medium, then transferred to 50mL fresh LB medium at 1% inoculum size and cultured at 37°C until the OD600 was about 0.6, adding IPTG with a final concentration of 0.5mM to induce After culturing at 30°C for 5 hours, the cells were collected, washed once with M9Y (M9 medium added with 0.125% Yeast extract), suspended in 50 mL of M9Y medium containing 1% glucose and cultured at 30°C for 20 hours. There were three groups of samples, MG1655fCK (control group), MG1655fA+T (experimental group, fed with 1 mM tyrosine), and MG1655fA (experimental group, not fed with tyrosine). The specific formula of M9Y medium is as follows:

[0048]

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Abstract

The invention relates to a method for biosynthesis of tyrosol in Escherichia coli and application of tyrosol, which belongs to the field of bioengineering technology. According to the method for biosynthesis of tyrosol in Escherichia coli, tyrosine or glucose is used as a substrate, (4-hydroxyphenyl)pyruvic acid is produced under the catalysis of ammonialyase coded by Escherichia coli; (4-hydroxyphenyl)acetaldehyde is produced under the action of the microzyme 4-hydroxyphenylpyruvate decarboxylase; (4-hydroxyphenyl)acetaldehyde is catalyzed by alcohol dehydrogenase so as to produce tyrosol; and a phenylacetaldehyde dehydrogenase gene of Escherichia coli is knocked out at the same time so as to block the transformation channel for (4-hydroxyphenyl)acetaldehyde into (4-hydroxyphenyl)acetic acid and promote accumulation of (4-hydroxyphenyl)acetaldehyde and transformation of (4-hydroxyphenyl)acetaldehyde into tyrosol. The invention provides a novel production approach for tyrosol; and the method lays a foundation for large-scale industrial production of tyrosol and has important economic values and social benefits.

Description

technical field [0001] The invention relates to a method for constructing a pathway for biosynthesizing tyrosol in Escherichia coli and its application, and belongs to the technical field of bioengineering. Background technique [0002] The biosynthesized tyrosol of the present invention has the following characteristics. English name Tyrosol, chemical name 4-(2-Hydroxyethyl)phenol, aliases p-Hydroxyphenethyl alcohol, 2-(4-Hydroxyphenyl)ethanol and 4-Hydroxyphenylethanol, molecular formula C 8 h 10 o 2 , molecular weight 138.164, CAS No. 501-94-0, structural formula [0003] Tyrosol is a phenolic compound with important industrial value, and tyrosol and its derivatives are the synthetic precursors of various organic compounds. Tyrosol can be used to prepare betasoprolol and metoprolol, which can treat hypertension, angina, heart failure and glaucoma; It can promote the recovery of some cancers, improve the effect of chemotherapy, and effectively reduce and inhibit the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22C12N1/21C12R1/19
Inventor 毕慧萍白艳芬庄以彬蔡韬李娥娥刘涛马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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