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Construction method and application of engineered strain for producing phenol through glycerol fermentation

A technology of engineering strains and construction methods, 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 production efficiency. The effect of economic benefits

Active Publication Date: 2015-12-02
QUFU NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Construction method and application of engineered strain for producing phenol through glycerol fermentation
  • Construction method and application of engineered strain for producing phenol through glycerol fermentation
  • Construction method and application of engineered strain for producing phenol through glycerol fermentation

Examples

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Embodiment 1

[0057] Example 1. Construction of recombinant Escherichia coli 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 full CDS sequence of tyrosine phenol lyase (tpl), and after design and repeated verification, an optimized tyrosine phenol lyase gene sequence suitable for expression in Escherichia coli is obtained, that is, the wild-type tyrosine phenol lyase gene The sequence is converted into a codon-optimized sequence preferred by Escherichia coli (high frequency usage) without changing the amino acid sequence, thereby improving the expression level of tyrosine phenol lyase in the culture environment of Escherichia coli.

[0061] The amino acid sequence of the tyrosine phenol lyase is specifically shown in Sequence 1 in the sequence listing, consisting of 456 amino acids in total;

[0062] The coding gene sequence of the tyrosine phenol ly...

Embodiment 2

[0115] Embodiment 2, take glycerol as substrate fermentation to produce phenol

[0116] 1. The fermentation process of producing phenol from glycerol

[0117] The preferred escherichia coli engineering strain:

[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 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 Amp100μg / mL to the medium if necessary;

[0123] Fermentation seed medium (g / L): peptone 10, sodium chloride 10, yeast powder 5, pH 7.0, liquid volume 10mL / 20mL, add Amp100μg / 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, NaCl0.5, NH 4 Cl1, MgSO 4 ·7H 2 O0.492, CaCl 2 ·6H 2 O0.022, the filling volume is 10mL / 20mL, if necessary, add Amp100μg / mL ...

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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 construction of engineering strains, in particular to a method for constructing engineering strains for producing phenol by fermentation of glycerin, and also relates to the application of the obtained engineering strains. 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, spices and various additives, etc., so the phenol market has a good development prospect. [0003] Glycerol, also known as glycerol, molecular formula C 3 h 5 (OH) 3 , is a viscous liquid with a sweet taste, miscible with water in any ratio, and has strong hygroscopicity. Glycerol, which is abundant and inexpensive, has become an attractive feedstock due to the hig...

Claims

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Application Information

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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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