CN112899319B

CN112899319B - Green synthesis method for converting field herbicide into theanine

Info

Publication number: CN112899319B
Application number: CN202110188799.1A
Authority: CN
Inventors: 李平; 冯骏晨; 赵美燚; 肖淳夫
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2021-02-19
Filing date: 2021-02-19
Publication date: 2023-07-04
Anticipated expiration: 2041-02-19
Also published as: CN112899319A

Abstract

The invention relates to a green synthesis method for converting a field herbicide into theanine, which takes the field herbicide containing an ethylamine group as a substrate, and generates ethylamine through dechlorination reaction and de-ethylamine reaction; then, theanine is synthesized by Glutamine Synthetase (GS). Further, a novel theanine synthesis pathway based on atrazine chlorohydrolase (AztA), hydroxy atrazine desethylamino enzyme (AtzB) and GS is designed. The invention also constructs the genetic engineering bacteria for expressing AtzA and AtzB, and the bacteria endogenously expresses GS, can synthesize theanine while degrading the field herbicide, and can synthesize 439.8 mu M theanine after 84 hours of fermentation experiment, and the atrazine conversion rate 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.

Description

Green synthesis method for converting field herbicide into theanine

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 with tea tree characteristics, has various health care effects of protecting nerves, improving cognition, calming nerves and the like, is widely applied to the industries of foods, health care products and medicines, and has larger market demands and wider application prospects.

Chinese patent CN112250592a discloses a method for extracting theanine from green tea dust, comprising the steps of: 1) Pulverizing tea dust, sieving, adding distilled water, heating in water bath, and leaching; 2) Carrying out ultrafiltration concentration on the tea soup by using an ultrafiltration centrifuge tube; 3) After ultrafiltration, adsorbing the solution by using resin, and adding ethanol for ethanol precipitation; 4) Separating theanine from the ethanol-precipitated tea decoction by dynamic column chromatography. The obtained crude theanine product contains no impurity such as tea polyphenols, caffeine, tea polysaccharide, etc., the extraction rate of the crude theanine product is 88%, and the purity of L-theanine in the crude theanine product is 91.23%.

Chinese patent CN109777763B discloses a novel high-efficiency gamma-glutamylmethylamine synthetase, a genetically engineered bacterium for producing L-theanine, its construction method and application. The patent provides a genetically engineered bacterium which is plasmid-free and takes cheap carbon sources such as glucose and the like as substrates to synthesize L-theanine, and takes escherichia coli as a host, and three copies of gamma-glutamylmethylamine synthetase gene gmas-Mu are integrated on the genome of the genetically engineered bacterium; a single copy of the glutamate dehydrogenase gene Cgl2079; a single copy of the pyruvate carboxylase gene Cgl0689; a single copy of the citrate synthase gene gltA was obtained. After the system metabolism is improved, the engineering bacteria can synthesize the L-theanine by taking glucose and ethylamine as raw materials, the highest yield of the L-theanine in the 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 Lactobacillus casei TH139 with high yield of gamma-glutamyl transpeptidase, which is preserved in the China general microbiological culture collection center with a preservation number of CGMCC No.18686; fermenting and producing gamma-glutamyl transpeptidase by using the strain at the temperature of 37 ℃ and the pH of 7.0; after centrifugally collecting bacterial cells, biologically converting L-glutamine and ethylamine at 40 ℃ and pH9.0 to obtain L-theanine; the reaction liquid is subjected to membrane separation, ion exchange resin separation, concentration and crystallization to obtain qualified L-theanine finished products.

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

Disclosure of Invention

The invention aims to provide a green synthesis 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, but takes the field herbicide (such as triazine herbicide) as a theanine synthesis substrate, avoids the toxicity of exogenous addition substrate ethylamine to microorganisms, synthesizes theanine while degrading environmental harmful substances, and is a brand-new method.

The invention introduces synthetic biological thinking, searches and predicts potential theanine metabolic pathways by using a bioinformatics method, is not limited to tea tree sources, and designs a novel theanine synthesis pathway consisting of AtzA, atzB, GS. And a genetic engineering strain is constructed based on the method, the genetic engineering strain can degrade triazine herbicide (especially atrazine) and synthesize theanine, and after 84 hours of fermentation experiment, 439.8 mu M theanine can be synthesized, and the atrazine conversion rate reaches 44.0%. The technical scheme breaks through the in-situ synthesis path of the tea tree and establishes a new theanine biosynthesis technology.

The aim of the invention can be achieved by the following technical scheme:

in a first aspect of the present invention, there is provided a green synthesis method for converting a field herbicide into theanine, wherein the field herbicide containing an ethylamino group (e.g., triazine herbicide) is used as a substrate, and the field herbicide undergoes a dechlorination reaction and a deethylamine reaction to produce ethylamine; then, theanine is synthesized under the action of glutamine synthetase.

In one embodiment of the invention, glutamic acid is also included in the substrate.

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

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

In one embodiment of the present invention, the deethylamine reaction of the triazine herbicide after dechlorination is carried out in the presence of a hydroxyatrazine deethylamine group enzyme (AtzB).

In one embodiment of the invention, the synthesis of theanine from ethylamine is performed by a glutamine synthetase.

In one embodiment of the invention, the green synthesis method for converting the field herbicide into theanine comprises the following specific processes: the method is characterized in that triazine herbicide and glutamic acid are used as substrates, and genetically engineered bacteria with high expression of atrazine chlorine hydrolase (AtzA), hydroxy atrazine de-ethylamino group enzyme (AtzB) and Glutamine Synthetase (GS) are utilized to synthesize theanine through fermentation.

In one embodiment of the invention, the genetically engineered bacteria highly expressing atrazine chlorine hydrolase (AtzA), hydroxyatrazine desethylamino enzyme (AtzB), glutamine Synthetase (GS) are obtained by: introducing one or more of atrazine chlorine hydrolase (AtzA), hydroxy atrazine de-ethylamino enzyme (AtzB) or Glutamine Synthetase (GS) into the strain to obtain genetically engineered bacteria;

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

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

In one embodiment of the invention, the fermentation refers to: inoculating 2-5% of endogenous high-expression atrazine chlorine hydrolase (AtzA), hydroxyl atrazine de-ethylamino enzyme (AtzB) and Glutamine Synthetase (GS) gene engineering bacteria into LB culture medium, culturing for 5-7h, adding 0.5-1mM triazine herbicide and 10-25mM glutamic acid, and fermenting for 84h.

In one embodiment of the present invention, a specific process of a theanine synthesis method is provided, comprising:

1) Using the donated pseudomonas kluyveromyces (p.knac kmussii, plant-derived endophytic bacteria isolated from rice roots, accession number ACCC 05571; laboratory detection shows that the strain has endogenous high expression GS and theanine synthesis capability by taking ethylamine as a substrate, a CFPS system (PkCFPS) is constructed, a novel theanine synthesis path is verified, and the theanine synthesis capability is detected;

2) The atrazine chlorine hydrolase (AtzA) and the hydroxy atrazine de-ethanamine group enzyme (AtzB) are introduced into the theanine-producing endophyte P.knac kmussii, the strain endogenously expresses Glutamine Synthetase (GS), the constructed genetic engineering bacteria take atrazine and glutamic acid as substrates, and theanine is synthesized through fermentation experiments, so that a novel theanine synthesis technology is established.

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

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

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 source tRNA solution, 2mM for each of 20 standard amino acids, 33mM PEP, 200ng expression plasmid, 4. Mu.L P.knackmussii source Extract. Then, pJL1-AtzA, pJL1-AtzB and pJL1-GS expression plasmids are used for respectively expressing three enzymes of AtzA, atzB and GS in a PkCFPS system, 10 mu L of each of the three PkCFPS systems is taken and mixed after protein expression is carried out for 4-8 hours, 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 for 24 hours at 30 ℃, and samples are taken at different time points to detect the synthesis amount of theanine.

In one embodiment of the invention, in step 2), the constructed genetically engineered bacterium is designated as P.knac kmussii-AtzAB, which is capable of expressing exogenously introduced AtzA, atzB and endogenously contains highly expressed Glutamine Synthetase (GS). The AtzA, atzB genes were inserted into uracil phosphoribosyl transferase (upp) sites in the p.knackmusssii genome using the pJQ200SK mediated gene insertion system, the Open Reading Frame (ORF) of the co-transcribed AtzAB gene being regulated by the promoter of the housekeeping gene rpsL, which is a constitutive expression promoter.

In one embodiment of the invention, in step 2), the fermentation experimentIs as follows: inoculating the genetically engineered strain into LB medium at a ratio of 2-5%, culturing for 5-7 hr (OD ₆₀₀ =2.0), atrazine (5% acetone-assisted) and glutamic acid were added at a final concentration of 0.5-1mM, fermented for 84h, and sampled at various time points to detect theanine synthesis amount in the fermented liquid.

The invention introduces the synthetic biology concept, breaks through the limitation of the in-situ synthesis path of theanine, 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 biological systems in existing nature, widely used in the fields of chemical synthesis, medicine, agriculture, environment, etc.

The invention also provides a genetic engineering bacterium which expresses atrazine chlorine hydrolase (AtzA), hydroxy atrazine de-ethylamino group enzyme (AtzB) and Glutamine Synthetase (GS) in a high degree.

The genetically engineered bacterium can be used for synthesizing theanine by taking a field herbicide containing an ethylamine group and glutamic acid as substrates through fermentation.

In one embodiment of the invention, the genetically engineered bacterium is the above-described P.knac kmussii-AtzAB.

The invention designs a theanine biosynthesis pathway which couples degradation of a field herbicide containing an ethylamino group with biosynthesis of theanine. The residue of the field herbicide containing the ethylamine group 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, the construction of the theanine biosynthesis pathway coupled with the degradation of the field herbicide containing the ethylamino group has certain environmental protection significance.

The invention verifies the novel synthesis path of the theanine in a CFPS system by a classical method for constructing genetically engineered bacteria and creatively establishes a novel theanine synthesis technology. CFPS is an in vitro system for synthesizing proteins by supplementing substrates and energy into the enzyme system of cell extracts using exogenous mRNA or DNA as templates, and can directly detect the biological functions of target genes, substrates, etc. without any obstacle by adding them to the reaction environment. CFPS, because it does not involve cell growth and division, exhibits good tolerance to toxic substrates, toxic products, etc., and more concentrated energy supply within the system and transcription and translation of the protein of interest. Based on the above advantages, CFPS has become an important platform for synthetic biology in recent years, and has been widely used in studies such as screening biological elements, designing genetic circuits, constructing biosynthetic pathways, and the like.

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

the invention introduces the thinking of synthetic biology and designs a new way for synthesizing theanine consisting of AtzA, atzB, GS. The genetically engineered strain constructed based on the method can degrade the field herbicide containing the ethylamine group and synthesize theanine at the same time, and after 84 hours of fermentation experiment, 439.8 mu M theanine can be synthesized, and the atrazine conversion rate reaches 44.0%. The patent breaks through the in-situ synthesis path of tea trees and establishes a new technology for theanine biosynthesis. 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 shows the potential theanine synthesis pathway obtained using bioinformatic analysis means in example 1 of the present invention. Cxxxxx numbering in the figures is the material number in the KEGG database; the solid arrows represent enzymes for which the reaction is known, and the two-way arrows are shown in the figure because most of the reactions in the KEGG database are labeled as reversible reactions, i.e., reactions catalyzed by the same enzyme or by both enzymes in one direction; the dashed line represents that the reaction has been confirmed, but the corresponding enzyme has not yet been identified.

FIG. 2 is a schematic diagram showing the path of atrazine degradation by the atrazine degradation model bacterium Pseudomonas sp.strain ADP in example 1 of the present invention. The degradation path mainly comprises the steps of dechlorination, de-ethylamino group, de-isopropylamine group, ring-opening reaction of s-triazine mother nucleus and the like.

FIG. 3 is a schematic diagram showing the novel approach for theanine synthesis designed after comparing the software predicted result with atrazine degradation pathway in example 1 of the present invention.

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

FIG. 5 is a novel pathway for the validation of theanine synthesis in the PkCFPS system in example 2 of the present invention. Wherein, A is the expression of AtzA, atzB and GS in a PkCFPS system. Panel B shows the reaction time versus theanine yield curve in the PkCFPS system.

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

FIG. 7 shows the detection of the ORF of AtzA and AtzB at the genomic level (panel A) and mRNA level (panel B), respectively, using PCR and reverse transcription-PCR in example 3 of the present invention.

FIG. 8 is a graph showing the time-yield of theanine produced by fermentation of genetically engineered bacteria, engineering bacteria P.knackmusssii-AtzAB in example 4 of the present invention. Wherein the substrates for fermentation experiments are atrazine and glutamic acid.

Detailed Description

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

(1) Searching potential theanine metabolic pathways by using bioinformatics means, and designing a novel theanine synthesis pathway;

(2) A novel theanine synthesis way is constructed and verified, and a novel theanine synthesis technology is established.

In the step (2), a novel theanine synthesis path is constructed and verified, and the process for establishing a novel theanine synthesis technology comprises the following steps: the predicted potential pathway of theanine synthesis is compared with atrazine degradation pathways of known mode bacteria, and a new pathway of theanine synthesis can be constructed in pseudomonas according to the comparison result. The final designed synthetic pathway is through 3 enzymes: atzA, atzB, GS, the biosynthesis from atrazine, hydroxy atrazine, ethylamine and theanine is realized.

1) A CFPS system (PkCFPS) based on the theanine-producing endophyte P.knakmussii was established, and a novel theanine synthesis pathway was 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 source tRNA solution, 2mM for each of 20 standard amino acids, 33mM PEP, 200ng expression plasmid, 4. Mu.L P.knackmussii source Extract. Then, pJL1-AtzA, pJL1-AtzB and pJL1-GS expression plasmids are used for respectively expressing three enzymes of AtzA, atzB and GS in a PkCFPS system, 10 mu L of each of the three PkCFPS systems is taken and mixed after protein expression is carried out for 4-8 hours, 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 for 24 hours at 30 ℃, and samples are taken at different time points to detect the synthesis amount of theanine.

2) Constructing genetic engineering bacteria P.knac kmussii-AtzAB, and realizing a novel technology of theanine biosynthesis. The engineering bacteria can express exogenously introduced AtzA and AtzB and endogenously contain high-expression Glutamine Synthetase (GS). The AtzA, atzB genes were inserted into uracil phosphoribosyl transferase gene (upp) sites in the p.knackmusssii genome using pJQ SK-mediated gene insertion system, the Open Reading Frame (ORF) of the co-transcribed AtzAB gene was regulated by the promoter of the housekeeping gene rpsL, which is a constitutive expression promoter, and the constructed genetically engineered strain was named p.knackmusssii-AtzAB. The ORFs of AtzA and AtzB were detected at the genomic and mRNA levels, respectively, using PCR and reverse transcription-PCR methods, confirming that AtzA and AtzB were successfully knocked into the p.knac kmussii genome and successfully completed expression. The P.knackmussii-AtzAB strain was inoculated at a ratio of 2-5% into LB medium and cultured for about 5-7 hours (OD ₆₀₀ =2.0), atrazine (5% acetone-co-solubilised) at a final concentration of 0.5-1mM and glutamic acid at 10-25mM were added, fermented for 84h, sampled at different time points and assayed for theanine concentration.

The invention will now be described in detail with reference to the drawings and specific examples.

Example 1

The software is used for predicting the potential synthesis path of theanine and designing a new path for synthesizing theanine.

Searching for potential theanine metabolic pathways using bioinformatic analysis means, the predicted results are shown in fig. 1, and analysis of the predicted results can be seen: 1) Ethylamine may be formed by deamination of N-ethylmelamine, but the enzyme required for the reaction has not been identified; 2) Ethylamine may be formed by de-ethylamino of N-ethylglycine, an enzyme involved in this reaction is known, but N-ethylglycine is present in very low levels in organisms and in the environment; 3) The ethylamine may be formed by deamination of hydroxy Atrazine, which is formed by dechlorination of Atrazine (also known as Atrazine), a widely used herbicide, which has a certain biohazard and has a problem of soil residue, so that Atrazine is used as a potential substrate for theanine synthesis.

Comparing the degradation pathway of atrazine degradation model strain Pseudomonas sp.strain ADP (fig. 2) with the predicted theanine synthesis potential pathway (fig. 1), the designed synthesis pathway was by 3 enzymes: atzA, atzB, GS, biosynthesis from atrazine- & gt hydroxyatrazine- & gt ethylamine- & gt theanine is achieved (FIG. 3).

Example 2

A CFPS (PkCFPS) system based on theanine-producing endophyte P.knakmussii (isolated from rice roots, accession number ACCC 05571) was established, and a novel theanine synthesis pathway was constructed and verified.

First, prepare an Extract from p.knackmussii, then place a standard 15 μl 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 source tRNA solution, 2mM for each of 20 standard amino acids, 33mM PEP, 200ng expression plasmid, 4. Mu.L Extract based on P.knackmussii.

The expression plasmids pJL1-AtzA, pJL1-AtzB and pJL1-GS are constructed, and the verification thinking is shown in figure 4. PkCFPS systems containing pJL1-AtzA, pJL1-AtzB and pJL1-GS plasmids are respectively prepared, the proteins are synthesized by reaction for 4 hours under the water bath condition of 30 ℃, and the expression conditions of AtzA, atzB and GS in the PkCFPS systems are shown in FIG. 5A. Then, 10. Mu.L of each of the 3 PkCFPS reaction solutions was mixed, atrazine (5% acetone-assisted dissolution) having a final concentration of 1mM and glutamic acid having a final concentration of 25mM were added as substrates to a final volume of 50. Mu.L, and the reaction was continued at 30℃in a water bath for 24 hours 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 yield curve was plotted (FIG. 5B).

Example 3

Constructing gene engineering bacteria P.knackmusssii-AtzAB for expressing AtzA and AtzB.

By comparing the P.knackmusssii genome data (GenBank: HG 322950.1) on Genbank, it was found that the bacterium did not contain the AtzA and AtzB genes, so that the selection gene synthesized AtzA and AtzB for the construction of metabolic pathways; whereas the conversion of ethylamine to theanine can be accomplished with endogenous high expression of GS in p.knakmusssii.

The AtzAB gene was inserted into uracil phosphoribosyl transferase (upp) site in p.knac kmussii genome using pJQ SK-mediated gene insertion system, the co-transcribed AtzAB ORF was regulated by the promoter of rpsL gene, the constructed strain was designated p.knac kmussii-AtzAB, and the genetically engineered strain construction process is shown in fig. 6. The ORFs of AtzA and AtzB were detected at the genomic level (fig. 7A) and at the mRNA level (fig. 7B), respectively, using PCR and reverse transcription-PCR methods, confirming that AtzA and AtzB were successfully knocked into the p.knakmusssii genome and successfully completed expression.

Example 4

Fermentation experiments for synthesizing theanine while degrading atrazine were performed by using the genetically engineered bacteria constructed in example 3.

The P.knac kmussii-AtzAB strain was inoculated at a ratio of 5% into LB medium and cultured for about 7 hours (OD ₆₀₀ =2.0), atrazine (5% acetone-assisted) and glutamic acid (25 mM) were added at a final concentration of 1mM, and after fermentation for 84h, samples were taken and assayed for theanine concentration; meanwhile, different time points are set in the process, and sampling and detection are carried out. The detection result shows that after 84 hours of fermentation, P.knakmussii-AtzAB can synthesize theanine with the concentration of about 439.8 mu M in a culture solution, and the atrazine conversion rate reaches 44.0%. Meanwhile, the P.knakmusssii has a high degradation rate of atrazine and synthesis of theanine, and after 45 hours of substrate addition, the theanine yield reaches 398.4 mu M, and the reaction is basically complete (FIG. 8).

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments 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-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims

1. A green synthesis method for converting a field herbicide into theanine is characterized by comprising the following specific steps:

the atrazine chlorine hydrolase (AtzA) and the hydroxy atrazine de-ethanamine group enzyme (AtzB) are led into pseudomonas klinella (P.knackmussii), the strain endogenously expresses Glutamine Synthetase (GS), and the constructed genetically engineered bacterium takes atrazine and glutamic acid as substrates to synthesize theanine through fermentation experiments; the pseudomonas klinensis (P.knac kmussii) is a plant-derived endophytic bacterium, is separated from the root of rice, has a bacterial deposit number ACCC05571, has endogenous high expression GS, and has theanine synthesis capacity with ethylamine as a substrate;

wherein atrazine is subjected to dechlorination reaction and de-ethylamine reaction to generate ethylamine, and then is used for theanine synthesis reaction.

2. The green synthesis method for converting a field herbicide into theanine according to claim 1, wherein the fermentation is: inoculating the constructed genetically engineered bacteria into LB culture medium at a ratio of 2-5%, culturing 5-7h, adding atrazine with a final concentration of 0.5-1mM and glutamic acid with a final concentration of 10-25mM, and fermenting 84-h.