A kind of construction method and application of engineering strain for producing phenol by fermentation of glycerol

A technology of engineering strains and glycerin, applied in the direction of microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve the problems of high production cost of phenol, large environmental pollution, and many by-products, so as to reduce production costs and improve economic efficiency. benefit effect

Active Publication Date: 2019-02-19
QUFU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of high production cost, large environmental pollution, and many by-products in the production of phenol by chemical methods in the above prior art, and the fact that there are few studies on the synthesis of phenol by biological methods, the present invention uses glycerin as a raw material to study a Construction method of an engineering strain producing phenol by fermentation of glycerol

Method used

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  • A kind of construction method and application of engineering strain for producing phenol by fermentation of glycerol
  • A kind of construction method and application of engineering strain for producing phenol by fermentation of glycerol
  • A kind of construction method and application of engineering strain for producing phenol by fermentation of glycerol

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Embodiment 1

[0057] Example 1. Construction of recombinant E. coli used for glycerol fermentation to produce phenol

[0058] 1. Construction of recombinant expression vector pTrc99a-tpl-p4m

[0059] 1. Selection and optimization of tpl gene

[0060] Select the complete CDS sequence of tyrosine phenol lyase (tpl), and obtain an optimized tyrosine phenol lyase gene sequence suitable for expression in E. coli after design and repeated verification, that is, the wild-type tyrosine phenol lyase gene The sequence is transformed into an E. coli preferred (high frequency use) codon optimized sequence without changing the amino acid sequence, thereby increasing the expression level of tyrosine phenol lyase in E. coli culture environment.

[0061] The amino acid sequence of the tyrosine phenol lyase is specifically shown in sequence 1 in the sequence table, and consists of 456 amino acids in total;

[0062] The coding gene sequence of the tyrosine phenol lyase is specifically shown in sequence 2 in the seque...

Embodiment 2

[0115] Example 2: Fermentation to produce phenol with glycerol as substrate

[0116] 1. The fermentation process of using glycerol to produce phenol

[0117] The E. coli engineered strain is preferably:

[0118] PH1: ATCC31884-pTrc99a-tpl-p4m;

[0119] PH2: ATCC31884-pTrc99a-tpl-p4m-trcglpX-tktA;

[0120] PH3: ATCC31884-pTrc99a-tpl-trcp4m-trcglpX-tktA;

[0121] The culture medium involved in the fermentation and production of phenol is preferably:

[0122] Solid plate medium (g / L): peptone 10, sodium chloride 10, yeast powder 5, agar 15, pH 7.0, add Amp 100μg / mL to the medium if necessary;

[0123] Fermentation seed medium (g / L): peptone 10, sodium chloride 10, yeast powder 5, pH 7.0, filling volume 10mL / 20mL, add Amp 100μg / mL to the medium if necessary;

[0124] Fermentation medium (g / L): glycerol 10, Na 2 HPO 4 ·12H 2 017.11, KH 2 PO 4 3. NaCl 0.5, NH 4 Cl 1, MgSO 4 ·7H 2 O0.492, CaCl 2 ·6H 2 O 0.022, filling volume 10mL / 20mL, add Amp 100μg / mL to the medium if necessary;

[0125] (1) Seed ...

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Abstract

The invention relates to the technical field of engineered strain construction, in particular to a construction method of an engineered strain for producing phenol through glycerol fermentation. The second base sequence and the third base sequence in a sequence list are inserted in a pTrc99a carrier to obtain recombinant plasmid; the recombinant plasmid is electrically transformed into an ATC31884 strain, the engineered strain for producing the phenol through glycerol fermentation is obtained. The constructed engineered strain efficiently presents tyrosine phenol lyase and phenylalanine4-monooxygenase, and content of the obtained escherichia coli engineered strain for producing the phenol through glycerol is 21.30-57.66 mg / L, so that the phenol is produced through glycerol fermentation through the biological method, production cost of the phenol is reduced, and economic benefits are improved.

Description

Technical field [0001] The invention relates to the technical field of engineering strain construction, in particular to a method for constructing an engineering strain that uses glycerol to ferment to produce phenol, and also relates to the application of the obtained engineering strain. Background technique [0002] Phenol is an important basic organic chemical raw material, mainly used in the production of phenolic resin, bisphenol A and caprolactam, adipic acid, aniline, etc.; phenol derivatives such as halogenated phenols, nitrophenols, and alkylphenols can be used in medicine, The production of dyes, fragrances and various additives, so the phenol market has a good development prospect. [0003] Glycerin is also called glycerol, molecular formula C 3 H 5 (OH) 3 , Is a viscous liquid with sweet taste, can be miscible with water in any ratio, and has strong hygroscopicity. Glycerin is rich in resources and low in price. Because of its high reduction of C atoms, it has a higher...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/22C12R1/19
Inventor 车程川王斌王莹袁雪娇李敏
Owner QUFU NORMAL UNIV
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