Engineered strain for high-yield of l-theanine, construction method and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and relates to an engineering strain for high-yield L-theanine, a construction method and application thereof. The application takes bacillus licheniformis DW2 as a starting strain, expresses alanine decarboxylase ALaDC and gamma-glutamylmethylamide synthetase GMAS through a plasmid to generate ethylamine from intracellular alanine through one-step decarboxylation, and then condenses the ethylamine and glutamic acid through GMAS to generate L-theanine. The L-theanine synthesis path provided by the application is shorter, has less metabolic interference, and has lower intermediate accumulation risk. In combination with a plurality of chromosome integration strategies such as strengthening of an alanine precursor supply and regulation of intracellular ATP level, the engineering strain has theanine synthesis capacity. Theanine yield is as high as 21.6 g / L after 60 h of fermentation in a 5 L fermenter, and efficient de novo synthesis of L-theanine without external addition of ethylamine is realized, thereby providing wide application support for green industrialized production of L-theanine.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and relates to an engineered strain that produces high levels of L-theanine, its construction method, and its applications. Background Technology

[0002] L-Theanine is a unique non-protein amino acid found in tea, possessing various physiological activities such as enhancing immunity, improving cognitive function, anti-inflammation, and anti-tumor activity. It is widely used in the food, pharmaceutical, and health product industries. Currently, the main production methods for theanine include chemical synthesis and microbial fermentation. Chemical synthesis involves highly toxic reagents such as hydrazine hydrate, resulting in numerous byproducts and high safety risks. Traditional microbial fermentation requires the exogenous addition of the toxic precursor ethylamine, which not only increases production costs but also poses fermentation safety hazards, severely limiting the industrial-scale green production of theanine.

[0003] Using *E. coli* as the starting strain is one of the important biotechnological pathways for the synthesis of L-theanine. This method has achieved good results by constructing genetically engineered strains that do not carry plasmids, do not require the addition of ethylamine, and can stably produce theanine using glucose. However, the ethylamine synthesis pathway used in this scheme is "acetyl-CoA → acetaldehyde → ethylamine," requiring the sequential expression of multiple enzymes such as phosphoryl ketonease (XFP), acetaldehyde dehydrogenase (EutE), and transaminase (SpuC). This results in a long metabolic pathway, and the intermediate product acetaldehyde is cytotoxic, posing a potential risk to cell growth and fermentation stability. Furthermore, *E. coli* is a non-GRAS strain, and its cell wall contains lipopolysaccharide (LPS) endotoxin, limiting its direct application in the food and pharmaceutical fields. Therefore, developing a de novo theanine synthesis process with a shorter synthetic pathway, lower intermediate toxicity, and higher host safety has significant commercial and research value. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an engineered strain that produces high levels of L-theanine, thereby solving the problems of long synthetic pathways, significant metabolic interference, and large accumulation of intermediates in existing methods for synthesizing L-theanine.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides an engineered strain that produces high levels of L-theanine.

[0007] Preferably, the genome of the engineered strain contains at least one of the following genetic modifications:

[0008] (A) Knock out the ydaP gene;

[0009] (B) Knock out the alsSD gene;

[0010] (C) Replace the promoter of the ald1 gene with P 43u12 ;

[0011] (D) Inserting ald at the oxdC site SD Gene;

[0012] (E) Inserting ald at the pflAB site SD Gene;

[0013] (F) Insert ALaDC at the xpt site E325K Gene;

[0014] (G) Insert ALaDC at the ydaP site E325K Gene;

[0015] (H) Insert the GMAS gene at the kipR site;

[0016] (I) Knock out the cyd gene;

[0017] (J) Replace the promoter of the ndh gene with P 43u12 ;

[0018] (K) Knock out the ggt gene.

[0019] More preferably, the NCBI accession number of the reference genome for the insertion site is NC_021362.1.

[0020] More preferably, the promoter P 43u12 The nucleotide sequence is shown in SEQ ID No. 27.

[0021] Preferably, the method for constructing the engineered strain includes the following steps:

[0022] SA-1, the first to eleventh target fragments were ligated to the T2 plasmid backbone to obtain the first to eleventh expression vectors;

[0023] SA-2, using Bacillus licheniformis DW2 as the starting strain, the first to eleventh expression vectors were sequentially transformed into the starting strain, and positive clones were screened to obtain the engineered strain.

[0024] More preferably, in step SA-1:

[0025] The first target fragment includes, from upstream to downstream, the upstream homologous arm KydaP-A and the downstream homologous arm KydaP-B sequences of the gene to be knocked out, and the nucleotide sequences of the upstream homologous arm KydaP-A and the downstream homologous arm KydaP-B are shown in SEQ ID No. 5-6, respectively.

[0026] The second target fragment includes, from upstream to downstream, the upstream homologous arm KalsSD-A and the downstream homologous arm KalsSD-B sequences of the gene to be knocked out, and the nucleotide sequences of the upstream homologous arm KalsSD-A and the downstream homologous arm KalsSD-B sequences are shown in SEQ ID No. 11-12, respectively.

[0027] The third target fragment, from upstream to downstream, includes the upstream homologous arm ald1-A of the gene to be edited, the promoter P, and so on. 43u12 The ald1 gene, the terminator TamyL, and the downstream homologous arm ald1-B sequence of the ald1 gene to be edited, wherein the nucleotide sequences of the upstream homologous arm ald1-A and the downstream homologous arm ald1-B are shown in SEQ ID No. 21-22, respectively;

[0028] The fourth target fragment, from upstream to downstream, includes, in sequence, the upstream homologous arm of the insertion site oxdC, KoxdC-A, and aldC, respectively. SD The gene and the downstream homologous arm KoxdC-B sequence of the insertion site oxdC, wherein the nucleotide sequences of the upstream homologous arm KoxdC-A and the downstream homologous arm KoxdC-B are shown in SEQ ID No. 33-34, respectively;

[0029] The fifth target fragment, from upstream to downstream, includes, in sequence, the upstream homologous arm KpflAB-A and ald of the insertion site pflAB. SD The gene and the downstream homologous arm KpflAB-B sequence of the insertion site pflAB, wherein the nucleotide sequences of the upstream homologous arm KpflAB-A and the downstream homologous arm KpflAB-B are shown in SEQ ID No. 39-40, respectively;

[0030] The sixth target fragment, from upstream to downstream, includes, in sequence, the upstream homologous arms Kxpt-A and ALaDC of the insertion site xpt. E325K The gene and the downstream homologous arm Kxpt-B sequence of the insertion site xpt, wherein the nucleotide sequences of the upstream homologous arm Kxpt-A and the downstream homologous arm Kxpt-B are shown in SEQ ID No. 46-47, respectively;

[0031] The seventh target fragment, from upstream to downstream, includes KydaP-A and ALaDC, the upstream homologous arms of the insertion site ydaP. E325K The gene and the downstream homologous arm KydaP-B sequence of the insertion site ydaP, wherein the nucleotide sequences of the upstream homologous arm KydaP-A and the downstream homologous arm KydaP-B are shown in SEQ ID No. 52-53, respectively;

[0032] The eighth target fragment includes, from upstream to downstream, the upstream homologous arm KkipR-A of the insertion site kipR, the GMAS gene, and the downstream homologous arm KkipR-B of the insertion site kipR. The nucleotide sequences of the upstream homologous arm KkipR-A and the downstream homologous arm KkipR-B are shown in SEQ ID No. 59-60, respectively.

[0033] The ninth target fragment includes, from upstream to downstream, the upstream homologous arm Kcyd-A and the downstream homologous arm Kcyd-B of the gene cyd to be knocked out. The nucleotide sequences of the upstream homologous arm Kcyd-A and the downstream homologous arm Kcyd-B are shown in SEQ ID No. 65-66, respectively.

[0034] The 10th target fragment, from upstream to downstream, includes, in sequence, the upstream homologous arm ndh-A of the gene to be edited, ndh, and the promoter P. 43u12 The sequence includes the ndh gene, the terminator TamyL, and the downstream homologous arm ndh-B of the gene to be edited, wherein the nucleotide sequences of the upstream homologous arm ndh-A and the downstream homologous arm ndh-B are shown in SEQ ID No. 73-74, respectively.

[0035] The 11th target fragment includes, from upstream to downstream, the upstream homologous arm Kggt-A and the downstream homologous arm Kggt-B sequences of the gene to be knocked out, and the nucleotide sequences of the upstream homologous arm Kggt-A and the downstream homologous arm Kggt-B are shown in SEQ ID No. 80-81, respectively.

[0036] More preferably, in step SA-1:

[0037] The synthesis of the first target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm KydaP-A and downstream homologous arm KydaP-B are synthesized using primers, and the upstream homologous arm KydaP-A and downstream homologous arm KydaP-B are sequentially ligated using overlap PCR technology to obtain the first target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KydaP-A are shown in SEQ ID No. 1-2, and the primer sequences for synthesizing the downstream homologous arm KydaP-B are shown in SEQ ID No. 3-4, respectively;

[0038] The synthesis of the second target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm KalsSD-A and downstream homologous arm KalsSD-B are synthesized using primers, and the upstream homologous arm KalsSD-A and downstream homologous arm KalsSD-B are sequentially ligated using overlap PCR technology to obtain the second target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KalsSD-A are shown in SEQ ID No. 7-8, and the primer sequences for synthesizing the downstream homologous arm KalsSD-B are shown in SEQ ID No. 9-10, respectively;

[0039] The synthesis of the third target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, primers are used to synthesize the upstream homologous arm ald1-A, the downstream homologous arm ald1-B, the ald1 gene, and the terminator TamyL; using the genome of Bacillus subtilis as a template, primers are used to synthesize the promoter P. 43u12 The upstream homologous arm ald1-A and promoter P were linked by overlap PCR technology. 43u12 The ald1 gene, terminator TamyL, and downstream homologous arm ald1-B are sequentially linked to obtain the third target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm ald1-A are shown in SEQ ID No. 13-14, and the promoter P is synthesized. 43u12 The primer sequences for synthesizing the ald1 gene are shown in SEQ ID No. 25-26, the primer sequences for synthesizing the terminator TamyL are shown in SEQ ID No. 19-20, and the primer sequences for synthesizing the downstream homologous arm ald1-B are shown in SEQ ID No. 15-16.

[0040] The synthesis of the fourth target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm KoxdC-A and the downstream homologous arm KoxdC-B are synthesized using primers; and ald is synthesized using standard gene synthesis techniques. SD Genes were synthesized using overlap PCR technology, which involved the upstream homologous arms KoxdC-A and ald. SD The gene and the downstream homologous arm KoxdC-B are sequentially linked to obtain the fourth target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KoxdC-A are shown in SEQ ID No. 29-30, and the primer sequences for synthesizing the downstream homologous arm KoxdC-B are shown in SEQ ID No. 31-32, respectively;

[0041] The synthesis of the fifth target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, synthesizing the upstream homologous arm KpflAB-A and the downstream homologous arm KpflAB-B using primers, and synthesizing ald using standard gene synthesis techniques. SD Genes, using overlap PCR technology, were used to connect the upstream homologous arms KpflAB-A and ald. SD The gene and the downstream homologous arm KpflAB-B are sequentially linked to obtain the fifth target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KpflAB-A are shown in SEQ ID No. 35-36, and the primer sequences for synthesizing the downstream homologous arm KpflAB-B are shown in SEQ ID No. 37-38, respectively;

[0042] The synthesis of the sixth target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm Kxpt-A and the downstream homologous arm Kxpt-B are synthesized using primers; and ALaDC is synthesized using standard gene synthesis techniques. E325K Genes, using overlap PCR technology, were used to combine the upstream homologous arms Kxpt-A and ALaDC. E325K The gene and the downstream homologous arm Kxpt-B were sequentially linked to obtain the sixth target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm Kxpt-A are shown in SEQ ID No. 42-43, and the primer sequences for synthesizing the downstream homologous arm Kxpt-B are shown in SEQ ID No. 44-45, respectively;

[0043] The synthesis of the seventh target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm KydaP-A and the downstream homologous arm KydaP-B are synthesized using primers; and ALaDCE is synthesized using standard gene synthesis techniques. 325K Genes were synthesized using overlap PCR technology, which involved the upstream homologous arms KydaP-A and ALaDCE. 325K The gene and the downstream homologous arm KydaP-B are sequentially linked to obtain the 7th target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KydaP-A are shown in SEQ ID No. 48-49, and the primer sequences for synthesizing the downstream homologous arm KydaP-B are shown in SEQ ID No. 50-51, respectively.

[0044] The synthesis of the eighth target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm KkipR-A and downstream homologous arm KkipR-B are synthesized using primers; the GMAS gene is synthesized using standard gene synthesis technology; and the upstream homologous arm KkipR-A, the GMAS gene, and the downstream homologous arm KkipR-B are sequentially ligated using overlap PCR technology to obtain the eighth target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KkipR-A are shown in SEQ ID No. 55-56, and the primer sequences for synthesizing the downstream homologous arm KkipR-B are shown in SEQ ID No. 57-58, respectively;

[0045] The synthesis of the 9th target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm Kcyd-A and downstream homologous arm Kcyd-B are synthesized using primers, and the upstream homologous arm Kcyd-A and downstream homologous arm Kcyd-B are sequentially ligated using overlap PCR technology to obtain the 9th target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm Kcyd-A are shown in SEQ ID No. 61-62, and the primer sequences for synthesizing the downstream homologous arm Kcyd-B are shown in SEQ ID No. 63-64, respectively;

[0046] The synthesis of the 10th target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, primers are used to synthesize the upstream homologous arm ndh-A, the downstream homologous arm ndh-B, the ndh gene, and the terminator TamyL; using the genome of Bacillus subtilis as a template, primers are used to synthesize the promoter P. 43u12 The upstream homologous arm ndh-A and promoter P were combined using overlap PCR technology. 43u12 The ndh gene, terminator TamyL, and downstream homologous arm ndh-B are sequentially linked to obtain the 10th target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm ndh-A are shown in SEQ ID No. 67-68, and the promoter P is synthesized. 43u12 The primer sequences for synthesizing the ndh gene are shown in SEQ ID No. 25-26, the primer sequences for synthesizing the ndh gene are shown in SEQ ID No. 71-72, the primer sequences for synthesizing the terminator TamyL are shown in SEQ ID No. 19-20, and the primer sequences for synthesizing the downstream homologous arm ndh-B are shown in SEQ ID No. 69-70.

[0047] The synthesis of the 11th target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm Kggt-A and downstream homologous arm Kggt-B are synthesized using primers, and the upstream homologous arm Kggt-A and downstream homologous arm Kggt-B are sequentially ligated using overlap PCR technology to obtain the 11th target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm Kggt-A are shown in SEQ ID No. 76-77, and the primer sequences for synthesizing the downstream homologous arm Kggt-B are shown in SEQ ID No. 78-79.

[0048] More preferably, the NCBI accession number of the genome of the Bacillus subtilis is NC_000964.3.

[0049] More preferably, the ald SD The nucleotide sequence of the gene is shown in SEQ ID No. 28;

[0050] The ALaDC E325K The nucleotide sequence of the gene is shown in SEQ ID No. 41;

[0051] The nucleotide sequence of the GMAS gene is shown in SEQ ID No. 54.

[0052] Secondly, the present invention also provides the application of the above-mentioned engineered strains that produce high levels of L-theanine in the preparation of L-theanine.

[0053] Thirdly, the present invention also provides a method for preparing L-theanine, comprising the following steps:

[0054] The above-mentioned engineered strains that produce high levels of L-theanine were inoculated into a fermentation medium and fermented to obtain a fermentation broth containing L-theanine.

[0055] Preferably, the fermentation medium comprises the following components: 6 g / L tryptone, 12 g / L yeast extract, 6 g / L (NH4)2SO4, 12.5 g / L K2HPO4•3H2O, 2.5 g / L KH2PO4, 10 mL / L buffer solution, and 60 g / L glucose.

[0056] More preferably, the buffer solution comprises the following components: FeSO4•7H2O 4 g / L, CaCl2 4 g / L, MnSO4•5H2O 1 g / L, CoCl2•6H2O 0.4 g / L, NaMoO4•2H2O 0.2 g / L, ZnSO4•7H2O 0.2 g / L, AlCl3•6H2O 0.1 g / L, CuCl2•H2O 0.1 g / L, and H3BO3 0.05 g / L.

[0057] Preferably, the fermentation conditions include: an inoculum size of 2%-10% for the engineered strain, a fermentation temperature of 35-40 ℃, and a rotation speed of 200-250 rpm.

[0058] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0059] (1) Shorter synthesis pathway: This invention requires only two enzymatic reactions (alanine → ethylamine → theanine), and the metabolic flux is easier to control;

[0060] (2) Lower intermediate toxicity: This invention does not use acetaldehyde intermediate, which is cytotoxic, and is more friendly to bacterial growth;

[0061] (3) More abundant precursor supply: Alanine is a naturally abundant amino acid in the cell, and there is no need to construct a complex acetyl-CoA bypass;

[0062] (4) Higher host safety: Bacillus licheniformis is a GRAS certified strain and has no endotoxin. Attached Figure Description

[0063] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0064] Figure 1 A schematic diagram of the metabolic pathway for constructing a high-yield L-theanine engineered strain using Bacillus licheniformis DW2 as the starting strain;

[0065] Figure 2 This is a diagram showing the fermentation scale-up verification test results of engineered strain L14. Detailed Implementation

[0066] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0067] The following specific embodiments further illustrate an engineered strain of high L-theanine production, its construction method, and its application. This section further explains the invention with reference to specific embodiments, but should not be construed as limiting the invention. Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art, such as the conditions described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or the conditions recommended by the manufacturer. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in the art.

[0068] Bacillus licheniformis is a GRAS-certified food-grade strain with advantages such as high biosafety, no endotoxins, rapid growth, simple genetic manipulation, and strong stress resistance, making it an excellent industrial substrate for synthesizing high-value amino acid derivatives. However, current technologies have not yet achieved efficient de novo synthesis of theanine from Bacillus licheniformis, mainly due to limitations such as insufficient alanine precursor supply, lack of ethylamine and theanine synthases, and insufficient intracellular energy supply. Therefore, this invention focuses on developing a de novo theanine synthesis process based on a food-grade host, without the need for exogenous precursor addition, antibiotics, or inducers, which has significant commercial and application value.

[0069] In the following embodiments, the Bacillus licheniformis DW2 was obtained with reference to the content described in the existing published patent (CN112226437A, Sequence Combination and Application for Gradient Regulation of Bacillus Promoter Startup Efficiency).

[0070] In the following embodiments, the Gene IDs of the ydaP gene, alsSD gene, cyd gene, and ggt gene in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) are 16053642, 92859576, 92859295, and 92859574, respectively.

[0071] In the following embodiments, the T2 plasmid was obtained with reference to the contents described in the existing published patent (CN119040235A, Engineered strains for synthesizing pyridoxal phosphate and their applications).

[0072] In the following embodiments, the expression plasmid was transformed using a calcium transfer method, which is based on the method described in the existing published patent (CN114921431A, Glycosyltransferase mutant and its application in fermentation production of aromatic alcohol glycosides).

[0073] In the following embodiments, the NCBI accession number of the Bacillus subtilis genome is NC_000964.3.

[0074] In the following embodiments, the NCBI accession number of the reference genome for the insertion site is NC_021362.1.

[0075] In the following embodiments, the standard gene synthesis of the target fragment was commissioned to Shanghai Sangon Biotech Co., Ltd.

[0076] In the following examples, the nucleotide and / or amino acid sequences involved are shown in Table 1.

[0077] Table 1: Nucleotide and / or amino acid sequences in the embodiments of the present invention

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] Example 1: Preparation of high-yield L-theanine engineered strain L14

[0084] This invention uses Bacillus licheniformis DW2 as the starting strain and involves knocking out the ydaP gene, knocking out the alsSD gene, and replacing the promoter of the ald1 gene with P. 43u12 Inserting ald at the oxdC site SD Gene, inserted at pflAB site. SD Gene, inserted at the xpt site into ALaDC E325K Gene, inserted at the ydaP site into ALaDC E325K Gene, insertion of the GMAS gene at the kipR site, knockout of the cyd gene, and replacement of the ndh gene promoter with P. 43u12 Genetic engineering operations such as knocking out the GGT gene (e.g.) Figure 1 As shown in the figure, the key pathways for L-theanine synthesis in engineered strains were gradually modified to increase the yield and accumulation of L-theanine, and finally a high-yield L-theanine engineered strain L14 was obtained.

[0085] This embodiment employs homologous recombination technology to manipulate the genes of Bacillus licheniformis DW2. Homologous recombination utilizes the homology between the target fragment (including at least the upstream and downstream homologous arms of the target sequence) and the target sequence. Through homologous recombination of the homologous portions of the two sequences, the target sequence of the host strain is replaced with the target fragment sequence, thereby editing the host strain's genome to obtain a recombinant strain. Gene editing types include, but are not limited to, gene knockout and gene replacement, depending on the structural form of the exogenous recombinant strain. Specifically, when the recombinant strain includes only the upstream homologous arm (A) and the downstream homologous arm (B), the target sequence can be deleted. When it includes not only the upstream and downstream homologous arm sequences of the target sequence but also the target gene to be knocked in located between the upstream and downstream homologous arm sequences, the target gene can replace the target sequence, thereby achieving gene replacement.

[0086] 1. Knock out the ydaP gene

[0087] Using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm KydaP-A (SEQ ID No. 5) and the downstream homologous arm KydaP-B (SEQ ID No. 6) of the gene to be knocked out, ydaP, were amplified using primers KydaP-AF (SEQ ID No. 1), KydaP-AR (SEQ ID No. 2), KydaP-BF (SEQ ID No. 3), and KydaP-BR (SEQ ID No. 4), respectively. These sequences were then sequentially ligated using overlap PCR to obtain the first target fragment. Using the T2 plasmid as a template, the T2 plasmid backbone was amplified using the upstream primer T2-GF (as shown in SEQ ID No. 82) and the downstream primer T2-GR (as shown in SEQ ID No. 83). The first target fragment was ligated into the T2 plasmid backbone using Exnase II T5 exonuclease (Wuhan Aibotek Biotechnology Co., Ltd., catalog number: RK21020). The fragment was then ligated into competent Escherichia coli DH5α (Thermopolymer) cells. The expression plasmid was transformed using Fisher Scientific Inc. (catalog number: 18265017). The plasmid was screened using LB plates containing kanamycin (Beijing Solarbio Science & Technology Co., Ltd., catalog number: L2030). The clone plasmid that was positive and correctly sequenced was named the first expression plasmid.

[0088] The first expression vector was transformed into Bacillus licheniformis DW2, and the resulting positive transformant was named engineered strain L1.

[0089] 2. Knockout of the alsSD gene

[0090] Using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm KalsSD-A (SEQ ID No. 11) and the downstream homologous arm KalsSD-B (SEQ ID No. 12) of the gene to be knocked out, alsSD, were amplified using primers KalsSD-AF (SEQ ID No. 7), KalsSD-AR (SEQ ID No. 8), KalsSD-BF (SEQ ID No. 9), and KalsSD-BR (SEQ ID No. 10), respectively. The above sequences were then sequentially ligated using overlap PCR to obtain the second target fragment. The second target fragment was ligated into the T2 plasmid backbone using the same method as above to obtain the second expression plasmid.

[0091] The second expression vector was transformed into Bacillus licheniformis DW2, and the resulting positive transformant was named engineered strain L2; the second expression vector was transformed into engineered strain L1, and the resulting positive transformant was named engineered strain L3.

[0092] 3. Replace the promoter of the ald1 gene with P. 43u12

[0093] Using the genome of Bacillus licheniformis DW2 as a template, primers ald1-AF (SEQ ID No. 13) and ald1-AR (SEQ ID No. 14), ald1-BF (SEQ ID No. 15) and ald1-BR (SEQ ID No. 16), ald1-F (SEQ ID No. 17) and ald1-R (SEQ ID No. 18), and TamyL-F (SEQ ID No. 19) and TamyL-R (SEQ ID No. 20) were used to amplify the upstream homologous arm ald1-A (SEQ ID No. 21), the downstream homologous arm ald1-B (SEQ ID No. 22), the ald1 gene (SEQ ID No. 23), and the terminator TamyL (SEQ ID No. 24) of the gene to be edited, ald1. Using the genome of Bacillus subtilis as a template, P... 43u12 -F (SEQ ID No. 25) and P 43u12 -R (SEQ ID No. 26) amplification yields promoter P 43u12 (SEQ ID No. 27); The upstream homologous arm ald1-A and promoter P of the gene to be edited, ald1, were inserted using overlap PCR technology. 43u12 The ald1 gene, the terminator TamyL, and the downstream homologous arm ald1-B sequence of the ald1 gene to be inserted are sequentially linked to obtain the third target fragment; the third target fragment is linked to the T2 plasmid backbone in the same way to obtain the third expression plasmid.

[0094] The third expression vector was transformed into engineered strain L3, and the resulting positive transformant was named engineered strain L4.

[0095] 4. ald SD Gene integration into the Bacillus licheniformis DW2 genome

[0096] ald SD The gene (as shown in SEQ ID No. 28) encodes an alanine dehydrogenase mutant derived from Bacillus subtilis, with ald inserted at the oxdC site and pflAB site, respectively. SD Genes can further enhance the supply of alanine, providing sufficient precursors for the synthesis of ethylamine.

[0097] 4.1 Inserting ald at the oxdC site SD Gene

[0098] Using the genome of Bacillus licheniformis DW2 as a template, primers KoxdC-AF (SEQ ID No. 29) and KoxdC-AR (SEQ ID No. 30), KoxdC-BF (SEQ ID No. 31) and KoxdC-BR (SEQ ID No. 32) were used to amplify the upstream homologous arm KoxdC-A (SEQ ID No. 33) and the downstream homologous arm KoxdC-B (SEQ ID No. 34) of the oxdC insertion site. ald was then synthesized using standard gene synthesis technology (Shanghai Sangon Biotech Co., Ltd.). SD Gene; using overlap PCR technology, the upstream homologous arms KoxdC-A and ald of the insertion site oxdC were inserted. SD The gene and the KoxdC-B sequence, the downstream homologous arm of the insertion site oxdC, were sequentially ligated to obtain the fourth target fragment; the fourth target fragment was then ligated to the T2 plasmid backbone in the same way to obtain the fourth expression plasmid.

[0099] The fourth expression vector was transformed into engineered strain L4, and the resulting positive transformant was named engineered strain L5.

[0100] 4.2. Insert ald at the pflAB site SD Gene

[0101] Using the genome of Bacillus licheniformis DW2 as a template, primers KpflAB-AF (SEQ ID No. 35), KpflAB-AR (SEQ ID No. 36), KpflAB-BF (SEQ ID No. 37), and KpflAB-BR (SEQ ID No. 38) were used to amplify the upstream homologous arm KpflAB-A (SEQ ID No. 39) and the downstream homologous arm KpflAB-B (SEQ ID No. 40) of the insertion site pflAB. ald was then synthesized using standard gene synthesis technology (Shanghai Sangon Biotech Co., Ltd.). SD Gene; using overlap PCR technology, the upstream homologous arms KpflAB-A and ald of the insertion site pflAB were inserted. SD The gene and the downstream homologous arm KpflAB-B sequence of the insertion site pflAB were sequentially ligated to obtain the fifth target fragment; the fifth target fragment was ligated to the T2 plasmid backbone in the same way to obtain the fifth expression plasmid.

[0102] The fifth expression vector was transformed into engineered strain L4, and the resulting positive transformant was named engineered strain L6; the fifth expression vector was transformed into engineered strain L5, and the resulting positive transformant was named engineered strain L7.

[0103] 5. ALaDC E325K Gene integration into the Bacillus licheniformis DW2 genome

[0104] ALaDC E325K The gene (as shown in SEQ ID No. 41) encodes an alanine decarboxylase mutant derived from the tea plant (Camellia sinensis), with ALaDC inserted at the xpt and ydaP sites, respectively. E325K Genes can further enhance the supply of ethylamine, providing sufficient precursors for theanine synthesis.

[0105] 5.1 Inserting ALaDC at the xpt site E325K Gene

[0106] Using the genome of Bacillus licheniformis DW2 as a template, primers Kxpt-AF (SEQ ID No. 42) and Kxpt-AR (SEQ ID No. 43), Kxpt-BF (SEQ ID No. 44) and Kxpt-BR (SEQ ID No. 45) were used to amplify the upstream homologous arm Kxpt-A (SEQ ID No. 46) and the downstream homologous arm Kxpt-B (SEQ ID No. 47) of the insertion site xpt. ALaDC was then synthesized using standard gene synthesis technology (Shanghai Sangon Biotech Co., Ltd.).E325K Gene; using overlap PCR technology, the upstream homologous arms Kxpt-A and ALaDC of the xpt insertion site were inserted. E325K The gene and the downstream homologous arm Kxpt-B sequence of the xpt insertion site are sequentially ligated to obtain the sixth target fragment; the sixth target fragment is ligated to the T2 plasmid backbone in the same way to obtain the sixth expression plasmid.

[0107] The sixth expression vector was transformed into engineered strain L7, and the resulting positive transformant was named engineered strain L8.

[0108] 5.2. Insert ALaDC at the ydaP site E325K Gene

[0109] Using the genome of Bacillus licheniformis DW2 as a template, primers KydaP-AF (SEQ ID No. 48) and KydaP-AR (SEQ ID No. 49), KydaP-BF (SEQ ID No. 50) and KydaP-BR (SEQ ID No. 51) were used to amplify the upstream homologous arm KydaP-A (SEQ ID No. 52) and the downstream homologous arm KydaP-B (SEQ ID No. 53) of the insertion site ydaP. ALaDC was then synthesized using standard gene synthesis technology (Shanghai Sangon Biotech Co., Ltd.). E325K Gene; using overlap PCR technology, the upstream homologous arms KydaP-A and ALaDC of the insertion site ydaP were inserted. E325K The gene and the KydaP-B sequence, the downstream homologous arm of the insertion site ydaP, were sequentially ligated to obtain the 7th target fragment; the 7th target fragment was then ligated to the T2 plasmid backbone in the same way to obtain the 7th expression plasmid.

[0110] The 7th expression vector was transformed into engineered strain L7, and the resulting positive transformant was named engineered strain L9; the 7th expression vector was transformed into engineered strain L8, and the resulting positive transformant was named engineered strain L10.

[0111] 6. The GMAS gene was integrated into the Bacillus licheniformis DW2 genome.

[0112] The GMAS gene (as shown in SEQ ID No. 54) encodes a γ-glutamyl methylamine synthase derived from the methyltrophic strain Methylovorusmays No. 9. Insertion of the GMAS gene at the kipR site can further enhance the synthesis and accumulation of theanine.

[0113] Using the genome of Bacillus licheniformis DW2 as a template, primers KkipR-AF (SEQ ID No. 55), KkipR-AR (SEQ ID No. 56), KkipR-BF (SEQ ID No. 57), and KkipR-BR (SEQ ID No. 58) were used to amplify the upstream homologous arm KkipR-A (SEQ ID No. 59) and the downstream homologous arm KkipR-B (SEQ ID No. 60) of the kipR to be inserted site. The GMAS gene was synthesized using standard gene synthesis technology (Shanghai Sangon Biotech Co., Ltd.). The sequences of the upstream homologous arm KkipR-A, the GMAS gene, and the downstream homologous arm KkipR-B of the kipR to be inserted site were sequentially ligated using overlap PCR to obtain the 8th target fragment. The 8th target fragment was ligated into the T2 plasmid backbone in the same way to obtain the 8th expression plasmid.

[0114] The eighth expression vector was transformed into engineered strain L10, and the resulting positive transformant was named engineered strain L11.

[0115] 7. Knock out the cyd gene

[0116] Using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm Kcyd-A (SEQ ID No. 65) and the downstream homologous arm Kcyd-B (SEQ ID No. 66) of the gene to be knocked out, cyd, were amplified using primers Kcyd-AF (SEQ ID No. 61), Kcyd-AR (SEQ ID No. 62), Kcyd-BF (SEQ ID No. 63), and Kcyd-BR (SEQ ID No. 64), respectively. The above sequences were then sequentially ligated using overlap PCR to obtain the 9th target fragment. The 9th target fragment was ligated into the T2 plasmid backbone using the same method to obtain the 9th expression plasmid.

[0117] The 9th expression vector was transformed into engineered strain L11, and the resulting positive transformant was named engineered strain L12.

[0118] 8. Replace the promoter of the ndh gene with P. 43u12

[0119] Using the genome of Bacillus licheniformis DW2 as a template, primers ndh-AF (SEQ ID No. 67) and ndh-AR (SEQ ID No. 68), ndh-BF (SEQ ID No. 69) and ndh-BR (SEQ ID No. 70), ndh-F (SEQ ID No. 71) and ndh-R (SEQ ID No. 72), and TamyL-F (SEQ ID No. 19) and TamyL-R (SEQ ID No. 20) were used to amplify the upstream homologous arm ndh-A (SEQ ID No. 73), the downstream homologous arm ndh-B (SEQ ID No. 74), the ndh gene (SEQ ID No. 75), and the terminator TamyL (SEQ ID No. 24) of the gene to be edited, ndh. The promoter P was then amplified according to the method described in Section 3 of this embodiment. 43u12 ; using overlap PCR technology, the upstream homologous arm ndh-A and promoter P of the gene to be edited, ndh, were... 43u12 The ndh gene, the terminator TamyL, and the downstream homologous arm ndh-B sequence of the gene to be inserted ndh are sequentially linked to obtain the 10th target fragment; the 10th target fragment is linked to the T2 plasmid backbone in the same way to obtain the 10th expression plasmid.

[0120] The 10th expression vector was transformed into engineered strain L12, and the resulting positive transformant was named engineered strain L13.

[0121] 9. Knock out the GGT gene

[0122] Using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm Kggt-A (SEQ ID No. 80) and the downstream homologous arm Kggt-B (SEQ ID No. 81) of the gene to be knocked out, ggt, were amplified using primers Kggt-AF (SEQ ID No. 76) and Kggt-AR (SEQ ID No. 77), Kggt-BF (SEQ ID No. 78) and Kggt-BR (SEQ ID No. 79), respectively. The above sequences were then sequentially ligated using overlap PCR to obtain the 11th target fragment. The 11th target fragment was ligated into the T2 plasmid backbone using the same method to obtain the 11th expression plasmid.

[0123] The 11th expression vector was transformed into engineered strain L13, and the resulting positive transformant was named engineered strain L14.

[0124] Example 2: L-theanine fermentation system and its scale-up verification

[0125] 1. Culture medium formulation

[0126] (1) LB medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L.

[0127] (2) LB solid medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, agar powder 15 g / L.

[0128] (3) A9 fermentation medium: tryptone 6 g / L, yeast extract 12 g / L, (NH4)2SO4 6 g / L, K2HPO4•3H2O 12.5 g / L, KH2PO4 2.5 g / L, A9 buffer 10 mL / L, glucose 60 g / L;

[0129] The A9 buffer is configured with the following proportions: FeSO4•7H2O 4 g / L, CaCl2 4 g / L, MnSO4•5H2O 1 g / L, CoCl2•6H2O 0.4 g / L, NaMoO4•2H2O 0.2 g / L, ZnSO4•7H2O 0.2 g / L, AlCl3•6H2O 0.1 g / L, CuCl2•H2O 0.1 g / L, and H3BO3 0.05 g / L.

[0130] 2. Shake-flask fermentation system

[0131] (1) Activation of strains: Select engineered bacterial cells and activate them by streaking on LB solid medium containing tetracycline (concentration of 20 μg / mL), and incubate upside down at 37 ℃ for 20 h;

[0132] (2) Seed culture: The activated strain was inoculated into 30 mL of LB medium containing tetracycline (concentration of 20 μg / mL) and cultured in a shaker at 37 ℃ and 230 rpm for 10 h until OD. 600 When the value is between 4 and 5, the strain seed solution is obtained;

[0133] (3) Shake flask fermentation culture: The strain seed liquid (3% inoculum) was inoculated into 30 mL of A9 fermentation medium and cultured at 37 ℃ and 230 rpm for 60 h. The fermentation pH was controlled based on the color development of phenol red indicator and dynamically adjusted by 50% (v / v) ammonia water to obtain the shake flask fermentation culture.

[0134] 3. Fermentation scale-up verification

[0135] (1) The activated strain and seed culture are carried out in accordance with the methods described in steps (1) to (2) of Section 2 of this embodiment to obtain the first-level seed liquid.

[0136] (2) Secondary expansion culture: The primary seed culture (inoculum amount 2%) was inoculated into 50 mL of LB medium containing tetracycline (concentration of 20 μg / mL) and cultured at 37 ℃ and 230 rpm for 10 h until OD. 600 The pH value was between 4 and 5. The fermentation pH was controlled based on the color development of phenol red indicator and dynamically adjusted by 50% (v / v) ammonia water to obtain the secondary seed culture.

[0137] (3) Fermentation scale-up: The secondary seed liquid (10% inoculation amount) was inoculated into 2.5 L of A9 fermentation medium, the initial pH value was adjusted to 7, and fermentation was carried out in a 5 L fermenter at 37 ℃ for 60 h to obtain L-theanine scale-up fermentation broth.

[0138] Table 1 shows the L-theanine yield data after shake-flask fermentation for each strain. Figure 2 The figure shows the fermentation scale-up verification test results of engineered strain L14; in the figure, "L-The" represents L-theanine, "L-Glu" represents L-glutamic acid, "Ethylamine" represents ethylamine, and "L-Ala" represents L-alanine. The results show that engineered strain L14 achieved the highest theanine yield (21.6 g / L) during fermentation in a 5 L fermenter for 60 h, which is the highest yield to date for the de novo fermentation production of theanine using Bacillus licheniformis DW2 as a microbial chassis.

[0139] Table 1: L-theanine yield after shake-flask fermentation of each strain

[0140]

[0141] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An engineered strain that produces high levels of L-theanine, characterized in that, The genome of the engineered strain contains at least one of the following genetic modifications: (A) Knock out the ydaP gene; (B) Knock out the alsSD gene; (C) Replace the promoter of the ald1 gene with P 43u12 ; (D) Inserting ald at the oxdC site SD Gene; (E) Inserting ald at the pflAB site SD Gene; (F) Insert ALaDC at the xpt site E325K Gene; (G) Insert ALaDC at the ydaP site E325K Gene; (H) Insert the GMAS gene at the kipR site; (I) Knock out the cyd gene; (J) Replace the promoter of the ndh gene with P 43u12 ; (K) Knock out the ggt gene; The NCBI accession number of the reference genome for the insertion site is NC_021362.

1.

2. The engineered strain as described in claim 1, characterized in that, The promoter P 43u12 The nucleotide sequence is shown in SEQ ID No.

27.

3. The engineered strain as described in claim 1, characterized in that, The method for constructing the engineered strain includes the following steps: SA-1, the first to eleventh target fragments were ligated to the T2 plasmid backbone to obtain the first to eleventh expression vectors; The first target fragment includes, from upstream to downstream, the upstream homologous arm KydaP-A and the downstream homologous arm KydaP-B sequences of the gene to be knocked out, and the nucleotide sequences of the upstream homologous arm KydaP-A and the downstream homologous arm KydaP-B are shown in SEQ ID No. 5-6, respectively. The second target fragment includes, from upstream to downstream, the upstream homologous arm KalsSD-A and the downstream homologous arm KalsSD-B sequences of the gene to be knocked out, and the nucleotide sequences of the upstream homologous arm KalsSD-A and the downstream homologous arm KalsSD-B sequences are shown in SEQ ID No. 11-12, respectively. The third target fragment, from upstream to downstream, includes the upstream homologous arm ald1-A of the gene to be edited, the promoter P, and so on. 43u12 The ald1 gene, the terminator TamyL, and the downstream homologous arm ald1-B sequence of the ald1 gene to be edited, wherein the nucleotide sequences of the upstream homologous arm ald1-A and the downstream homologous arm ald1-B are shown in SEQ ID No. 21-22, respectively; The fourth target fragment, from upstream to downstream, includes, in sequence, the upstream homologous arm of the insertion site oxdC, KoxdC-A, and aldC. SD The gene and the downstream homologous arm KoxdC-B sequence of the insertion site oxdC, wherein the nucleotide sequences of the upstream homologous arm KoxdC-A and the downstream homologous arm KoxdC-B are shown in SEQ ID No. 33-34, respectively; The fifth target fragment, from upstream to downstream, includes, in sequence, the upstream homologous arm KpflAB-A and ald of the insertion site pflAB. SD The gene and the downstream homologous arm KpflAB-B sequence of the insertion site pflAB, wherein the nucleotide sequences of the upstream homologous arm KpflAB-A and the downstream homologous arm KpflAB-B are shown in SEQ ID No. 39-40, respectively; The sixth target fragment, from upstream to downstream, includes, in sequence, the upstream homologous arms Kxpt-A and ALaDC of the insertion site xpt. E325K The gene and the downstream homologous arm Kxpt-B sequence of the insertion site xpt, wherein the nucleotide sequences of the upstream homologous arm Kxpt-A and the downstream homologous arm Kxpt-B are shown in SEQ ID No. 46-47, respectively; The seventh target fragment, from upstream to downstream, includes KydaP-A and ALaDC, the upstream homologous arms of the insertion site ydaP. E325K The gene and the downstream homologous arm KydaP-B sequence of the insertion site ydaP, wherein the nucleotide sequences of the upstream homologous arm KydaP-A and the downstream homologous arm KydaP-B are shown in SEQ ID No. 52-53, respectively; The eighth target fragment includes, from upstream to downstream, the upstream homologous arm KkipR-A of the insertion site kipR, the GMAS gene, and the downstream homologous arm KkipR-B of the insertion site kipR. The nucleotide sequences of the upstream homologous arm KkipR-A and the downstream homologous arm KkipR-B are shown in SEQ ID No. 59-60, respectively. The ninth target fragment includes, from upstream to downstream, the upstream homologous arm Kcyd-A and the downstream homologous arm Kcyd-B of the gene to be knocked out, and the nucleotide sequences of the upstream homologous arm Kcyd-A and the downstream homologous arm Kcyd-B are shown in SEQ ID No. 65-66, respectively. The 10th target fragment, from upstream to downstream, includes, in sequence, the upstream homologous arm ndh-A of the gene to be edited, ndh, and the promoter P. 43u12 The sequence includes the ndh gene, the terminator TamyL, and the downstream homologous arm ndh-B of the gene to be edited, wherein the nucleotide sequences of the upstream homologous arm ndh-A and the downstream homologous arm ndh-B are shown in SEQ ID No. 73-74, respectively. The 11th target fragment includes, from upstream to downstream, the upstream homologous arm Kggt-A and the downstream homologous arm Kggt-B sequences of the gene to be knocked out, and the nucleotide sequences of the upstream homologous arm Kggt-A and the downstream homologous arm Kggt-B are shown in SEQ ID No. 80-81, respectively. SA-2, using Bacillus licheniformis DW2 as the starting strain, the first to eleventh expression vectors were sequentially transformed into the starting strain, and positive clones were screened to obtain the engineered strain.

4. The engineered strain as described in claim 3, characterized in that, In step SA-1: The synthesis of the first target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm KydaP-A and downstream homologous arm KydaP-B are synthesized using primers, and the upstream homologous arm KydaP-A and downstream homologous arm KydaP-B are sequentially ligated using overlap PCR technology to obtain the first target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KydaP-A are shown in SEQ ID No. 1-2, and the primer sequences for synthesizing the downstream homologous arm KydaP-B are shown in SEQ ID No. 3-4, respectively; The synthesis of the second target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm KalsSD-A and downstream homologous arm KalsSD-B are synthesized using primers, and the upstream homologous arm KalsSD-A and downstream homologous arm KalsSD-B are sequentially ligated using overlap PCR technology to obtain the second target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KalsSD-A are shown in SEQ ID No. 7-8, and the primer sequences for synthesizing the downstream homologous arm KalsSD-B are shown in SEQ ID No. 9-10, respectively; The synthesis of the third target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, primers are used to synthesize the upstream homologous arm ald1-A, the downstream homologous arm ald1-B, the ald1 gene, and the terminator TamyL; using the genome of Bacillus subtilis as a template, primers are used to synthesize the promoter P. 43u12 The upstream homologous arm ald1-A and promoter P were linked by overlap PCR technology. 43u12 The ald1 gene, terminator TamyL, and downstream homologous arm ald1-B are sequentially linked to obtain the third target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm ald1-A are shown in SEQ ID No. 13-14, and the promoter P is synthesized. 43u12 The primer sequences for synthesizing the ald1 gene are shown in SEQ ID No. 25-26, the primer sequences for synthesizing the terminator TamyL are shown in SEQ ID No. 19-20, and the primer sequences for synthesizing the downstream homologous arm ald1-B are shown in SEQ ID No. 15-16. The synthesis of the fourth target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm KoxdC-A and the downstream homologous arm KoxdC-B are synthesized using primers; and ald is synthesized using standard gene synthesis techniques. SD Genes were synthesized using overlap PCR technology, which involved the upstream homologous arms KoxdC-A and ald. SD The gene and the downstream homologous arm KoxdC-B are sequentially linked to obtain the fourth target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KoxdC-A are shown in SEQ ID No. 29-30, and the primer sequences for synthesizing the downstream homologous arm KoxdC-B are shown in SEQ ID No. 31-32, respectively; The synthesis of the fifth target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, synthesizing the upstream homologous arm KpflAB-A and the downstream homologous arm KpflAB-B using primers, and synthesizing ald using standard gene synthesis techniques. SD Genes, using overlap PCR technology, were used to connect the upstream homologous arms KpflAB-A and ald. SD The gene and the downstream homologous arm KpflAB-B are sequentially linked to obtain the fifth target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KpflAB-A are shown in SEQ ID No. 35-36, and the primer sequences for synthesizing the downstream homologous arm KpflAB-B are shown in SEQ ID No. 37-38, respectively; The synthesis of the sixth target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm Kxpt-A and the downstream homologous arm Kxpt-B are synthesized using primers; and ALaDC is synthesized using standard gene synthesis techniques. E325K Genes, using overlap PCR technology, were used to combine the upstream homologous arms Kxpt-A and ALaDC. E325K The gene and the downstream homologous arm Kxpt-B were sequentially linked to obtain the sixth target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm Kxpt-A are shown in SEQ ID No. 42-43, and the primer sequences for synthesizing the downstream homologous arm Kxpt-B are shown in SEQ ID No. 44-45, respectively; The synthesis of the seventh target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, the upstream homologous arm KydaP-A and the downstream homologous arm KydaP-B are synthesized using primers; and ALaDCE is synthesized using standard gene synthesis techniques. 325K Genes were synthesized using overlap PCR technology, which involved the upstream homologous arms KydaP-A and ALaDCE. 325K The gene and the downstream homologous arm KydaP-B are sequentially linked to obtain the 7th target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KydaP-A are shown in SEQ ID No. 48-49, and the primer sequences for synthesizing the downstream homologous arm KydaP-B are shown in SEQ ID No. 50-51, respectively. The synthesis of the eighth target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm KkipR-A and downstream homologous arm KkipR-B are synthesized using primers; the GMAS gene is synthesized using standard gene synthesis technology; and the upstream homologous arm KkipR-A, the GMAS gene, and the downstream homologous arm KkipR-B are sequentially ligated using overlap PCR technology to obtain the eighth target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm KkipR-A are shown in SEQ ID No. 55-56, and the primer sequences for synthesizing the downstream homologous arm KkipR-B are shown in SEQ ID No. 57-58, respectively; The synthesis of the 9th target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm Kcyd-A and downstream homologous arm Kcyd-B are synthesized using primers, and the upstream homologous arm Kcyd-A and downstream homologous arm Kcyd-B are sequentially ligated using overlap PCR technology to obtain the 9th target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm Kcyd-A are shown in SEQ ID No. 61-62, and the primer sequences for synthesizing the downstream homologous arm Kcyd-B are shown in SEQ ID No. 63-64, respectively; The synthesis of the 10th target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, primers are used to synthesize the upstream homologous arm ndh-A, the downstream homologous arm ndh-B, the ndh gene, and the terminator TamyL; using the genome of Bacillus subtilis as a template, primers are used to synthesize the promoter P. 43u12 The upstream homologous arm ndh-A and promoter P were combined using overlap PCR technology. 43u12 The ndh gene, terminator TamyL, and downstream homologous arm ndh-B are sequentially linked to obtain the 10th target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm ndh-A are shown in SEQ ID No. 67-68, and the promoter P is synthesized. 43u12 The primer sequences for synthesizing the ndh gene are shown in SEQ ID No. 25-26, the primer sequences for synthesizing the ndh gene are shown in SEQ ID No. 71-72, the primer sequences for synthesizing the terminator TamyL are shown in SEQ ID No. 19-20, and the primer sequences for synthesizing the downstream homologous arm ndh-B are shown in SEQ ID No. 69-70. The synthesis of the 11th target fragment includes the following steps: using the genome of Bacillus licheniformis DW2 as a template, upstream homologous arm Kggt-A and downstream homologous arm Kggt-B are synthesized using primers, and the upstream homologous arm Kggt-A and downstream homologous arm Kggt-B are sequentially ligated using overlap PCR technology to obtain the 11th target fragment; wherein, the primer sequences for synthesizing the upstream homologous arm Kggt-A are shown in SEQ ID No. 76-77, and the primer sequences for synthesizing the downstream homologous arm Kggt-B are shown in SEQ ID No. 78-79, respectively; The NCBI accession number for the genome of Bacillus subtilis is NC_000964.

3.

5. The engineered strain as described in claim 4, characterized in that, The ald SD The nucleotide sequence of the gene is shown in SEQ ID No. 28; The ALaDC E325K The nucleotide sequence of the gene is shown in SEQ ID No. 41; The nucleotide sequence of the GMAS gene is shown in SEQ ID No.

54.

6. The use of the engineered strain with high L-theanine production as described in any one of claims 1 to 5 in the preparation of L-theanine.

7. A method for preparing L-theanine, characterized in that, Includes the following steps: The engineered strain that produces high levels of L-theanine as described in any one of claims 1 to 5 is inoculated into a fermentation medium and fermented to obtain a fermentation broth containing L-theanine.

8. The method as described in claim 7, characterized in that, The fermentation medium contains the following components: 6 g / L tryptone, 12 g / L yeast extract, 6 g / L (NH4)2SO4, 12.5 g / L K2HPO4•3H2O, 2.5 g / L KH2PO4, 10 mL / L buffer solution, and 60 g / L glucose.

9. The method as described in claim 8, characterized in that, The buffer solution contains the following components: FeSO4•7H2O 4 g / L, CaCl2 4 g / L, MnSO4•5H2O 1 g / L, CoCl2•6H2O 0.4 g / L, NaMoO4•2H2O 0.2 g / L, ZnSO4•7H2O 0.2 g / L, AlCl3•6H2O 0.1 g / L, CuCl2•H2O 0.1 g / L, and H3BO3 0.05 g / L.

10. The method as described in claim 7, characterized in that, The conditions for the fermentation process include: the inoculum size of the engineered strain is 2%-10%, the fermentation temperature is 35-40 ℃, and the rotation speed is 200-250 rpm.

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