γ-Glutamine synthetase mutant and its application in the production of theanine

Abstract

The present invention discloses a γ-glutamylformamide synthetase mutant, which has higher theanine synthesis activity and can improve the yield and conversion rate of theanine. The present invention also discloses a theanine production chassis strain obtained by introducing a gene construction such as tea tree alanine decarboxylase, which can endogenously synthesize ethylamine and can avoid the problems of safety and high cost caused by exogenous addition of ethylamine in the production process of theanine. The strain is used to express a highly active γ-glutamylformamide synthetase mutant, which is applied to the fermentation production of theanine, which can greatly improve the yield and conversion rate of theanine, improve safety, and reduce production costs, which is of great significance to the large-scale production of theanine.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and fermentation engineering, and specifically relates to a mutant of gamma-glutamylformamide synthetase and a theanine production strain capable of endogenously synthesizing ethylamine and applications thereof in the production of theanine. Background Art

[0002] Theanine (γ-glutamylethylamide) is a unique non-protein amino acid that occurs naturally in tea plants. In tea plants, theanine is synthesized by a special theanine synthase in the roots of tea plants using glutamate, ethylamine, and ATP as substrates, where ethylamine is generated from alanine under the action of alanine decarboxylase. Theanine is the most abundant free amino acid in tea leaves, accounting for more than 50% of the total free amino acids. Theanine has the effects of promoting relaxation, improving concentration, preventing vascular diseases, and lowering blood pressure. It has been approved as a safe (GRAS) ingredient by the U.S. Food and Drug Administration (FDA) and has been commercially developed as a valuable ingredient for use in food and beverages to improve or maintain human health.

[0003] At present, the production methods of theanine mainly include tea extraction, chemical synthesis and biological methods. The separation and extraction of high-purity theanine from tea requires a large amount of tea raw materials, the operation process is complicated, time-consuming and cost-effective. The chemical synthesis of theanine has problems such as difficulty in isomer separation. In addition, it is difficult to be accepted by consumers for safety reasons and is not allowed to be used in the food industry. The biological preparation of theanine includes in vivo microbial fermentation and in vitro enzymatic conversion. The enzymatic conversion method synthesizes L-theanine, which includes at least two processes: the cultivation of enzyme-producing strains and the preparation of L-theanine by enzymatic reaction. It also requires the use of expensive glutamate and ATP as raw materials. Therefore, the production cost is difficult to reduce, and it has no advantages over the extraction method and chemical method. The microbial fermentation method has the advantages of simple culture conditions, short cycle, simple operation, green environmental protection, low raw materials and production costs, and has better industrial application value and prospects.

[0004] Theanine is prepared by microbial fermentation. Theanine is synthesized in microbial cells using glutamate and ethylamine as precursors under the action of enzymes. γ-glutamylmethylamide synthetase (GMAS) was originally an enzyme that catalyzes the synthesis of N-methyl-L-glutamine from L-glutamate and methylamine, but studies have found that some GMAS also have the catalytic function of combining ethylamine with the γ-amino group of L-glutamate to form L-theanine. Yamamoto S et al. found that in the presence of ATP, Methylovorus maysThe affinity of GMAS from NO.9 for ethylamine is 100 times higher than that for ammonia (Yamamoto S, Wakayama M. Characterization of theanine-forming enzyme from Methylovorus mays no. 9 in respect to utilization of theanine production.Biosci Biotechnol Biochem. 2007;71(2):545-552. doi:10.1271 / bbb.60590), the encoding gene was recombinantly expressed in Escherichia coli, and the enzyme production capacity of the recombinant strain was higher than that of the original strain. Methylovorus mays No.9 is 23 times higher, and the enzymatic properties of recombinant GMAS are not significantly different from those of natural GMAS, which makes it possible to use GMAS for large-scale preparation of L-theanine. At present, wild-type GMAS is used in fermentation production of theanine, but there are still problems such as insufficient yield and conversion rate. Therefore, molecular modification of GMAS can increase the yield and conversion rate of theanine and reduce costs, which is of great significance for large-scale fermentation production of theanine.

[0005] Theanine synthesis requires glutamate and ethylamine as precursors, among which the synthesis of ethylamine is the rate-limiting step in the production of theanine. The addition of ethylamine in vitro during the fermentation process can increase the production of theanine to a certain extent. However, the low boiling point of ethylamine makes it extremely volatile, which not only poses problems and hidden dangers in terms of safety and storage, but its toxicity to microorganisms also leads to a decrease in enzyme activity, making it difficult to increase conversion rate and yield. Therefore, by means of genetic engineering, an endogenous ethylamine synthesis engineered strain capable of synthesizing a large amount of ethylamine intracellularly is constructed as a base strain for theanine production, which can not only avoid the various risks and cytotoxicity caused by the addition of exogenous ethylamine, but also simplify the fermentation process, greatly reduce costs, and has the potential for large-scale promotion and application.

[0006] Therefore, on the one hand, GMAS is molecularly modified to increase the synthesis of theanine by increasing the activity of GMAS enzymes; on the other hand, an engineered strain that can endogenously synthesize ethylamine is constructed as the base bacteria for theanine production, which increases and accelerates the synthesis and conversion of ethylamine in the cell, removes the rate limit, and provides sufficient precursors for theanine synthesis. The above two points are of great significance for increasing the yield and conversion rate of theanine and reducing costs. Summary of the invention

[0007] One of the purposes of the present invention is to improve the yield and substrate conversion rate of theanine produced by microbial fermentation, and to improve the yield and substrate conversion rate of theanine produced by microbial fermentation. Methylovorus maysNO.9) was mutated and screened to obtain a GMAS mutant with high enzyme activity (enzyme activity, enzyme activity, reaction enzyme activity, enzyme function, catalytic function, etc. or similar expressions with the same meaning mentioned later in the present invention, unless otherwise specified, all refer to the activity or function of catalyzing the synthesis of theanine from glutamate and ethylamine).

[0008] In order to achieve the above object, the present invention provides a γ-glutamylformamide synthetase mutant, which is derived from Methylophaga ( Methylovorus mays NO.9) by performing an amino acid substitution mutation on the γ-glutamylformamide synthetase GMAS, wherein the amino acid sequence of the mutant is based on the amino acid sequence shown in SEQ ID NO: 1, and a mutation is performed on the following sites:

[0009] 1) The mutation of amino acid 126 from E to A;

[0010] 2) The mutation of amino acid 186 from E to K;

[0011] 3) The mutation of amino acid 193 from E to A;

[0012] 4) The mutation of amino acid 321 from R to A.

[0013] The present invention provides a nucleic acid molecule encoding the γ-glutamylformamide synthetase mutant.

[0014] The present invention provides biological materials containing the nucleic acid molecule, and the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, virus vectors or engineered bacteria.

[0015] The present invention provides a recombinant microorganism comprising the mutant or the nucleic acid molecule.

[0016] The recombinant microorganism can be constructed by introducing a nucleic acid molecule encoding the γ-glutamylformamide synthetase mutant into Escherichia coli via a plasmid or integrating the nucleic acid molecule into the chromosome of Escherichia coli via genetic engineering.

[0017] Alanine decarboxylase CsAlaDC, which is unique to the tea tree (Camellia sinensis), can convert alanine into ethylamine, which is a precursor for theanine synthesis and is crucial to the synthesis of theanine in tea trees. Although this ethylamine synthesis pathway does not exist in most microorganisms, the alanine synthesis pathway exists naturally in bacterial cells such as Escherichia coli. If CsAlaDC, which can decarboxylate alanine to produce ethylamine, is introduced through genetic engineering, the problem of endogenous synthesis of ethylamine can be solved, thereby improving the safety of fermentation production of theanine and reducing costs.

[0018] Therefore, another object of the present invention is to provide an Escherichia coli strain expressing alanine decarboxylase CsAlaDC gene as a theanine producing strain.

[0019] The invention provides a theanine production strain, which is an Escherichia coli strain expressing an alanine decarboxylase CsAlaDC gene and further expressing the gamma-glutamylformamide synthetase mutant.

[0020] The invention provides a recombinant Escherichia coli strain comprising the above-mentioned γ-glutamylformamide synthetase mutant or a nucleic acid molecule encoding the mutant, and the strain expresses an alanine decarboxylase CsAlaDC gene.

[0021] The nucleotide sequence of the alanine decarboxylase CsAlaDC gene is shown in SEQ ID NO: 5.

[0022] Furthermore, in order to increase the endogenous synthesis of ethylamine, the ability of cells to synthesize alanine can also be enhanced by genetic engineering. The alanine synthesis and metabolic pathways involve numerous catalytic enzymes. After many experiments, the present invention found that by introducing Bifidobacterium Bifidobacterium adolescentis phosphoketolase gene xfp and Bacillus sphaericus Lysinibacillus sphaericus Alanine dehydrogenase gene alD , which can greatly increase the production of alanine.

[0023] Therefore, in order to achieve the above further object, the present invention provides an Escherichia coli strain expressing alanine decarboxylase CsAlaDC gene, wherein the strain further expresses a phosphoketolase gene xfp and alanine dehydrogenase gene alD。

[0024] The invention provides a theanine production strain, which expresses the phosphoketolase gene xfp、 Alanine dehydrogenase gene alD and an Escherichia coli strain carrying the alanine decarboxylase CsAlaDC gene, the strain also expressing the above-mentioned γ-glutamylformamide synthetase mutant.

[0025] The present invention provides a recombinant Escherichia coli strain comprising the above-mentioned γ-glutamylformamide synthetase mutant or a nucleic acid molecule encoding the mutant, wherein the strain is integrated with a phosphoketolase gene xfp and alanine dehydrogenase gene alD And express alanine decarboxylase CsAlaDC gene.

[0026] The nucleotide sequence of the alanine decarboxylase CsAlaDC gene is shown in SEQ ID NO: 5.

[0027] The phosphoketolase gene xfp The nucleotide sequence is shown in SEQ ID NO: 3.

[0028] Alanine dehydrogenase gene alD The nucleotide sequence is shown in SEQ ID NO: 4.

[0029] Glutathione hydrolase gene from Escherichia coli ggt (nucleotide sequence as shown in SEQ ID NO: 6) can hydrolyze molecules containing γ-glutamyl including theanine. In order to further increase the yield of theanine, the present invention further knocks out the above-mentioned Escherichia coli or theanine production strain ggt Gene to reduce theanine degradation, increase theanine accumulation and improve yield.

[0030] The present invention provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli expresses a γ-glutamylformamide synthetase gene, a phosphoketolase gene, an alanine dehydrogenase gene and an alanine decarboxylase gene, and knocks out a glutathione hydrolase gene, wherein the nucleotide sequence of the phosphoketolase gene is shown in SEQ ID NO: 3, the nucleotide sequence of the alanine dehydrogenase gene is shown in SEQ ID NO: 4, the nucleotide sequence of the alanine decarboxylase gene is shown in SEQ ID NO: 5, and the nucleotide sequence of the glutathione hydrolase gene is shown in SEQ ID NO: 6.

[0031] The present invention provides use of the γ-glutamylformamide synthetase mutant, the biological material, the recombinant microorganism, the Escherichia coli strain or the theanine production strain in producing theanine.

[0032] The present invention provides a method for producing theanine by fermentation, characterized in that the method comprises using the γ-glutamylformamide synthetase mutant, the biological material, the recombinant microorganism, the Escherichia coli strain or the theanine producing strain.

[0033] The theanine production method does not require exogenous addition or supplementation of ethylamine during the fermentation process.

[0034] In the above-mentioned method for producing theanine by fermentation, the fermentation medium used is composed of the following: glucose 10-50 g / L, peptone 1-5 g / L, yeast powder 2-8 g / L, glutamic acid 0.2-2.0 g / L, citric acid 0.2-2.0 g / L, potassium hydroxide 1-4 g / L, 85%, phosphoric acid 0.4-3.3 g / L, magnesium sulfate 0.2-1.2 g / L, ferrous sulfate 2-20 mg / L, manganese sulfate 1-20 mg / L, CaCl2 1-20 mg / L, and the rest is water, pH 7.0-7.2.

[0035] The fermentation culture has the following conditions: the inoculation amount is 5-20%, the pH is stabilized at 6.5-7.5, the temperature is maintained at 30-45°C, the dissolved oxygen is between 0-10%, and the fermentation period is 20-60 hours.

[0036] The present invention utilizes a γ-glutamylformamide synthetase mutant and / or a recombinant Escherichia coli capable of endogenously generating ethylamine to ferment and produce theanine, which not only improves the yield and conversion rate of theanine, but also solves the safety problem caused by exogenous addition of ethylamine, effectively improves production efficiency and reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the relative enzyme activity of γ-glutamylformamide synthetase mutants;

[0038] Figure 2 It is the fermentation tank growth curve;

[0039] Figure 3 Theanine production results for fermenters. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific examples. Where specific experimental conditions are not indicated, they are all based on conventional conditions well known to those skilled in the art.

[0041] Table 1. Plasmids used and constructed in the present invention

[0042]

[0043] Table 2. Strains used and constructed in the present invention

[0044]

[0045] Table 3. Primers used for constructing plasmids and gene editing in the present invention

[0046]

[0047] Example 1 Construction of γ-glutamylformamide synthetase mutant

[0048] (1) The E. coli expression vector pTrc99a with a promoter was used to express the gene from Methylophaga Methylovorus maysNO.9) was expressed by expressing γ-glutamylformamide synthetase GMAS (amino acid sequence as shown in SEQ ID NO: 1) to obtain the vector pTrc99a-GMAS, and the nucleotide coding sequence of the GMAS is shown in SEQ ID NO: 2.

[0049] (2) Using pTrc99a-GMAS as a template, the nucleotide coding sequence of GMAS was amplified by PCR using the four pairs of mutant primers shown in Table 1 (SEQ ID NO: 7-14). The amplified products were PCR amplified using Dpn I After enzyme digestion, the gene was transferred into E. coli DH5α competent cells and spread on LB solid plate medium (Kan, 50 µg / mL) for screening.

[0050] (3) Colony PCR verification was performed using primers pTrc99a-JD-F and pTrc99a-JD-R, and the PCR products were sequenced. Single colonies with correct sequencing results were selected for activation and plasmid extraction to obtain plasmids containing mutant genes, pTrc99a-GMAS E126A , pTrc99a-GMAS E186K , pTrc99a-GMAS E193A , pTrc99a-GMAS R321A .

[0051] (4) The plasmids containing the mutant genes were respectively transferred into Escherichia coli W3110 to obtain the mutant recombinant strain W3110-GMAS E126A 、W3110-GMAS E186K 、W3110-GMAS E193A 、W3110-GMAS R321A At the same time, the unmutated vector pTrc99a-GMAS was transformed into Escherichia coli W3110 to obtain the recombinant strain W3110-GMAS expressing wild-type GMAS as a control.

[0052] Example 2 Activity test of γ-glutamylformamide synthetase mutant

[0053] (1) Expression of recombinant proteins

[0054] The mutant recombinant strain and the control recombinant strain constructed in Example 1 were inoculated in LB medium containing 50 μg / mL kanamycin, respectively, and cultured at 37°C with shaking. When the bacterial solution concentration reached OD 600 When the concentration was 0.6-0.8, the cells were cultured at 30℃ for 20 hours and then collected by centrifugation.

[0055] (2) Activity test of γ-glutamylformamide synthetase mutant

[0056] 10 g of the γ-glutamylformamide synthetase bacterial cells collected above were taken respectively, resuspended in water, and then disrupted by a high-pressure homogenizer at 800-1000 bar. The obtained cell disrupted liquid was used as the crude enzyme liquid of γ-glutamylformamide synthetase and added into 1 L enzyme activity reaction system for enzyme activity determination.

[0057] The enzyme activity reaction system consists of: Tris-HCl 100 mM, glutamate 30 mM, ethylamine 30 mM, ATP 30 mM, magnesium sulfate 10 mM, sodium hexametaphosphate 20 mM, crude enzyme solution (cell disruption solution obtained by adding 10 g of bacteria to each liter of reaction system), and the reaction conditions are 32°C and pH 8.0. After the reaction is completed, an equal volume of 1M HCl is added to terminate the reaction. The reaction samples are detected by HPLC and the relative enzyme activity of each mutant is calculated.

[0058] The results are as follows Figure 1 As shown, compared with the wild type, mutant GMAS E126A and GMAS R321A The enzyme activities of the mutant GMAS were significantly improved. E126A The enzyme activity was increased by about 30% compared with the wild type.

[0059] Example 3 Construction of ethylamine-synthesizing strain

[0060] tea tree( Camellia sinensis ) can convert alanine into ethylamine, which is a precursor for theanine synthesis and is crucial to the synthesis of theanine in tea trees. Although this ethylamine synthesis pathway does not exist in most microorganisms, alanine synthesis pathways exist naturally in bacterial cells such as Escherichia coli. If CsAlaDC, which can decarboxylate alanine to produce ethylamine, is introduced through genetic engineering, the problem of endogenous synthesis of ethylamine can be solved.

[0061] Furthermore, in order to increase the endogenous synthesis of ethylamine, the ability of cells to synthesize alanine can also be enhanced by genetic engineering. The alanine synthesis and metabolic pathways involve numerous catalytic enzymes. After many experiments, the present invention found that by introducing Bifidobacterium Bifidobacterium adolescentis phosphoketolase gene xfp (nucleotide sequence as shown in SEQ ID NO: 3) and Bacillus sphaericus Lysinibacillus sphaericus Alanine dehydrogenase gene alD (The nucleotide sequence is shown in SEQ ID NO: 4), which can greatly increase the production of alanine. Based on this, by expressing CsAlaDC from tea tree (the nucleotide coding sequence is shown in SEQ ID NO: 5), a strain capable of endogenously synthesizing a large amount of ethylamine can be constructed. One of the specific implementations is as follows:

[0062] 1. Integration using a CRISPR / Cas9-based scarless editing method xfp Genes and alD Gene

[0063] (1) Construction of linear recombinant fragments

[0064] pTrc- alD -12345 linear recombinant fragment construction: using the E. coli W3110 genome as a template, using primers pTrc- alD -UF / pTrc- alD -UR and pTrc- alD -DF / pTrc- alD -DR respectively amplified the upstream and downstream homology arms, and recorded them as fragments pTrc- alD -1 and pTrc- alD -5; using plasmid pSTC as template, using primers pTrc- alD -Tet-F / T2 and T1 / pTrc- alD -Tet-R segmentally amplified the Tet resistance gene fragment, which was recorded as pTrc- alD -2 and pTrc- alD -3; then with Bacillus sphaericus Lysinibacillus sphaericus Alanine dehydrogenase gene alD (nucleotide sequence as shown in SEQ ID NO: 4) as a template, using primers pTrc- alD -F1 / pTrc- alD -F2 / pTrc- alD -R was amplified in two steps alD Gene fragment, denoted as fragment pTrc- alD -4; then fragment pTrc- alD -1 and pTrc- alD -2 was obtained by fusion PCR to obtain the fragment pTrc- alD -12, fragment pTrc- alD -3 and pTrc- alD -4 obtained fragment pTrc- alD -34, fragment pTrc- alD -34 and pTrc- alD -5 was obtained by fusion PCR to obtain the fragment pTrc- alD -345; the last fragment pTrc- alD -12 and pTrc- alD -345 was obtained by fusion PCR to obtain the linear recombinant fragment pTrc- alD -12345.

[0065] The PCR reaction system and parameters were referred to the instruction manual of Takara Primer STAR Max DNA Polymerase. Specifically, the Touch-Down PCR program was used: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 30 s, annealing at 65°C for 15 s, and extension at 72°C for 1 min (15 s / kb, depending on the fragment size), with the annealing temperature decreasing by 1°C in each cycle for 10 cycles until it reached 55°C; denaturation at 98°C for 30 s, annealing at 65°C for 15 s, and extension at 72°C for 1 min for 20 cycles; extension at 72°C for 10 min, and storage at 4°C.

[0066] pTrc- xfp -12345 linear recombinant fragment construction: refer to the above pTrc- alD -12345, using the corresponding primers in Table 3, a triple fusion PCR was performed to obtain a gene carrying Bifidobacterium Bifidobacterium adolescentis phosphoketolase gene xfp (nucleotide sequence as shown in SEQ ID NO: 3) linear recombinant fragment pTrc- xfp -12345. The PCR reaction system and parameters were the same as above.

[0067] (2) Transformation of recombinant fragments

[0068] The auxiliary plasmid pKD46 was electroporated into the W3110 competent cells. The culture medium and preparation process required for competent cells were prepared according to the conventional standard operation. alD -12345 was electrotransformed into W3110 recombinant electrocompetent cells containing the auxiliary plasmid pKD46, and spread on LB solid plate medium (Tet, 10 µg / mL; Spc, 50 µg / mL), and cultured in a 30 ℃ incubator for 12-16 h. Single colonies were picked and cloned using primers pTrc- alD -UF / pTrc- alD -DR colony PCR was performed to verify the positive clone strains.

[0069] (3) Pop-up of screening marker (Tet)

[0070] Inoculate the verified positive single colony into 3 mL LB liquid medium (IPTG, 2 mM; arabinose, 4 g / L; Spc, 50 µg / mL) and place it in a 30 ℃ shaker for induction for about 6-8 hours. After the induction, dip a small amount of bacterial liquid and streak it on LB solid plate medium (arabinose, 4 g / L; Spc, 50 µg / mL) for rescreening. Then pick the single colony on the plate and perform a spot plate experiment on the LB plate medium with or without Tet resistance to screen the colony that successfully pops out the selection marker Tet, that is, the colony that grows in the medium without Tet resistance but does not grow in the medium containing Tet resistance (integrated alD gene and containing the helper plasmid pKD46 W3110-pTrc- alD ).

[0071] (4) Elimination of auxiliary plasmids

[0072] The colony popped out by the screening marker was inoculated into LB liquid medium and placed in a 42 ℃ shaker for overnight culture. Take the overnight cultured bacterial liquid and perform streak culture on LB solid plate medium. After a single colony grows, pick a single colony and perform a spot plate experiment on LB solid plate medium containing 50 µg / mL Spc resistance. Compare and observe the growth of the colonies in the two culture media, and screen out the colonies that grow in LB but not in LB medium containing Spc, that is, the colonies that have successfully eliminated the helper plasmid. Activate and preserve the strain in a glycerol tube, and you will get the integrated alD Escherichia coli strain W3110-pTrc- alD .

[0073] (5) pTrc- xfp Integration of the linear recombinant fragment pTrc-12345: Refer to the operation of steps (2) to (4) and integrate the linear recombinant fragment pTrc-12345 constructed in step (1) into xfp -12345 was electrotransformed into W3110-pTrc-containing auxiliary plasmid pKD46 obtained in step (3) alD The electroporated competent cells prepared by the recombinant strains were spread on LB solid plate medium (Tet, 10 µg / mL; Spc, 50 µg / mL) and cultured in a 30 ℃ incubator for 12-16 h. xfp -UF / pTrc- xfp -DR was used for colony PCR verification to obtain positive clone strains. The clone strains that have been verified to be positive were popped out of the screening marker (Tet) and the auxiliary plasmid was eliminated to obtain the integrated alD and xfp Escherichia coli strain W3110-pTrc- alD -pTrc-xfp .

[0074] 2. Construction of ethylamine-synthesizing strain

[0075] (1) Construction of pRB1C-CsAlaDC plasmid

[0076] Using tea plant CsAlaDC (nucleotide coding sequence as shown in SEQ ID NO: 5) as a template, PCR amplification was performed using the primer pair CsAlaDC-F / CsAlaDC-R in Table 3 to obtain the CsAlaDC gene fragment, and the gel block was recovered; using the pRB1C vector as a template, the primer pair pRB1C-ZT-F / pRB1C-ZT-R was used to amplify the assembly vector, and the amplified product was purified by Dpn I After enzyme digestion, the DNA was recovered and cloned using the Hieff Clone® Universal II One Step Cloning Kit. The DNA was then transferred into E. coli DH5α competent cells and plated on LB solid plate medium (Cm, 24 µg / mL) for screening. Colony PCR was performed using primers pRB1C-JD-F and pRB1C-JD-R, and the PCR product was sequenced. The single colony with the correct sequencing result was selected for activation and plasmid extraction to obtain pRB1C-CsAlaDC.

[0077] (2) Electroporate the pRB1C-CsAlaDC plasmid into W3110-pTrc- alD -pTrc- xfp strains, and spread them on LB solid plate medium (Cm, 24 µg / mL), and cultured in a 37 ℃ incubator overnight. Single colonies were picked and colony PCR was performed using primers CsAlaDC-JD-F / CsAlaDC-JD-R to obtain the positive clone strain W3110-pTrc- alD -pTrc- xfp (pRB1C-CsAlaDC), denoted as T1.

[0078] Example 4 Construction of basic strains for reducing theanine degradation

[0079] Escherichia coli ggt The glutathione hydrolase encoded by the gene (nucleotide sequence as shown in SEQ ID NO: 6) can hydrolyze molecules containing γ-glutamyl, including L-theanine. Therefore, in order to further increase the production of theanine, the present invention knocked out the Escherichia coli ggt Gene to reduce the degradation reaction of theanine.

[0080] (1) Δ ggt-1234 linear recombinant fragment construction: using W3110 genome as template, using primers Δ ggt -UF / Δ ggt -UR and Δ ggt -DF / Δ ggt -DR amplifies the upstream and downstream homology arms respectively, and is recorded as fragment Δ ggt -1 and Δ ggt -4. Using plasmid pSTC as template, primer Δ ggt -Tet-F / Tet-R and Tet-F / Δ ggt -Tet-R segmentally amplifies the Tet resistance gene fragment, denoted as Δ ggt -2 and Δ ggt -3. Then the fragment Δ ggt -1 and Δ ggt -2 obtained by fusion PCR ggt -12, fragment Δ ggt -3 and Δ ggt -4 was obtained by fusion PCR ggt -34. Last segment Δ ggt -12 and Δ ggt -34 was subjected to fusion PCR to obtain a linear recombinant fragment Δ ggt The PCR reaction system and parameters were the same as those in Example 3.

[0081] (2) Δ ggt -1234 linear recombinant fragment integration: refer to Example 3 pTrc- alD -12345 transformation and integration method (based on CRISPR / Cas9 scarless editing method), the constructed linear recombinant fragment Δ ggt -1234 was electrotransformed into W3110-pTrc- alD -pTrc- xfp (pRB1C-CsAlaDC) recombinant electroporated competent cells and spread on LB solid plate medium (Tet, 10 µg / mL; Spc, 50 µg / mL, Cm, 24 µg / mL), and cultured in a 30°C incubator for 12-16 h. Pick a single colony and use primers Δ ggt -UF / Δ ggt -DR colony PCR verification was performed to obtain positive clone strains. The clone strains that have been verified positive were popped out of the screening marker (Tet) and the auxiliary plasmid was eliminated to obtain knockout ggt Escherichia coli strain W3110 - pTrc- alD -pTrc- xfp -Δ ggt(pRB1C-CsAlaDC), denoted as T2, was used as the fermentation strain for theanine production.

[0082] Example 5 Fermentation production of theanine

[0083] 1. Construction of fermentation strain:

[0084] The wild-type GMAS expression plasmid pTrc99a-GMAS and the mutant expression plasmid pTrc99a-GMAS obtained in Example 1 were E126A and pTrc99a-GMAS R321A They were electrotransformed into theanine-producing strain T2 and plated on LB solid plate medium (Cm, 24 µg / mL; Kan, 50 µg / mL) for screening. After overnight culture, single colonies were picked and GMAS-JD-F / GMAS-JD-R and CsAlaDC-JD-F / CsAlaDC-JD-R were verified by colony PCR using identification primers. Single colonies with correct results were selected for activation and stored in glycerol tubes, which were recorded as T3, T4, and T5, respectively (see Table 2).

[0085] 2. Fermentation in fermenter:

[0086] (1) Seed preparation: Take out the preserved strains T3, T4, and T5, first streak them on LB solid plate medium (Cm, 24 µg / mL; Kan, 50 µg / mL) and place them in a 37 ℃ incubator for culture. The next day, pick out several single colonies with good growth and streak them again for subculture, and place them in a 37 ℃ incubator for overnight growth for about 16 hours. Then draw 10 mL of sterile water onto the solid plate medium of the second-generation culture to wash off all colonies (no abnormal colonies), thereby obtaining a bacterial suspension;

[0087] (2) Seed culture: Inoculate the bacterial suspension into LB liquid medium (Cm, 24 µg / mL; Kan, 50 µg / mL) at a 5% inoculum volume. After culturing for 4 h, transfer it to the seed culture medium. Keep the pH stable at around 7.0, the temperature constant at 37°C, and the dissolved oxygen at around 30% for seed culture.

[0088] The seed culture medium comprises: 2 g / L yeast powder, 6 g / L peptone, 1 g / L beef extract, 1 g / L magnesium sulfate, 3 g / L potassium dihydrogen phosphate, 1 g / L potassium hydrogen phosphate, 50 mg / L ferrous sulfate, 0.5 mg / L VB1, 1 mg / L VB3, 1 mg / L VB5, 0.5 mL / L defoamer, 24 µg / mL Cm, and 50 µg / mL Kan.

[0089] (3) Fermentation: Inoculate 7 L of fresh fermentation medium with 10% of the seed culture to start fermentation. During the fermentation process, the pH was kept stable at 7.0, the temperature was maintained at 37°C, and the dissolved oxygen was between 10% and 20%. 600 =15, 0.5 g / L inducer arabinose was added, and the fermentation period was 40 h. After the fermentation, the theanine content was measured. No exogenous ethylamine was added during the entire fermentation process.

[0090] The fermentation medium is composed of: 40 g / L glucose, 1 g / L peptone, 2 g / L yeast powder, 0.5 g / L glutamic acid, 1.5 g / L citric acid, 1.8 g / L potassium hydroxide, 3.2 g / L 85% phosphoric acid, 0.6 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 1 mg / L manganese sulfate, 20 mg / L CaCl2, 0.5 mL / L defoamer, and the rest is water, with a pH of 7.0-7.2.

[0091] During the fermentation process, the growth was monitored based on the readings of the dissolved oxygen electrode and the offline UV spectrophotometer. Concentrated ammonia was added during the fermentation process to maintain the pH at around 7.0. Before 20 h of fermentation, the glucose concentration was maintained at 2-5 g / L, and the glucose concentration was maintained at 0.5-1 g / L during 20-40 h of fermentation. L-theanine was detected using Shimadzu liquid HPLC equipped with a UV detector. The chromatographic conditions were as follows: chromatographic column: YMC-C18 column (4.6×250 mm, 5 μm); mobile phase: 5 mM sulfuric acid, isocratic elution, flow rate 0.6 mL / min, detection wavelength 210 nm; injection volume: 2 µL. The fermentation results are shown in the figure. Figure 2 , Figure 3 shown.

[0092] from Figure 3 It can be seen that after 40 h of fermentation, the mutant GMAS was expressed E126A The production strain T4 can produce 112.3 g / L of theanine, expressing mutant GMAS R321A The theanine production of the production strain T5 can reach 96.3g / L, which is 59.7% and 37% higher than the theanine production of 70.3 g / L of the control strain T3 expressing wild-type GMAS; in addition, the sugar-acid conversion rates of T4 and T5 are also increased by 19% and 10.3% respectively compared with T3 expressing wild-type GMAS, which not only greatly improves the theanine production conversion rate, but also effectively reduces the production cost.

[0093] from Figure 2 The fermentation growth curve combined with Figure 3 The yield results show that the control strain T3 expressing wild-type GMAS grew too vigorously between 24 and 40 h, and the final OD600 As high as about 80, but its theanine production was much lower than that of T4 and T5, which indicates that the metabolic flow of the control strain is more directed to the accumulation of biomass (bacterial growth and reproduction) rather than the synthesis and accumulation of products. E126A and GMAS R321A The production strains T4 and T5 gradually entered the stable period after about 15 hours of culture. The bacterial concentration tended to be stable between 20 hours and 40 hours, and the main material and energy metabolism was concentrated on the synthesis and accumulation of the target product theanine. When the fermentation reached 40 hours, the OD of the T4 and T5 strains was 600 Maintained at around 50, much lower than T3, lower bacterial concentration and higher product synthesis and accumulation capabilities, the amount of impurities such as bacterial fragments and endogenous proteins in the final fermentation system of T4 and T5 is also much lower than that of T3, which is more conducive to the separation and purification of subsequent products.

[0094] In addition, the entire fermentation process does not require exogenous supplementation of ethylamine, which not only greatly reduces the cost but also effectively avoids the dangers caused by the storage and use of ethylamine.

[0095] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A γ-glutamylformamide synthetase mutant, characterized in that The mutant is obtained by performing the following mutations based on the amino acid sequence shown in SEQ ID NO: 1: the 126th amino acid is mutated from E to A or the 321st amino acid is mutated from R to A.

2. A nucleic acid molecule encoding the mutant according to claim 1.

3. A biological material containing the nucleic acid molecule according to claim 2, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector or engineered bacteria.

4. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli comprises the mutant according to claim 1 or the nucleic acid molecule according to claim 2.

5. The recombinant Escherichia coli according to claim 4, characterized in that The Escherichia coli expresses an alanine decarboxylase gene, and the nucleotide sequence of the alanine decarboxylase gene is shown in SEQ ID NO:

5.

6. The recombinant Escherichia coli according to claim 5, characterized in that The phosphoketolase gene and the alanine dehydrogenase gene are also expressed. The nucleotide sequence of the phosphoketolase gene is shown in SEQ ID NO: 3, and the nucleotide sequence of the alanine dehydrogenase gene is shown in SEQ ID NO:

4.

7. The recombinant Escherichia coli according to claim 6, characterized in that The glutathione hydrolase gene is further knocked out, and the nucleotide sequence of the glutathione hydrolase gene is shown in SEQ ID NO:

6.

8. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli expresses a γ-glutamylformamide synthetase gene, a phosphoketolase gene, an alanine dehydrogenase gene and an alanine decarboxylase gene, and knocks out a glutathione hydrolase gene, wherein the nucleotide sequence of the phosphoketolase gene is shown in SEQ ID NO: 3, the nucleotide sequence of the alanine dehydrogenase gene is shown in SEQ ID NO: 4, the nucleotide sequence of the alanine decarboxylase gene is shown in SEQ ID NO: 5, and the nucleotide sequence of the glutathione hydrolase gene is shown in SEQ ID NO:

6.

9. Use of the γ-glutamylformamide synthetase mutant according to claim 1, the nucleic acid molecule according to claim 2, the biological material according to claim 3 or the recombinant Escherichia coli according to any one of claims 4 to 8 in the production of theanine.

10. A method for producing theanine, characterized in that: Use the mutant according to claim 1, the nucleic acid molecule according to claim 2, the biological material according to claim 3 or the recombinant Escherichia coli according to any one of claims 4 to 8.

Images