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Gene engineer bacterium for producing protocatechuic acid (3, 4-dihydroxybenzoic acid) by using phenol and construction method thereof

A technology of dihydroxybenzoic acid and genetically engineered bacteria, which is applied in the directions of genetic engineering, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of not yet producing protocatechuic acid and the like

Inactive Publication Date: 2019-01-25
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the production of protocatechuic acid (3,4-dihydroxybenzoic acid) genetically engineered bacteria

Method used

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  • Gene engineer bacterium for producing protocatechuic acid (3, 4-dihydroxybenzoic acid) by using phenol and construction method thereof
  • Gene engineer bacterium for producing protocatechuic acid (3, 4-dihydroxybenzoic acid) by using phenol and construction method thereof
  • Gene engineer bacterium for producing protocatechuic acid (3, 4-dihydroxybenzoic acid) by using phenol and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0023] A kind of construction that utilizes phenol as raw material to produce the genetically engineered bacterium EYP of protocatechuic acid (3,4-dihydroxybenzoic acid):

[0024] In vitro amplification of the target gene:

[0025] The genomes of Bacillus subtilis 168 and Pseudomonas aeruginosa PAO1 were extracted using a bacterial genome extraction kit, using SEQ ID NO.1 and SEQ ID NO.2 in the sequence table as upstream and downstream primers, and using the genome of Bacillus subtilis 168 as a template, The p-hydroxybenzoic acid decarboxylase gene yclBCD is obtained by PCR amplification, and the sequence of the gene yclBCD is shown in SEQ ID NO.5. Using SEQ ID NO.3 and SEQ ID NO.4 in the sequence table as upstream and downstream primers, and using the genome of Pseudomonas aeruginosa PAO1 as a template, the hydroxylase gene pobA is obtained by PCR amplification, and the sequence of the gene pobA is Shown in SEQ ID NO.6. The steps are: take 10ul of genome solution, 50μl of P...

Embodiment 2

[0031] Construction of conditions for producing protocatechuic acid in genetically engineered strain EYP

[0032] The constructed strain EYP was cultured in LB medium, and the setting parameters of the shaker were temperature 37°C and rotation speed 200rpm. When the OD600nm of the bacterial solution reached 0.6-0.8, it was induced by adding isopropylthiogalactoside (IPTG), and the final concentration of IPTG was 0.1mM, the shaker setting parameters are temperature 18°C, rotation speed 200rpm, induction time 15h, after which the bacteria are collected by centrifugation, and the collected bacteria are transferred into M9Y medium under sterile conditions for fermentation and culture, the shaker setting The parameters are temperature 30° C., rotation speed 200 rpm, and fermentation time 48 hours.

Embodiment 3

[0034] Hydroxylase expression identification

[0035] The cells were collected, centrifuged at 8000rpm for 10min, and the cells were taken in a boiling water bath at 100°C for 10min, followed by SDS-PAGE electrophoresis ( image 3 ). From image 3 Comparing the total protein of the constructed strain with the wild-type strain, it can be observed that the hydroxylase is expressed, and its size is 57KD.

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Abstract

The invention discloses a genetically engineered bacterium for producing protocatechuic acid (3, 4-dihydroxybenzoic acid) by using phenol as raw material and a construction method thereof. The engineering strain contains p-hydroxybenzoic acid decarboxylase gene yclBCD and hydroxylase gene pobA, phenol is used as raw material, phenol is carboxylated to obtain p-hydroxybenzoic acid by biological transformation, and then 3-hydroxybenzoic acid is oxidized to obtain protocatechuic acid (3, 4-dihydroxybenzoic acid). Compared with the traditional methods such as plant extraction and chemical synthesis, this method is convenient and quick, and will not pollute the environment, and can reasonably utilize phenol to save energy and protect the environment. It is of great significance to the production of protocatechuic acid.

Description

technical field [0001] The invention belongs to the field of synthetic biology, and specifically relates to a genetically engineered bacterium that uses phenol as a raw material to produce protocatechuic acid (3,4-dihydroxybenzoic acid) and its construction method and application Background technique [0002] 3,4-Dihydroxybenzoic acid (3,4-Dihydroxybenzoic acid), also known as protocatechuic acid, is a substance with extremely strong pharmacological effects. The substance has an antibacterial effect, and has different degrees of antibacterial effects on Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Shigella, Alcaligenes, Bacillus subtilis and Staphylococcus aureus in vitro. Protocatechuic acid has a positive effect on the cardiovascular system, and also has the effects of eliminating phlegm and relieving asthma. It is often used in the treatment of chronic bronchitis in clinical practice. In addition, it is also used as dyes, pharmaceutical intermediates, anal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/60C12N15/53C12P7/42C12R1/19
CPCC12N9/0071C12N9/88C12N15/70C12P7/42C12Y401/01061
Inventor 戴玉杰缪颖琪凌君贾士儒钟成张雅婷缴鹏何博韬
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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