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Escherichia coli genetically engineered bacterium and method for producing L-theanine by fermenting escherichia coli genetically engineered bacterium

A technology of genetically engineered bacteria and theanine, applied in the field of bioengineering, can solve the problems of low yield and product purity, and achieve the effects of improving product yield and product quality, reducing pollution risks, and reducing use.

Pending Publication Date: 2021-12-10
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large amount of acid-base reagents or flocculants are used in the above-mentioned extraction methods, and the yield and product purity are low. Therefore, a method for separation and purification of high-purity L-theanine is also badly needed

Method used

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  • Escherichia coli genetically engineered bacterium and method for producing L-theanine by fermenting escherichia coli genetically engineered bacterium
  • Escherichia coli genetically engineered bacterium and method for producing L-theanine by fermenting escherichia coli genetically engineered bacterium
  • Escherichia coli genetically engineered bacterium and method for producing L-theanine by fermenting escherichia coli genetically engineered bacterium

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Experimental program
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Effect test

Embodiment 1

[0072] Embodiment 1: the construction of genetically engineered bacterial strain Escherichia coli E.coli THEE (transformation strategy sees figure 1 ):

[0073] 1. Methods of gene editing

[0074] 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.

[0075] 2. The specific process of strain construction

[0076] L-theanine-producing genetic engineering bacteria constructed with ZL...

Embodiment 2

[0095] Example 2: Fermentation of strain E.coli THEE 5L fermenter to produce L-theanine

[0096] The composition of the slant medium is: glucose 2g / L, peptone 6g / L, beef extract 6g / L, yeast powder 3g / L, sodium chloride 2g / L, agar 18g / L, pH 7.0.

[0097] The composition of the seed medium is: glucose 25g / L, yeast extract 6g / L, peptone 15g / L, sodium chloride 15g / L, the rest is water, pH 7.0.

[0098] The composition of the fermentation medium is: glucose 30g / L, yeast powder 6g / L, corn steep liquor 8ml / L, citric acid 1.5g / L, potassium dihydrogen phosphate 2.5g / L, dipotassium hydrogen phosphate 2.0g / L, magnesium sulfate 1.0g / L, the rest is water, pH 7.2.

[0099] (1) Incline culture: Scrape a ring of strains from the bacteria-preserving tube of the -80°C refrigerator, spread evenly on the activated slope, incubate at 37°C for 14 hours, transfer to an eggplant-shaped bottle and continue to cultivate for 14 hours;

[0100] (2) Seed culture: Take an appropriate amount of sterile wate...

Embodiment 3

[0107] Embodiment 3 Determination of OD linkage ethylamine supplementation strategy

[0108] Fermentation and production of L-theanine in a 5L fermenter, when OD 600nm When it reaches above 10, start to add ethylamine. According to the degree of glucose consumption by the bacteria, adjust the flow rate of ethylamine to different degrees every hour to ensure that the sugar consumption rate is maintained at 7-8g L -1 h -1 . Collect 50 batches of fermentation batches whose L-theanine yield reaches 80g / L, and calculate the average OD at different fermentation times 600nm Value, average fermentation broth volume, and average ethylamine flow acceleration rate are shown in the following table:

[0109]

[0110]

[0111] According to the data in the above table, use ORIGIN software to perform linear fitting, Y is the average ethylamine flow acceleration rate, and X is the average OD 600nm value / (average fermentation broth volume (L)×fermentation time (h). Fitting results are...

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Abstract

The invention belongs to the field of bioengineering, and particularly relates to a method for producing L-theanine by fermenting escherichia coli genetically engineered bacteria. The engineering bacterium is obtained by taking a strain obtained by single copy of T7RNAP, double copy of gmas, knockout of xylR and knockout of sucCD on an escherichia coli W3110 genome as a starting strain, integrating xfp, pta, acs, gltA and ppc on the genome and knockout of ackA. According to the present invention, the L-theanine is efficiently synthesized from the beginning by using the cheap carbon source such as glucose as the substrate by using the OD-linked ethylamine supplementing strategy, the saccharic acid conversion rate is high, the production performance is stable, the yield of the L-theanine can achieve 75-80 g / L after 20-25 h, and the saccharic acid conversion rate achieves 52-55%; the fermentation liquor is purified by adopting a membrane separation and anion-cation resin tandem technology, the primary crystallization yield is 72.3%, and the purity of the finished product of L-theanine reaches 99%.

Description

Technical field: [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for producing L-theanine by fermenting Escherichia coli genetically engineered bacteria. [0002] technical background: [0003] L-theanine (N-ethyl-γ-L-glutamine) is a unique non-protein amino acid in tea. It was isolated and named from Gyokuro tea by Japanese scholar Yajiro Sato in 1950. L-theanine mainly exists in tea plants, usually in free form, does not participate in protein synthesis, and accounts for 40%-70% of the total free amino acids in tea plants. The brewing rate of L-theanine in tea soup can reach 80%. It is the main component of tea soup showing refreshing taste. The positive correlation coefficient with green tea grade is 0.787-0.876. It is also an important substance to evaluate the quality and flavor of other teas. With the in-depth research on the physiological activity of L-theanine, it has broad application prospects in the fields of food...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12N15/54C12N15/60C12N15/52C12P13/04C07C231/24C07C237/06C12R1/19
CPCC12N15/70C12N9/1029C12N9/88C12N9/1217C12N9/93C12N9/1025C12N15/52C12P13/04C12Y401/02022C12Y203/01008C12Y401/01031C12Y207/02001C12Y602/01003C12Y203/03001C07C231/24C07B2200/07C07C237/06C12N2310/20C12Y401/00C12Y602/01001C12Y203/03C12P13/14Y02A50/30C12N1/00C12N2510/00
Inventor 范晓光季圆清周宇航李静刘博超徐庆阳
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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