阅读说明：本技术 一种田园除草剂转化为茶氨酸的绿色合成方法 (Green synthesis method for converting field herbicide into theanine ) 是由 李平 冯骏晨 赵美燚 肖淳夫 于 2021-02-19 设计创作，主要内容包括：本发明涉及一种田园除草剂转化为茶氨酸的绿色合成方法,以含有乙胺基团的田园除草剂为底物,经过脱氯反应、脱乙胺反应,生成乙胺；之后在谷氨酰胺合成酶(GS)的作用下,被合成茶氨酸。进而设计出基于莠去津氯水解酶(AztA)、羟基莠去津脱乙胺基团酶(AtzB)、GS组成的茶氨酸新型合成途径。本发明同时还构建了表达AtzA、AtzB的基因工程菌,且该菌内源性高表达GS,能够在降解田园除草剂的同时合成茶氨酸,发酵实验进行84h后可以合成439.8μM的茶氨酸,莠去津转化率达到44.0％。本发明提供的茶氨酸合成新技术在降解环境有害物的同时,有望提高茶树茶氨酸含量,具有良好的应用价值。(The invention relates to a green synthesis method for converting a rural herbicide into theanine, which takes the rural herbicide containing ethylamine as a substrate and generates ethylamine through dechlorination reaction and ethylamine removal reaction; then, theanine is synthesized by Glutamine Synthetase (GS). And then a novel theanine synthesis way based on the composition of atrazine chlorine hydrolase (AztA), hydroxyatrazine deaminase (AtzB) and GS is designed. The invention also constructs the genetic engineering bacteria expressing the AtzA and the AtzB, endogenous high-expression GS of the genetic engineering bacteria can synthesize theanine while degrading the pastoral herbicide, and the theanine with 439.8 mu M can be synthesized after 84 hours of fermentation experiments, and the conversion rate of atrazine reaches 44.0%. The novel theanine synthesis technology provided by the invention is expected to improve the theanine content of tea trees while degrading environmental harmful substances, and has good application value.)

1. A green synthesis method for converting a rural herbicide into theanine is characterized in that the rural herbicide containing ethylamine groups is used as a substrate, and the rural herbicide is subjected to dechlorination reaction and ethylamine removal reaction to generate ethylamine, and then the ethylamine is used for the theanine synthesis reaction.

2. The green synthetic method for converting a herbicide to theanine as claimed in claim 1, wherein the substrate further comprises glutamic acid.

3. The green synthesis method for converting the farmland herbicide into the theanine as claimed in claim 1, wherein the farmland herbicide containing the ethylamine group is a triazine herbicide containing the ethylamine group.

4. The green synthesis method for converting the field herbicide into theanine as claimed in claim 3, wherein the triazine herbicide is atrazine.

5. The green synthesis method for converting the field herbicide into theanine as claimed in claim 3, wherein the triazine herbicide dechlorination reaction is carried out in the presence of Atrazine chlorohydrohydrolase (AtzA).

6. The green synthesis method for converting herbicides into theanine as claimed in claim 3, wherein the dechlorination of triazine herbicide followed by the deethanization reaction is carried out in the presence of Hydroxyatrazine N-ethylaminohydrosilase (AtzB).

7. The green synthesis method for converting the herbicide into theanine as claimed in claim 3, wherein the theanine synthesis reaction is carried out in the presence of Glutamine Synthetase (GS).

8. The green synthesis method for converting the field herbicide into the theanine as claimed in claim 1, which is characterized by comprising the following steps:

taking triazine herbicide and glutamic acid as substrates, and utilizing genetic engineering bacteria of high-expression atrazine chlorine hydrolase (AtzA), hydroxyatrazine deaminase (AtzB) and Glutamine Synthetase (GS) to synthesize the theanine by fermentation.

9. The green synthesis method for converting field herbicides into theanine as claimed in claim 8, wherein the genetically engineered bacteria highly expressing atrazine chloride hydrolase (AtzA), atrazine deaminase (AtzB) and Glutamine Synthetase (GS) are obtained by: introducing one or more of atrazine chlorine hydrolase (AtzA), hydroxyatrazine deaminase (AtzB) or Glutamine Synthetase (GS) into the strain to obtain genetically engineered bacteria;

if the strain expresses one of atrazine chlorohydrase (AtzA), hydroxyatrazine deaminase (AtzB) or Glutamine Synthetase (GS), no additional introduction of such an enzyme is required.

10. The green synthetic method for converting a field herbicide into theanine as claimed in claim 9, wherein the fermentation is: inoculating 2-5% of genetically engineered bacteria with high expression atrazine chlorine hydrolase (AtzA), hydroxyatrazine deaminase (AtzB) and Glutamine Synthetase (GS) into LB culture medium, culturing for 5-7h, adding triazine herbicide with final concentration of 0.5-1mM and glutamic acid with final concentration of 10-25mM, and fermenting for 84 h.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a green synthesis method for converting a field herbicide into theanine.

Background

Theanine is a non-protein amino acid which is characterized by tea trees, has various health care effects of protecting nerves, improving cognition, calming and soothing nerves and the like, is widely applied to food, health care products and pharmaceutical industries, and has larger market demand and wider application prospect.

Chinese patent CN112250592A discloses a method for extracting theanine from bamboo leaf green tea dust, which comprises the following steps: 1) pulverizing tea dust, sieving, adding distilled water, and heating in water bath for leaching; 2) ultrafiltering the tea soup with ultrafiltration centrifuge tube to concentrate; 3) after ultrafiltration, adsorbing the solution by using resin, and adding ethanol for alcohol precipitation; 4) precipitating the tea soup with ethanol, and separating theanine by dynamic column chromatography. The obtained theanine crude product does not contain tea polyphenol, caffeine, tea polysaccharide and other impurities, the extraction rate of the theanine crude product is 88%, and the purity of the L-theanine content in the theanine crude product is 91.23%.

Chinese patent CN109777763B discloses a novel efficient gamma-glutamyl methylamine synthetase, a genetically engineered bacterium for producing L-theanine, and a construction method and application thereof. The patent provides a gene engineering bacterium which is plasmid-free and takes cheap carbon sources such as glucose and the like as substrates to synthesize L-theanine, and the gene engineering bacterium takes escherichia coli as a host and integrates three copies of gamma-glutamyl methylamine synthetase gene gmas-Mu on the genome; a single copy of the glutamate dehydrogenase gene Cgl 2079; single copy pyruvate carboxylase gene Cgl 0689; a single copy of the citrate synthase gene gltA. After the system metabolism is modified, the engineering bacteria can synthesize the L-theanine by taking glucose and ethylamine as raw materials, the highest yield of the L-theanine in fermentation of a 5L fermentation tank can reach 60g/L, and the sugar-acid conversion rate can reach 40%.

Chinese patent CN111073830A discloses a Lactobacillus casei TH139 with high yield of gamma-glutamyltranspeptidase, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 18686; the strain is used for producing gamma-glutamyl transpeptidase by fermentation at 37 ℃ and pH7.0; after centrifugal collection of the somatic cells, biologically converting L-glutamine and ethylamine at 40 ℃ and pH9.0 to obtain L-theanine; and (3) carrying out membrane separation, ion exchange resin separation and concentration crystallization on the reaction solution to obtain a qualified L-theanine finished product.

As described above, the conventional theanine production methods mainly include a tea extraction method and a fermentation method, and thus there is still room for improvement in terms of cost and yield.

Disclosure of Invention

The invention aims to provide a green synthetic method for converting a field herbicide into theanine. The method provided by the invention is different from the traditional tea extraction method or fermentation method, takes a rural herbicide (such as triazine herbicide) as a theanine synthesis substrate, avoids the toxicity of exogenously added substrate ethylamine to microorganisms, synthesizes theanine while degrading environmental harmful substances, and is a brand-new method.

The invention introduces synthetic biology thinking, searches and predicts a potential theanine metabolic pathway by using a bioinformatics method, is not limited to tea tree sources, and designs a new theanine synthesis pathway consisting of AtzA, AtzB and GS. And a genetic engineering strain is constructed on the basis, the genetic engineering strain can be used for synthesizing theanine while degrading triazine herbicides (especially atrazine), the fermentation experiment can be carried out for 84h, and then the theanine with the purity of 439.8 mu M can be synthesized, and the conversion rate of the atrazine reaches 44.0%. The technical scheme of the invention breaks through the in-situ synthesis path of tea trees and establishes a new theanine biosynthesis technology.

The purpose of the invention can be realized by the following technical scheme:

in the first aspect of the invention, a green synthesis method for converting a rural herbicide into theanine is provided, wherein the rural herbicide (such as triazine herbicide) containing an ethylamine group is used as a substrate, and the rural herbicide is subjected to dechlorination and deethylation to generate ethylamine; then, theanine is synthesized by the action of glutamine synthetase.

In one embodiment of the invention, the substrate further comprises glutamic acid.

In one embodiment of the present invention, the herbicide containing an ethylamine group is a triazine herbicide containing an ethylamine group, preferably atrazine.

In one embodiment of the present invention, the dechlorination of the triazine herbicide is desirably carried out in the presence of atrazine chlorohydrolase (AtzA).

In one embodiment of the invention, the dechlorination of the triazine herbicide followed by the deethanization reaction is carried out in the presence of the hydroxyatrazine deethanase (AtzB).

In one embodiment of the invention, theanine synthesis from ethylamine is carried out under the action of glutamine synthetase.

In one embodiment of the invention, the green synthesis method for converting the pastoral herbicide into theanine comprises the following specific processes: taking triazine herbicide and glutamic acid as substrates, and utilizing genetic engineering bacteria of high-expression atrazine chlorine hydrolase (AtzA), hydroxyatrazine deaminase (AtzB) and Glutamine Synthetase (GS) to synthesize the theanine by fermentation.

In one embodiment of the invention, the genetically engineered bacteria highly expressing atrazine chloride hydrolase (AtzA), atrazine hydroxyethylamine transferase (AtzB), Glutamine Synthetase (GS) are obtained by: introducing one or more of atrazine chlorine hydrolase (AtzA), hydroxyatrazine deaminase (AtzB) or Glutamine Synthetase (GS) into the strain to obtain genetically engineered bacteria;

if the strain expresses one of atrazine chlorohydrase (AtzA), hydroxyatrazine deaminase (AtzB) or Glutamine Synthetase (GS), no additional introduction of such an enzyme is required.

The strain is preferably tea tree endophyte with high theanine yield.

In one embodiment of the present invention, the fermentation refers to: inoculating 2-5% of genetically engineered bacteria of endogenous highly expressed atrazine chlorine hydrolase (AtzA), hydroxyatrazine deaminase (AtzB) and Glutamine Synthetase (GS) into LB culture medium, culturing for 5-7h, adding triazine herbicide with final concentration of 0.5-1mM and glutamic acid with final concentration of 10-25mM, and fermenting for 84 h.

In one embodiment of the present invention, a specific process of a method for synthesizing theanine is provided, which comprises:

1) using a donated pseudomonas klebsiella (p.knackmussii, a plant-derived endophytic bacterium isolated from rice roots, strain accession No. ACCC 05571; laboratory detection shows that endogenous high-expression GS has theanine synthesis capacity with ethylamine as a substrate) to construct a CFPS system (PkCFPS), verify a novel theanine synthesis way and detect the theanine synthesis capacity;

2) the method comprises the steps of introducing atrazine chlorine hydrolase (AtzA) and hydroxyatrazine deaminase (AtzB) into theanine-producing endophyte P.knackmussi, enabling the strain to endogenously and highly express Glutamine Synthetase (GS), and using atrazine and glutamic acid as substrates to synthesize theanine through fermentation experiments by the constructed genetic engineering bacteria to establish a novel theanine synthesis technology.

In one embodiment of the present invention, in step 1), the present invention performs a validation experiment in a cell-free protein synthesis system, specifically: atrazine chlorine hydrolase (AtzA), atrazine deaminase (AtzB) and Glutamine Synthetase (GS) are respectively expressed in a CFPS system, 10 mu L of each of three PkCFPS systems are mixed after protein expression is carried out for 4-8h, atrazine (5% acetone for assisting dissolution) with final concentration of 0.5-1mM and glutamic acid with final concentration of 10-25mM are added, water bath reaction is carried out at 30 ℃, samples are taken at different time points, and the synthesis amount of theanine in reaction liquid is detected.

In one embodiment of the present invention, in step 1), the present invention performs a validation experiment in a cell-free protein synthesis system, specifically: respectively expressing atrazine chlorine hydrolase (AtzA), hydroxyatrazine deaminase (AtzB) and Glutamine Synthetase (GS) in a PkCFPS system, after protein expression is carried out for 4-8h, respectively mixing 10 mu L of the three PkCFPS systems, adding atrazine (5% acetone for dissolution) with final concentration of 0.5-1mM and glutamic acid with final concentration of 10-25mM, carrying out water bath reaction at 30 ℃, sampling at different time points, and detecting the synthesis amount of theanine in a reaction solution.

In one embodiment of the present invention, in step 1), the PkCFPS system comprises: 12mM magnesium acetate, 10mM ammonium glutamate, 130mM potassium glutamate, 1.2mM ATP, 0.85mM GTP, 0.85mM UTP, 0.85mM CTP, 34. mu.g/mL folinic acid, 170. mu.g/mL E.coli-derived tRNA solution, 2mM each of 20 standard amino acids, 33mM PEP, 200ng expression plasmid, 4. mu.L P.knackmussi-derived Extract. Then, pJL1-AtzA, pJL1-AtzB and pJL1-GS expression plasmids are used for respectively expressing three enzymes AtzA, AtzB and GS in a PkCFPS system, 10 mu L of each of the three PkCFPS systems are mixed after 4-8h of protein expression, atrazine (5% acetone is used as a cosolvent of atrazine) with the final concentration of 0.5-1mM and glutamic acid with the final concentration of 10-25mM are added, water bath reaction is carried out at 30 ℃ for 24h, and samples are taken at different time points for detecting the synthesis amount of theanine.

In one embodiment of the invention, the constructed genetically engineered bacterium in step 2) is named as p.knackmussi-AtzAB, and can express exogenously introduced AtzA and AtzB and endogenously contains highly expressed Glutamine Synthetase (GS). The AtzA, AtzB genes were inserted into the uracil phosphoribosyl transferase gene (upp) site in the p. knackmussi genome using pJQ200SK mediated gene insertion system, and the Open Reading Frame (ORF) of the co-transcribed AtzAB gene was regulated by the promoter of the housekeeping gene rpsL, which is a constitutively expressing promoter.

In one embodiment of the present invention, in step 2), the fermentation experiment refers to: inoculating the genetic engineering strain into LB culture medium at a ratio of 2-5%, and culturing for 5-7h (OD)₆₀₀2.0), atrazine (5% acetone solubilizing) and glutamic acid were added to a final concentration of 0.5-1mM, fermented for 84h, and sampled at different time points to detect the amount of theanine synthesized in the fermentation broth.

The invention introduces a synthetic biology concept, breaks through the limit of a theanine in-situ synthesis path, designs a novel theanine synthesis path and further develops a novel theanine synthesis technology.

Synthetic biology refers to the design and construction of biological elements, devices and systems, and the purposeful redesign of existing natural biological systems, widely used in the fields of chemical synthesis, medicine, agriculture, the environment, etc.

The invention also provides a genetically engineered bacterium, which highly expresses atrazine chlorine hydrolase (AtzA), atrazine hydroxyethylamine dehydrogenase (AtzB) and Glutamine Synthetase (GS).

The genetic engineering bacteria can synthesize theanine by fermentation by taking a rural herbicide containing an ethylamine group and glutamic acid as substrates.

In one embodiment of the invention, the genetically engineered bacterium is the above-mentioned p.

The invention designs a theanine biosynthesis pathway which couples degradation of a rural herbicide containing an ethylamine group and theanine biosynthesis. The residue of the herbicide containing the ethylamine group in the field is harmful to animals and plants, but still has larger market volume and stable market demand in view of the advantages of price, effect and the like. Therefore, a theanine biosynthesis way for coupling degradation of the herbicide containing the ethylamine group in the field is constructed, and the method has certain environmental protection significance.

The invention verifies the novel theanine synthesis way through a classical method for constructing genetic engineering bacteria and creatively in a CFPS system, thereby establishing a novel theanine synthesis technology. CFPS is an in vitro system for synthesizing proteins by supplementing substrates and energy to an enzyme system of a cell extract using exogenous mRNA or DNA as a template, and can directly detect biological functions by adding a target gene, substrate, and the like to a reaction environment without any trouble. Since the growth and division of the bacterial cells are not involved, CFPS shows good tolerance to toxic substrates, toxic products and the like, and the energy in the system is supplied more intensively to the transcription and translation of the target protein. Based on the above advantages, CFPS has become an important platform in synthetic biology in recent years, and has become more and more widely used in research such as screening biological elements, designing gene circuits, constructing biosynthetic pathways, and the like.

Compared with the prior art, the invention has the beneficial effects that:

the invention introduces synthetic biology thinking and designs a new theanine synthesis way consisting of AtzA, AtzB and GS. The gene engineering strain constructed on the basis can synthesize theanine while degrading the herbicide containing the ethylamine group in a field and garden, and the theanine with the concentration of 439.8 mu M can be synthesized after a fermentation experiment is carried out for 84 hours, and the conversion rate of atrazine reaches 44.0%. The method breaks through the in-situ synthesis pathway of tea trees and establishes a new theanine biosynthesis technology. The novel theanine synthesis technology provided by the invention is expected to improve the theanine content of tea trees while degrading environmental harmful substances, and has good application value.

Drawings

FIG. 1 is a schematic diagram of a potential theanine synthesis pathway obtained by using bioinformatic analysis means in example 1 of the present invention. Cxxxxx numbering in the figure is the material numbering in the KEGG database; the solid arrows represent the known enzymes corresponding to the reaction, and since most of the reactions in the KEGG database are labeled as reversible reactions, i.e., reactions catalyzed by the same enzyme or two enzymes each catalyzing one direction, the two-way arrows are shown in the figure; the dotted line indicates that the reaction has been confirmed, but the corresponding enzyme has not been identified.

Fig. 2 is a schematic diagram of a pathway for degrading atrazine by the atrazine degradation mode bacterium Pseudomonas sp. The degradation pathway mainly comprises the steps of dechlorination, ethylamine removal, isopropylamine removal, ring opening reaction of s-triazine mother nucleus and the like.

FIG. 3 is a schematic diagram showing the comparison between the predicted results of the software and the new theanine synthesis pathway designed after the atrazine degradation pathway in example 1 of the present invention.

FIG. 4 is a schematic diagram showing the validation of a novel pathway for theanine synthesis in the PkCFPS system in example 2 of the present invention.

FIG. 5 is a diagram showing the verification of the novel theanine synthesis pathway in the PkCFPS system in example 2 of the present invention. Wherein, A is the expression of AtzA, AtzB and GS in the PkCFPS system. Panel B is a reaction time-theanine production curve in the PkCFPS system.

FIG. 6 is a schematic diagram of a method for constructing a genetically engineered bacterium P.knackmussii-AtzAB in example 3 of the present invention.

FIG. 7 is a method of detecting the ORF of AtzA and AtzB at the genome level (panel A) and the mRNA level (panel B), respectively, using PCR and reverse transcription-PCR in example 3 of the present invention.

FIG. 8 is a time-yield curve of theanine production by fermentation of engineering bacteria P.knackmussii-AtzAB in example 4 of the present invention. Wherein the substrates of the fermentation experiment are atrazine and glutamic acid.

Detailed Description

In one embodiment of the present invention, a novel technology for synthesizing theanine is provided, which comprises the following steps:

(1) using a bioinformatics approach to search potential theanine metabolic pathways and designing a novel theanine synthesis pathway;

(2) constructing and verifying a novel theanine synthesis way and establishing a novel theanine synthesis technology.

In the step (2), a novel theanine synthesis way is constructed and verified, and the process for establishing a novel theanine synthesis technology is as follows: and comparing the predicted potential theanine synthesizing path with the atrazine degrading path of the known model bacteria, and obtaining that a new theanine synthesizing path can be constructed in the pseudomonas according to the comparison result. The synthetic pathway finally designed was via 3 enzymes: AtzA, AtzB, GS, effecting biosynthesis from atrazine → hydroxyatrazine → ethylamine → theanine.

1) A CFPS system (PkCFPS) based on theanine-producing endophyte P.knackmussi is established, and a novel theanine synthesis path is constructed and verified. Wherein the PkCFPS system comprises: 12mM magnesium acetate, 10mM ammonium glutamate, 130mM potassium glutamate, 1.2mM ATP, 0.85mM GTP, 0.85mM UTP, 0.85mM CTP, 34. mu.g/mL folinic acid, 170. mu.g/mL E.coli-derived tRNA solution, 2mM each of 20 standard amino acids, 33mM PEP, 200ng expression plasmid, 4. mu.L P.knackmussi-derived Extract. Then, pJL1-AtzA, pJL1-AtzB and pJL1-GS expression plasmids are used for respectively expressing three enzymes AtzA, AtzB and GS in a PkCFPS system, 10 mu L of each of the three PkCFPS systems are mixed after 4-8h of protein expression, atrazine (5% acetone is used as a cosolvent of atrazine) with the final concentration of 0.5-1mM and glutamic acid with the final concentration of 10-25mM are added, water bath reaction is carried out at 30 ℃ for 24h, and samples are taken at different time points for detecting the synthesis amount of theanine.

2) A new technology for realizing theanine biosynthesis by constructing a genetic engineering bacterium P.knackmussii-AtzAB. The engineering bacteria can express exogenously introduced AtzA and AtzB and endogenously contain high-expression Glutamine Synthetase (GS). Uracil phosphoribosyltransferase Gene having AtzA and AtzB genes inserted into P.knackmussii genome Using pJQ200 SK-mediated Gene insertion System(upp) site, Open Reading Frame (ORF) of co-transcribed AtzAB gene is regulated and controlled by promoter of housekeeping gene rpsL, the promoter is constitutive expression promoter, and the constructed gene engineering strain is named as P.knackmussii-AtzAB. Using the PCR and reverse transcription-PCR methods, ORFs of AtzA and AtzB were detected at the genome level and mRNA level, respectively, confirming that AtzA and AtzB were successfully knocked into the p. Inoculating P.knackmussii-AtzAB strain at a ratio of 2-5% to LB medium, and culturing for about 5-7h (OD)₆₀₀2.0), atrazine (5% acetone co-solubilisation) was added to a final concentration of 0.5-1mM and glutamic acid 10-25mM, fermented for 84h, sampled at different time points and tested for theanine concentration.

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1

Software is used for predicting a potential synthesis path of theanine and designing a new path for theanine synthesis.

The potential theanine metabolic pathway is searched by using bioinformatics analysis means, the prediction result is shown in figure 1, and the analysis of the prediction result shows that: 1) ethylamine may be formed from the deethylamine group of N-ethylmelamine, but the enzyme required for the reaction has not been identified; 2) ethylamine may be generated by deethylamine group of N-ethylglycine, and the enzyme involved in the reaction is known, but the content of N-ethylglycine in organisms and environment is extremely low; 3) ethylamine may be generated from a hydroxyatrazine deamination group, hydroxyatrazine is dechlorinated by Atrazine (also called Atrazine), Atrazine is a widely used herbicide, has certain biological hazard and has a soil residue problem, so that Atrazine is used as a potential substrate for theanine synthesis.

Comparing the degradation pathway of atrazine degradation mode strain Pseudomonas sp.strain ADP (fig. 2) with the predicted potential pathway for theanine synthesis (fig. 1), the designed synthetic pathway is via 3 enzymes: AtzA, AtzB, GS, effecting biosynthesis from atrazine → hydroxyatrazine → ethylamine → theanine (fig. 3).

Example 2

A CFPS (PkCFPS) system based on theanine-producing endophyte P.knackmussi (separated from the root of rice and with the strain preservation number of ACCC05571) is established, and a novel theanine synthesis path is established and verified.

Prepare Extract from p.knackmussii, then prepare a standard 15 μ L PkCFPS system in a 1.5mL centrifuge tube, the reaction system contains: 12mM magnesium acetate, 10mM ammonium glutamate, 130mM potassium glutamate, 1.2mM ATP, 0.85mM GTP, 0.85mM UTP, 0.85mM CTP, 34. mu.g/mL folinic acid, 170. mu.g/mL E.coli-derived tRNA solution, 2mM each of 20 standard amino acids, 33mM PEP, 200ng expression plasmid, 4. mu.L P.knackmussii-based Extract.

Expression plasmids pJL1-AtzA, pJL1-AtzB and pJL1-GS were constructed, and the verification concept is shown in FIG. 4. PkCFPS systems containing pJL1-AtzA, pJL1-AtzB and pJL1-GS plasmids are respectively prepared and reacted for 4 hours under the condition of water bath at 30 ℃ to synthesize protein, and FIG. 5A shows the expression conditions of AtzA, AtzB and GS in the PkCFPS systems respectively. Then, 10. mu.L of each of the 3 PkCFPS reaction solutions was mixed, 1mM atrazine (5% acetone solubilizing agent) and 25mM glutamic acid were added as substrates to a final volume of 50. mu.L, and the reaction was continued for 24 hours in a water bath at 30 ℃ to synthesize theanine. Samples were taken at different time points and 3 volumes of ethanol were added, followed by centrifugation at 12000rpm for 30min, and the supernatant was aspirated as a sample for examination and a time-theanine production curve was plotted (FIG. 5B).

Example 3

And constructing a genetic engineering bacterium P.knackmussii-AtzAB for expressing AtzA and AtzB.

Comparing P.knackmussii genome data (GenBank: HG322950.1) on Genbank, finding that the strain does not contain AtzA and AtzB genes, and selecting genes to synthesize AtzA and AtzB for constructing metabolic pathways; the conversion of ethylamine into theanine can be completed by endogenous high-expression GS in P.knackmussii.

The gene insertion system of pJQ200SK is used to insert the AtzAB gene into the uracil phosphoribosyl transferase gene (upp) site in the P.knackmussi genome, the co-transcribed AtzAB ORF is regulated by the promoter of rpsL gene, the constructed strain is named as P.knackmussi-AtzAB, and the construction process of the genetic engineering strain is shown in FIG. 6. Using the PCR and reverse transcription-PCR methods, the ORFs of AtzA and AtzB were detected at the genomic level (fig. 7A) and mRNA level (fig. 7B), respectively, confirming that AtzA and AtzB were successfully knocked into the p.

Example 4

A fermentation experiment for synthesizing theanine while degrading atrazine was performed using the genetically engineered bacterium constructed in example 3.

The P.knackmussii-AtzAB strain was inoculated at a ratio of 5% to LB medium and cultured for about 7h (OD)₆₀₀2.0), adding atrazine (5% acetone for solubilization) with the final concentration of 1mM and glutamic acid with the final concentration of 25mM, fermenting for 84h, sampling and detecting the concentration of theanine; meanwhile, different time points are set in the process, and sampling and detection are carried out. The detection result shows that P.knackmussi-AtzAB can synthesize theanine with the concentration of about 439.8 mu M in the culture solution after 84h of fermentation, and the conversion rate of atrazine reaches 44.0%. Meanwhile, the speed of degrading atrazine and synthesizing theanine by P.knackmussii is higher, the yield of the theanine reaches 398.4 mu M after 45 hours of adding the substrate, and the reaction is almost completely carried out (figure 8).

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.