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Method for production of phenol by heterogenous metabolism path and application thereof

A metabolic pathway, phenol technology, applied in the field of clean production of phenol, can solve problems such as serious environmental pollution and non-renewability

Inactive Publication Date: 2016-04-20
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this method, propylene and benzene are catalyzed by aluminum trichloride to generate cumene, then cumene is oxidized to generate cumene peroxide, and then decomposed with sulfuric acid to obtain phenol. Renewable, and more serious environmental pollution

Method used

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  • Method for production of phenol by heterogenous metabolism path and application thereof
  • Method for production of phenol by heterogenous metabolism path and application thereof
  • Method for production of phenol by heterogenous metabolism path and application thereof

Examples

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Effect test

specific Embodiment 1

[0027] Construction of Metabolic Pathway for Microbial Heterologous Synthesis of Phenol

[0028] Aromatic compounds are naturally synthesized by many bacteria. The pathway for the synthesis of aromatic amino acids is called the shikimate pathway. In the shikimate pathway, glucose undergoes a central metabolic pathway to synthesize phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). These two compounds are enzymatically formed into 3-deoxy-D-arabinosyl-7-phosphate (DAHP), which after several steps of reaction forms chorismate. Starting from chorismate, the pathway branches into several branches to form a variety of aromatic compounds, including phenylalanine, tyrosine, tryptophan, folic acid, etc. So far, the shikimate pathway has been used to synthesize a variety of aromatic compounds, some of which have been commercialized. The seven enzymes of the shikimate pathway were originally discovered through studies on bacteria, mainly Escherichia coli and Salmonella typhim...

specific Embodiment 2

[0030] Introduction of heterologous metabolic pathways into phenol-producing microorganisms

[0031] The genomes of Escherichia coli, Pseudomonas fluorescens, and Klebsiella pneumoniae were extracted respectively, and the target genes, entC, pchB, and kpBDC were obtained by PCR from the genomes using the designed primers, as shown in Table 2. Then use the corresponding enzymes to digest the fragments and vectors, and perform gel recovery or column recovery on the digested fragments, and then insert the target gene into the plasmid pZE12-luc (high copy) and plasmid pCS27 (medium copy) to obtain The connecting fluid is spare.

[0032] Preparation of competent cells by electroporation: Inoculate single colonies of Escherichia coli BW25113 and QH4 into 3 mL of liquid LB medium, culture overnight at 37° C. and 250 rpm. Then inoculate 500 μL of bacterial solution into 50 mL of LB medium, culture at 37° C. with shaking at 250 rpm until OD=0.4-0.6. Centrifuge the bacterial solution ...

specific Embodiment 3

[0039] Fermentation of phenol-producing microorganisms

[0040] Pick a single colony from the plate in the genetic engineering of Escherichia coli, connect it to 3mL liquid LB, add ampicillin and kana antibiotics to the medium, the final concentration of antibiotics is 100mg / L, shake culture at 37°C, 250rpm, and then Transfer the bacterial liquid to 50mL liquid M9Y medium, add ampicillin and kana antibiotics to the medium, the final concentration of antibiotics is 100mg / L, shake the culture until OD600 reaches 0.6, add IPTG, IPTG to the medium The final concentration in the medium was 0.25 mM, induced at 30°C. After induction, samples were taken every 12hrs to measure OD, and the concentrations of phenol and salicylic acid were determined by high performance liquid chromatography.

[0041] The fermentation results showed that the plasmid pZE-EP-BDC was introduced into Escherichia coli BW25113, and the strain was defined as BP1. The BP1 strain could only produce 67.65±1.65mg / ...

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Abstract

A method for production of phenol by a heterogenous metabolism path and application thereof belong to the field of biological synthesis of the phenol. A phenol production metabolism path is introduced into a microorganism for phenol production. More precisely, isochorismate synthase, isochorismate pyruvate lyase and salicylate decarboxylase are highly expressed in escherichia coli. Genes encoded by the isochorismate synthase, the isochorismate pyruvate lyase and the salicylate decarboxylase are constructed on antibiotic-resistant expression plasmids with Lac promoter. The constructed plasmids are introduced into the escherichia coli to obtain a producing strain for production of the phenol from glucose, by addition of a carbon source into the path, the method for production of the phenol is more effective, and by the choice of phenylalanine high-yielding strain derived bacteria as host bacteria, the production of the phenol is facilitated.

Description

technical field [0001] The present invention relates to the field of biotechnology, more precisely, the present invention relates to the metabolic pathway of heterologous biosynthesis of phenol, by enhancing the carbon flux, and selecting the derivative bacteria of the high-yield strain of phenylalanine as the host cell, finally improving the yield of phenol , to achieve clean production of phenol. Background technique [0002] Phenol, also known as carbolic acid, is an important benzene series intermediate, and as a bulk chemical, it has a wide range of applications. It is mainly used as a precursor for the synthesis of polycarbonate, epoxy and phenolic resins. In addition, it can also be used to synthesize herbicides and drugs. In 2008, more than eight million tons of phenol were produced worldwide. At present, the main way to produce phenol is to use benzene as raw material to produce phenol by cumene method. In this method, propylene and benzene are catalyzed by alum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22C12R1/19
Inventor 袁其朋孙新晓任彦仙杨森
Owner BEIJING UNIV OF CHEM TECH
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