Genetically engineered bacterium for producing L-theanine by utilizing glucose de novo fermentation, method and application

A technology of genetically engineered bacteria and genetically engineered strains, applied in the field of genetic engineering, can solve the problems of weakened ATP regeneration, harmful external environment, and low yield of tea theanine, so as to avoid damage to the human body, reduce equipment occupancy, and simplify The effect of the fermentation process

Pending Publication Date: 2022-08-09
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2014, my country approved tea theanine (theanine made from tea leaves through extraction, filtration, concentration and other processes) as a new food raw material, but the yield of tea theanine obtained through plant extraction is low, High cost, unable to meet the growing market demand
[0004] Although supplementing sufficient amount of ethylamine during the fermentation process can effectively produce theanine, ethylamine itself is cytotoxic, and its boiling point is 16.6°C. After gasification, it is not good for human health and harmful to the external environment, so special equipment is required for supplementation. material
In addition, ethylamine that has not been completely metabolized will accumulate in a large amount outside the cell, restricting the growth of bacteria, weakening ATP regeneration, and thus inhibiting the synthesis of theanine

Method used

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  • Genetically engineered bacterium for producing L-theanine by utilizing glucose de novo fermentation, method and application
  • Genetically engineered bacterium for producing L-theanine by utilizing glucose de novo fermentation, method and application
  • Genetically engineered bacterium for producing L-theanine by utilizing glucose de novo fermentation, method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1: Construction of gene engineering bacteria E.Coli E5

[0103] 1. The method of gene editing

[0104] In the present invention, the gene editing method mediated by CRISPR / CAS 9 refers to the literature (MetaboliceNGINEERING, 2015, 31: 13-21.). The two plasmids used in this method are PGRB and Predcas9, respectively. Among them, Predcas9 carries the GRNA plasmid elimination system, the red restructuring system of the λ phage and the CAS9 protein expression system, Qiithromycin resistance (work concentration: 100mg / L), 32 ℃ cultivation; PGRB plasmid, PUC18 as the skeleton, including promoter J23100000000 , GRNA-CAS9 combines regional sequences and terminate subeenters, ammonia gryimonicin resistance (working concentration: 100mg / L), 37 ℃ cultivation.

[0105] The specific steps of this method are as follows:

[0106] 1.1 PGRB plasmid construction

[0107] The purpose of constructing the plasmid PGRB is to transcribe the corresponding GRNA, so as to form a complex for...

Embodiment 2

[0162] Example 2: Use the genetic engineering bacteria E.Coli E5 fermentation to produce belline

[0163] (1) Shaking bottle fermentation:

[0164] The fermentation medium for fermentation of bottle fermentation becomes: glucose 10g / L, yeast powder 3g / L, protein 胨 4g / L, sodium citrate 1g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 1g / L, iron sulfate iron 15mg / L, manganese sulfate 15mg / L, VB 1 , VB 3 , VB 5 , VB 12 V H Each 1mg / L, the rest is water, pH 7.0-7.2.

[0165] Fermentation process: Take -80 ° C to preserve the bacteria species and inoculate it in the active slope, cultivate 12 hours at 37 ° C, and spread it once. Use the inoculating ring to take a ring -to -ring seed in the 500ml triangle bottle equipped with a 30ml seed medium, seal the nine layers of gauze, 37 ° C, and 220rpm cultivation for 10h. The inoculation amount of 15 % of the seed culture liquid accumulation was inoculated to the 500ml triangular bottle (30ml of the final volume) equipped with a fe...

Embodiment 3

[0169] Example 3: Use the genetic engineering bacteria E.Coli E5 fermentation to produce belline

[0170] (1) Shaking bottle fermentation:

[0171] The fermentation medium for fermentation of bottle fermentation becomes: glucose 40g / L, yeast powder 4g / L, protein 胨 6g / L, sodium citrate 2g / L, potassium dihydrogen phosphate 6g / L, magnesium sulfate 2g / L, iron sulfate 20mg 20mg / L, manganese sulfate 20 mg / L, VB 1 , VB 3 , VB 5 , VB 12 V H 3mg / L each, the rest are water, pH 7.0-7.2.

[0172] Fermentation process: Take -80 ° C to preserve the bacteria species and inoculate it in the active slope, cultivate 12 hours at 37 ° C, and spread it once. Use the inoculating ring to take a ring -to -ring seed in the 500ml triangle bottle equipped with a 30ml seed medium, seal the nine layers of gauze, 37 ° C, and 220rpm cultivation for 10h. The inoculation amount of 20 % of the seed culture liquid accumulation is inoculated to a 500ml triangular bottle equipped with a fermented medium (the final v...

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Abstract

The invention discloses a genetically engineered bacterium for producing L-theanine by utilizing glucose de novo fermentation, and the genetically engineered bacterium contains gamma-glutamyl methylamine synthetase for synthesizing theanine and a gene for coding a path enzyme for de novo synthesis of theanine. The theanine de novo synthetic pathway enzymes comprise phosphoketolyase, transaminase, acetaldehyde dehydrogenase and alanine dehydrogenase. A complete anabolism pathway from glucose to theanine is constructed, a genetic engineering strain which does not carry plasmids, does not need to add ethylamine and can stably produce theanine by using glucose is obtained, after the strain is fermented for 33 hours, the yield of theanine can reach 46 g / L, the conversion rate can reach 0.16 g theanine / g glucose, the production intensity can reach 1.4 g theanine / L / h, and the production cost can be greatly reduced. The highest yield, the conversion rate and the production intensity of the theanine produced by directly utilizing the glucose for de novo fermentation without adding ethylamine are reported at present, and the method has a good industrial application prospect.

Description

Technical field [0001] The present invention is a genetic engineering technology field, especially a genetic engineering bacteria, methods and applications that use glucose to fermented from the beginning. Background technique [0002] L-Caacide (hereinafter referred to as Caacin) is a special amino acid that exists in the tea tree plant, which has many useful effects on human health, such as relieving stress, improving sleep quality, antioxidant, regulating neurotransmitter transmission, and regulating neurotransmitters. Inhibit hypertension, anti -tumor, and improve memory. Catemaine, as a natural sedative, antidepressant, has a high degree of recognition in Japan and the United States, and has formed hundreds of functional foods, beverages and health products. In 2014, my country approved tea catechide (catexine made of tea as a raw material, categinine made of extraction, filtration, concentration and other processes) as new food raw materials, but the tea catexine obtained t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/54C12N15/53C12N15/60C12N15/70C12P13/04C12R1/19
CPCC12N9/93C12Y603/04012C12N9/1096C12Y206/01C12N9/0008C12Y104/01001C12N9/0016C12N9/88C12N15/70C12P13/04
Inventor 范晓光季圆清温昊妍周宇航徐庆阳
Owner TIANJIN UNIV OF SCI & TECH
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