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Genetic engineering bacterium for L-theanine production and fermentation method thereof

A technology of genetically engineered bacteria and theanine is applied in the field of bioengineering to achieve the effects of simple ingredients, short fermentation cycle and significant cost advantages

Active Publication Date: 2019-02-22
TIANJIN UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the preparation of L-theanine by microbial fermentation using glucose as raw material

Method used

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  • Genetic engineering bacterium for L-theanine production and fermentation method thereof
  • Genetic engineering bacterium for L-theanine production and fermentation method thereof
  • Genetic engineering bacterium for L-theanine production and fermentation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction method of genetically engineered bacteria with high L-theanine production

[0037] 1. Integrate the RNA polymerase gene derived from T7 phage into the lacI-lacZ site of the genome of E.coli W3110

[0038] Using the E.coli W3110 (ATCC27325) genome as a template, design upstream homology arm primers UP-lacI-lacZ-S (see Sequence Table 5 for the nucleotide sequence) and UP-lacI-lacZ according to the upper and lower sequences of its lacI-lacZ gene -A (see Sequence Table 6 for the nucleotide sequence) and downstream homology arm primers DN-lacI-lacZ-S (see Sequence Table 7 for the nucleotide sequence), DN-lacI-lacZ-A (see Sequence Table 7 for the nucleotide sequence) list 8), and PCR amplification of its upstream and downstream homology arm fragments; obtain the T7RNAP gene sequence by gene synthesis, which contains xylose promoter P xylF , design primers T7RNAP-S (see Sequence Table 9 for nucleotide sequence) and T7RNAP-A (see Sequence Table 10 for n...

Embodiment 2

[0051] Example 2: Production of L-theanine by fermentation in a 5L fermenter

[0052] 1. Medium

[0053] The composition of the slant medium is: glucose 1g / L, peptone 10g / L, beef extract 10g / L, yeast powder 5g / L, sodium chloride 2.5g / L, agar 25g / L, pH 7.0;

[0054] The composition of the seed medium is: glucose 25g / L, yeast extract 10g / L, peptone 15g / L, sodium chloride 15g / L, pH 7.2.

[0055] The composition of the fermentation medium is: glucose 30g / L, yeast extract 10g / L, peptone 15g / L, potassium dihydrogen phosphate 5g / L, magnesium sulfate 2g / L, pH 7.2.

[0056] 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;

[0057] 2. Fermentation method

[0058] Seed culture: Take an appropriate amount of sterile water in an eggplant-shaped bottle, insert the bacterial susp...

Embodiment 3

[0063] Effects of different feeding methods of ethylamine on the fermentation of L-theanine in a 5L fermenter

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Abstract

The invention provides a genetic engineering bacterium for L-theanine production and a fermentation method thereof. A construction method of the genetic engineering bacterium comprises the steps of carrying out single copying on an ribonucleic acid (RNA) polymerase gene T7RNAP derived from T7 phage on an Escherichia coli W3110 genome, wherein the gene is controlled by a xylose promoter; carrying out dual copying on a gamma-glutamyl methylamine synthetase gene gmas derived from methylovorusmays, wherein the gene is controlled by a T7 promoter; knocking out a xylose operon repressor protein genexylR; and knocking out a succinyl CoA synthetase gene sucCD. The genetic engineering bacterium has the beneficial effects that the yield of L-theanine fermented by using the genetic engineering bacterium can be up to 40g / L and the sugar acid conversion rate can be up to 25%; and the L-theanine production method provided by the invention has the advantages of being low in raw material price, shortin cycle, simple and convenient to operate, green and friendly to environment and high in yield and has good industrial application value.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a genetically engineered bacterium used for the production of L-theanine and a fermentation method thereof. Background technique [0002] L-Theanine (L-Theanine), also known as glutamic acid γ-ethylamide, is a unique free amino acid in tea, accounting for more than 50% of the total free amino acids in tea. As the main component of the sweet taste of tea, theanine determines the quality of tea to a certain extent. Theanine has many beneficial effects on human health, including promoting the learning and memory functions of the brain, preventing cardiovascular diseases, and inhibiting fat accumulation. In addition, theanine can also enhance the flavor, suppress the bitter and spicy taste in food, and reduce the astringency, so it has been widely used in the pharmaceutical and food industries. [0003] At present, the main preparation methods of theanine mainly include extraction met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/04C12R1/19
CPCC12N9/1247C12N9/93C12N15/70C12P13/04C12Y207/07006
Inventor 范晓光朱新雅曹华杰徐庆阳张通谢沛刘博超陈宁谢希贤张成林
Owner TIANJIN UNIV OF SCI & TECH
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