Plasmid for theanine production and construction and application method of corresponding engineering bacteria of plasmid

A construction method and technology of theanine, which is applied in the field of theanine production of plasmids, can solve the problems of low theaninase activity, low plasmid expression efficiency, and unstable strains, etc., and achieve stable yield, high cost-effective substrate, Easy to Grow Effects

Active Publication Date: 2015-12-30
绵阳晟氏健康科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The first object of the present invention is to provide an Escherichia coli expression plasmid of γ-glutamine synthetase, which has high expression efficiency in Escherichia coli, wherein the γ-glutamine synthetase gene expresses The γ-glutamyl methylamine synthetase itself has a high activity, which can solve the problem of low activity of the existing enzymes used for theanine; and its catalyzed substrates are low-cost glutamic acid and ethylamine , which can solve the problem of high substrate cost in the prior art
[0009] The second object of the present invention is to provide a method for constructing an Esch

Method used

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  • Plasmid for theanine production and construction and application method of corresponding engineering bacteria of plasmid
  • Plasmid for theanine production and construction and application method of corresponding engineering bacteria of plasmid
  • Plasmid for theanine production and construction and application method of corresponding engineering bacteria of plasmid

Examples

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Embodiment 1

[0058] A method for constructing an Escherichia coli expression plasmid of γ-glutamine synthetase, comprising the following steps:

[0059] 1) Optimize the original sequence of the γ-glutamine synthetase gene of GenBank Accession No. AB333782.1 from Methylovorusmays NO.9 for the E. coli expression system, and convert the low-frequency codons in the original sequence to synonymous large intestine Bacillus high-frequency codons, the terminator in the original sequence was replaced with a TAAT strong terminator, and restriction endonuclease sites were added to both ends of the original sequence to obtain an optimized gmas gene sequence;

[0060] 2), using the optimized gmas gene sequence as a template, the synthetic base sequence is the gmas gene fragment shown in SEQ ID NO:1;

[0061] 3), connecting the optimized gmas gene fragment to the prokaryotic expression vector to obtain the Escherichia coli expression plasmid of γ-glutamine synthetase.

Embodiment 2

[0063] A method for constructing an Escherichia coli expression plasmid of γ-glutamine synthetase, comprising the following steps:

[0064] 1) Optimize the original sequence of the γ-glutamine synthetase gene of GenBank Accession No. AB333782.1 from Methylovorusmays NO.9 for the E. coli expression system, and convert the low-frequency codons in the original sequence to synonymous large intestine Bacillus high-frequency codons, the terminator in the original sequence was replaced with a TAAT strong terminator, and restriction endonuclease sites were added to both ends of the original sequence to obtain an optimized gmas gene sequence;

[0065] 2), using the optimized gmas gene sequence as a template, the synthetic base sequence is the gmas gene fragment shown in SEQ ID NO:1;

[0066] 3), connecting the gmas gene fragment to the pUC57 cloning vector to obtain pUC57-gmas;

[0067] Amplify pUC57-gmas;

[0068] The gmas gene fragment was excised from pUC57-gmas, connected to the ...

Embodiment 3

[0070] Transform the plasmid described in Example 2 into the theanine genetically engineered bacteria obtained in the Escherichia coli expression strain Transetta.

[0071] A method for the production of L-theanine by the above-mentioned theanine genetically engineered bacteria, comprising the steps of:

[0072] 1), inoculate the genetically engineered bacteria on the LB solid selection medium containing the antibiotics corresponding to the antibiotics that the engineered bacteria have transferred to, and screen out resistant strains;

[0073] 2), the resistant strain is carried out inductive culture, to induce gmas gene expression; In the described culture process, detect the expression situation of gamma-glutamine synthetase with SDS-PAGE method, pass through low temperature after the detection is qualified centrifuging the fermentation medium to obtain wet thallus;

[0074] 3) Utilize the wet bacterium in the biotransformation reaction system to catalyze the substrate to g...

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Abstract

The invention relates to the field of genetic engineering, in particular to a plasmid for theanine production and a construction and application method of corresponding engineering bacteria of the plasmid. The invention provides an escherichia coli expression plasmid of gamma-glutamyl-methylamine synthetase. A target gene sequence in the plasmid is optimized, so that the expression efficiency in escherichia coli is high; the gamma-glutamyl-methylamine synthetase expressed by a gamma-glutamyl-methylamine synthetase gene is high in activity; and the problem of low activity of an existing enzyme for the theanine can be solved. The catalytic substrate is low-cost glutamic acid and ethylamine, so that the problem of relatively high cost of the substrate in the prior art can be solved. The invention further provides theanine engineering bacteria and a matched culture method thereof. The engineering bacteria are obtained by the escherichia coli expression plasmid of the gamma-glutamyl-methylamine synthetase, can be applied to production of the theanine, and are easy to culture, so that the defects that an existing bacterial strain is not stable enough can be solved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a plasmid for the production of theanine and a method for constructing and using the corresponding engineering bacteria. Background technique [0002] Theanine (L-Theanine) is a unique free amino acid in tea, which belongs to amide compounds, and its scientific name is N-ethyl-γ-L-glutamine (5-N-ethyl-γ-L-glutamine). Theanine is similar in chemical structure to glutamine and glutamic acid, which are the active substances in the brain, and is the main ingredient in tea that promotes body fluids and moistens sweetness. [0003] Theanine is mainly used in the fields of medicine and food. In medicine, theanine has the effect of lowering blood pressure; used together with anticancer drugs, it can enhance the curative effect of antitumor drugs; it has the effect of relaxing nervous tension and anxiety; Learning and memory functions, and can prevent diseases such as Parkinson's dise...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N1/21C12P13/04
Inventor 李晚军王钦芳范明陈纹锐
Owner 绵阳晟氏健康科技有限公司
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