A genetically engineered bacterium for the production of l-theanine and its construction and application

A technology of genetically engineered bacteria and theanine, applied in the field of genetic engineering, can solve the problems of high price and no advantage in production cost, etc.

Active Publication Date: 2020-06-30
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme-catalyzed synthesis of L-theanine includes two processes: the cultivation of enzyme-producing strains and the preparation of L-theanine by enzymatic reaction, and the need to use glutamate and ATP with higher prices as raw materials, so the production cost No advantage over extraction and chemical methods

Method used

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  • A genetically engineered bacterium for the production of l-theanine and its construction and application
  • A genetically engineered bacterium for the production of l-theanine and its construction and application
  • A genetically engineered bacterium for the production of l-theanine and its construction and application

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Experimental program
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Effect test

Embodiment 1

[0058] Embodiment 1: the construction of genetically engineered bacterial strain Escherichia coli (Escherichia coli) THE:

[0059] 1 Methods of gene editing

[0060]In the present invention, the gene editing method mediated by CRISPR / Cas 9 is carried out with reference to literature (Metabolic Engineering, 2015, 31:13-21.), and the two plasmids used in this method are pGRB and pREDCas9 respectively. Among them, pREDCas9 carries gRNA plasmid elimination system, Red recombination system of lambda phage and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg / L), cultured at 32°C; pGRB plasmid, with pUC18 as the backbone, including promoter J23100 , gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg / L), cultured at 37°C.

[0061] 2 Specific process of strain construction

[0062] 2.1 will P trc - gmas-Mu (fragment containing gmas-Mu gene and trc promoter) integrated at the pseudogene ...

Embodiment 2

[0085] Embodiment 2: Fermentation production of L-theanine by bacterial strain E.coli THE fermenter

[0086] Fermentation experiment of strain E.coli THE on 5L fermenter:

[0087] Slope activation culture: Scrape a ring of strains from the bacteria preservation tube of the -80°C refrigerator, spread evenly on the activation slope, incubate at 37°C for 12 hours, transfer to an eggplant-shaped bottle and continue to cultivate for 12 hours;

[0088] Seed culture: Take an appropriate amount of sterile water in an eggplant-shaped bottle, insert the bacterial suspension into the seed medium, keep the pH at about 7.0, the temperature at 37°C, and the dissolved oxygen at 25-35%, and cultivate for 6 hours;

[0089] Fermentation culture: Add fresh fermentation medium according to 15% inoculation amount, and the liquid filling volume is 60% (v medium / v fermentation tank). Between 25-35%; when the glucose in the medium is consumed, add 800g / L glucose solution and maintain the glucose con...

Embodiment 3

[0094] Example 3: Fermentation production of L-theanine by bacterial strain E.coli THE fermenter

[0095] Fermentation experiment of strain E.coli THE on 5L fermenter:

[0096] Slope activation culture: Scrape a ring of strains from the bacteria preservation tube of the -80°C refrigerator, spread evenly on the activation slope, incubate at 37°C for 12 hours, transfer to an eggplant-shaped bottle and continue to cultivate for 12 hours;

[0097] Seed culture: Take an appropriate amount of sterile water in an eggplant-shaped bottle, insert the bacterial suspension into the seed medium, keep the pH at about 7.0, the temperature at 37°C, and the dissolved oxygen at 25-35%, and cultivate for 6 hours;

[0098] Fermentation culture: Add fresh fermentation medium according to 15% inoculation amount, and the liquid filling volume is 60% (v medium / v fermentation tank). Between 25-35%; when the glucose in the medium is consumed, add 700g / L glucose solution and maintain the glucose concen...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to novel high-efficiency gamma-glutamyl methylamine synthetase and a plasmid-free genetic engineering bacterium for L-theanine production and construction and application thereof. The plasmid-free genetic engineering bacterium which performs denovo synthesis on L-theanine efficiently by taking cheap carbon sources such as glucose as a substrate is provided, escherichia coli serves as a host, and gamma-glutamyl methylamine synthase genes gmas-Mu copied three times are integrated on a genome of the escherichia coli; a glutamate dehydrogenase gene Cgl2079 is copied once; a pyruvate carboxylase gene Cgl0689 is copied once; a citrate synthase gene gltA is copied once, and the genetic engineering bacterium is obtained. After metabolic transformation of a system, the engineering bacterium can perform denovo synthesis on the L-theanine by taking the glucose as the raw material, the fermentation yieldand sugar-acid conversion rate are the highest values reported so far, in fermentation of a 5 L fermentor, the maximum production of the L-theanine can reach 60 g / L, and the sugar-acid conversion ratecan reach 40%.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a novel high-efficiency gamma-glutamine synthetase, a plasmid-free genetically engineered bacterium for L-theanine production, a construction method and application thereof. Background technique: [0002] L-theanine (N-ethyl-L-glutamine) belongs to amide compounds, which is unique in tea and is also the most abundant free amino acid, accounting for more than 50% of the total free amino acid content. It is the main component of tea aroma and taste, so its content plays a key role in the flavor and quality of tea. A large number of research results show that L-theanine has many important physiological functions, such as promoting the learning and memory function of the brain, preventing cardiovascular diseases, inhibiting fat accumulation, etc., so it is widely used in food, health products and pharmaceutical industries. [0003] The existing main preparati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/04C12R1/19
Inventor 范晓光张通田俊宇徐庆阳谢希贤陈宁张成林李燕军马倩
Owner TIANJIN UNIV OF SCI & TECH
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