L-theanine producing strain and construction method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN UNIV OF SCI & TECH
- Filing Date
- 2026-06-11
- Publication Date
- 2026-07-14
AI Technical Summary
Existing microbial fermentation processes suffer from problems such as insufficient ethylamine supply, unreasonable carbon flow distribution, byproduct accumulation, and low yield, resulting in high production costs and significant safety risks for L-theanine, making it difficult to achieve high-yield industrial production without exogenous ethylamine.
L-theanine-producing strain THE-17 was constructed. By knocking out glutathione hydrolase and glutamine permease genes and introducing γ-glutamylmethylamine synthase, Entner-Doudoroff pathway, phosphoryl ketolase, alanine dehydrogenase, alanine decarboxylase and transaminase genes, an endogenous ethylamine synthesis system was established, carbon flux and energy supply were optimized, and the L-theanine synthesis capacity was enhanced.
It achieves efficient L-theanine fermentation production without the need for exogenous ethylamine, significantly reducing safety risks and raw material costs, increasing yield, conversion rate and production intensity, adapting to high-density fermentation, and possessing potential for industrial application.
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Figure CN122381987A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of bioengineering and fermentation engineering technology, and in particular to an L-theanine producing strain, its construction method, and its application. Background Technology
[0002] L-Theanine (N-ethyl-L-glutamine) is a non-protein amino acid unique to tea. It possesses physiological functions such as sedation, lowering blood pressure, and improving sleep, and is widely used in food, beverages, health products, and cosmetics. There are two main types of existing microbial processes for producing L-theanine: One type is the enzymatic conversion process: This utilizes L-theanine synthase to catalyze the reaction of L-glutamate (or L-glutamine) with ethylamine in vitro to produce L-theanine. This includes L-glutaminase (GLS), gamma-glutamyl transpeptidase (GGT), L-glutamine synthase (GS), gamma-glutamylmethylamide synthase (GMAS), and gamma-glutamylcysteine synthase (GCS). However, this method requires the addition of large amounts of exogenous ethylamine, which is a flammable, explosive, and toxic chemical, resulting in high storage and transportation costs, and potential residues that could affect product safety.
[0003] Another type is microbial fermentation technology: L-theanine is synthesized using genetically engineered strains, but it still generally relies on exogenous ethylamine, resulting in complex processes, high safety risks, and high production costs.
[0004] It is evident that while existing technologies have attempted to construct ethylamine synthesis pathways (such as the alanine decarboxylase pathway), they generally suffer from problems such as insufficient ethylamine supply, unreasonable carbon flow distribution, byproduct accumulation, and low yield. High-yield industrial production of L-theanine without the need for exogenous ethylamine has not yet been achieved. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide an L-theanine producing strain.
[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned L-theanine producing strain.
[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned L-theanine producing strain.
[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A strain of L-theanine-producing bacteria, named strain THE-17, is derived from wild-type Escherichia coli. Escherichia coli W3110 lacI Obtained through targeted modification of the starting strain: glutathione hydrolase gene knockout ggtand glutamine permease gene glnP Overexpression of γ-glutamyl methylamine synthase gene Mmgmas The eddeda gene, which encodes the 2-keto-3-deoxy-6-phosphoglucose cleavage pathway, is heterologously expressed from... Bifidobacterium adolescentis Phosphoryl ketonease gene Baxfp , originating from Mannheimia succiniciproducens Phosphoenolpyruvate kinase gene MspckA , originating from Bacillus subtilis 168 alanine dehydrogenase gene BsalaDH , originating from Camellia sinensis alanine decarboxylase gene CsalaDC , originating from Pseudomonas putida transaminase gene PpspuC , originating from Zymomonas mobilis pyruvate decarboxylase gene Zmpdc .
[0009] Preferably, the above-mentioned L-theanine producing strain, wherein Mmgmas The nucleotide sequence of the gene is shown in SEQ ID NO.1. ggt The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing. glnP The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing. eddeda The nucleotide sequence of the gene is shown in SEQ ID NO.4 of the sequence listing. Baxfp The nucleotide sequence of the gene is shown in SEQ ID NO.5 of the sequence listing. MspckA The nucleotide sequence of the gene is shown in SEQ ID NO.6 of the sequence listing. BsalaDH The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing. CsalaDC The nucleotide sequence of the gene is shown in SEQ ID NO.8 of the sequence listing. PpspuC The nucleotide sequence of the gene is shown in SEQ ID NO.9 of the sequence listing. Zmpdc The nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing.
[0010] The above method for constructing L-theanine-producing strains involves using *Escherichia coli* as the starting strain. Escherichia coli W3110 lacI Based on this foundation, targeted modifications will be carried out, with the specific steps as follows: (1) The integration of three copies originates from Methylovorus mays γ-glutamyl methylamine synthase gene Mmgmas ; (2) To prevent L-theanine from being broken down after entering the cell, the glutathione hydrolase gene was knocked out. ggt and glutamine permease gene glnP ; (3) To enhance the supply of precursors pyruvate and L-glutamate, a copy of the eddeda gene encoding the Entner-Doudoroff pathway (2-keto-3-deoxy-6-phosphogluconic acid cleavage pathway) was first integrated. This pathway can directly generate glyceraldehyde-3-phosphate and pyruvate from 6-phosphogluconic acid, which is shorter than the glycolytic synthesis pathway. Then, a gene from... Bifidobacterium adolescentis Phosphoryl ketonease gene Baxfp This avoids the carbon loss caused by the decarboxylation step in the formation of acetyl-CoA from pyruvate; then, a carbon source derived from... Mannheimia succiniciproducens Phosphoenolpyruvate kinase gene MspckA It increased the supply of ATP; (4) In order to achieve the autonomous synthesis of intracellular ethylamine, a copy of the substance derived from the cell was first integrated. Bacillus subtilis 168 alanine dehydrogenase gene BsalaDH It provides the precursor L-alanine for the synthesis of ethylamine; then it integrates three copies derived from Camellia sinensis alanine decarboxylase gene CsalaDC This allows for the synthesis of L-theanine without the addition of ethylamine. (5) To enhance the supply of ethylamine, a transamination pathway is introduced to synthesize ethylamine, specifically by integrating a copy derived from Pseudomonas putida transaminase gene PpspuC The acetaldehyde and the amino group provided by L-alanine are converted into ethylamine; then, the amino group derived from... Zymomonas mobilis pyruvate decarboxylase gene Zmpdc The pyruvate is converted to acetaldehyde; to enhance transaminase expression, two copies of the enzyme are then integrated. Pseudomonas putida transaminase gene PpspuC .
[0011] Preferably, the construction method of the above-mentioned L-theanine-producing strain includes the following specific steps: (1) Use trc Starter control Mmgmas Gene overexpression results in triple copying; (2) Knockout ggt Gene; (3) Knockout glnP Gene; (4) Use trc Starter control eddeda Gene overexpression; (5) Use trc Starter controlBaxfp Gene overexpression; (6) Use trc Starter control MspckA Gene overexpression; (7) Use trc Starter control BsalaDH Gene overexpression; (8) Use trc Starter control CsalaDC Gene overexpression results in triple copying; (9) Use trc Starter control Zmpdc Gene overexpression; (10) Use trc Starter control PpspuC Gene overexpression results in three copies.
[0012] Preferably, in the method for constructing the above-mentioned L-theanine-producing strain, the... trc The nucleotide sequence of the promoter is shown in SEQ ID NO.11 of the sequence listing.
[0013] Preferably, in the method for constructing the above-mentioned L-theanine producing strain, to prevent acetaldehyde accumulation from inhibiting the expression of pyruvate decarboxylase, the strain undergoes acetaldehyde tolerance evolution after construction.
[0014] Application of the above-mentioned L-theanine producing strains in the production of L-theanine.
[0015] Preferably, the above-mentioned application involves producing L-theanine using a fermentation method in a shake flask or fermenter.
[0016] Beneficial effects: The aforementioned L-theanine-producing strains achieve L-theanine accumulation by introducing γ-glutamylmethylamine synthetase, knocking out the L-theanine degradation pathway, enhancing the flux of the Entner-Doudoroff pathway, and introducing a phosphate ketonease pathway to optimize energy and carbon flux, constructing a dual-pathway endogenous ethylamine synthesis system. Combined with adaptive evolution to select acetaldehyde-tolerant strains, this achieves highly efficient L-theanine fermentation production without exogenous ethylamine. These strains use glucose as the sole carbon source and produce L-theanine through the endogenous ethylamine synthesis pathway, which can be widely used in the industrial preparation of food, cosmetics, and pharmaceutical raw materials. Specifically: 1. Completely eliminates the addition of exogenous ethylamine, thereby eliminating dependence on exogenous ethylamine and significantly reducing safety risks and raw material costs; 2. Construct an efficient endogenous ethylamine synthesis system. The dual-pathway ethylamine synthesis system ensures a continuous supply of ethylamine and improves the synthesis rate of L-theanine. 3. Optimize carbon flow and energy supply through the phosphoryl ketolase pathway, reduce byproduct accumulation, and increase L-theanine yield, conversion rate, and production intensity; 4. Improve the strain's tolerance to intermediate products (such as acetaldehyde) during fermentation to adapt to high-density fermentation and significantly improve the stability of the strain during fermentation. 5. Its output, conversion rate, and production intensity are all superior to existing technologies, and it has the potential for industrial application. Attached Figure Description
[0017] Figure 1 A diagram illustrating the process of modifying the de novo synthesis pathway for L-theanine-producing strains.
[0018] Figure 2 This is a diagram illustrating the evolution of acetaldehyde tolerance in THE-17.
[0019] Figure,3 The graph shows the feed-in fermentation process curve of the evolved THE-17. Detailed Implementation
[0020] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.
[0021] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.
[0022] The promoters and genes involved in the strain construction process in the examples are listed in the sequence listing, the strains involved are listed in Table 1, the primers involved are listed in Table 2, and the plasmids involved are listed in Table 3. The starting strain involved is wild-type *Escherichia coli*. Escherichia coli W3110 lacI References: Zhou, X.; Zhang, J.; Ren, M.; Li, J.; Wang, F.; Tian, S.; Zhao, G.; Zhang, C.; Li, Y. Systems Metabolic Engineering of Escherichia coli for Efficient de novo Biosynthesis of 2,5-Dimethylpyrazine from Glucose. J.Agric.Food Chem. 2026, 74 (11), 9628–9640. strain, named THE-0.
[0023] Table 1
[0024] Table 2
[0025]
[0026]
[0027]
[0028]
[0029]
[0030] Table 3
[0031] Example 1 This embodiment aims to illustrate the specific construction steps of strain THE-17, using wild-type Escherichia coli. Escherichia coli W3110 lacI As the starting strain, the metabolic pathway was modified as follows: Figure 1 As shown: (1) In yghX Pseudogene loci use trc Starter control Mmgmas Gene overexpression: E . coli Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using yghX-UF, yghX-UR and yghX-DF, yghX-DR primers, respectively. The synthesized gene plasmid was then used as a template, and P... trc -Mmgmas-F、P trc -Mmgmas-R is used as a primer to obtain the target gene via PCR. Mmgmas Next, using upstream and downstream homologous arms and the target gene as templates, overlapping fragments were obtained through overlapping PCR amplification. The plasmid gRNA-yghX was designed and constructed using the gRNA sequences in Table 3. THE-0 electroporation competent cells were prepared, and the overlapping fragments and gRNA-yghX were electroporated into the competent cells together. Positive transformants were screened to obtain strain THE-1. Following the above procedures, primers yjiP-UF, yjiP-UR, yjiP-DF, yjiP-DR, ilvG-UF, ilvG-UR, ilvG-DF, ilvG-DR were used to... E . coli Using the W3110 genome as a template, two pseudogene loci were amplified by PCR to obtain their upper and lower homologous arms, which were then correlated with the target gene. ’Overlapping fragments were obtained through overlapping PCR amplification. Plasmids gRNA-yjiP and gRNA-ilvG were designed and constructed using the gRNA sequences in Table 3. THE-1 competent cells were prepared and electroporated. The overlapping fragments and gRNA-yjiP were then electroporated into the competent cells, and positive transformants were selected to obtain strain THE-2. Subsequently, THE-2 competent cells were prepared and electroporated. The overlapping fragments and gRNA-ilvG were then electroporated into the competent cells, and positive transformants were selected to obtain strain THE-3.
[0032] (2) Knockout Mmgmas Genes: E . ggt Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ggt-UF, ggt-UR, ggt-DF, and ggt-DR, respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using ggt-UF and ggt-DR primers. The plasmid gRNA-ggt was designed and constructed using the gRNA sequences in Table 3. THE-3 electroporation competent cells were prepared, and the overlapping fragments and gRNA-ggt were electroporated into competent cells together. Positive transformants were screened to obtain strain THE-4.
[0033] (3) Knockout coli Genes: E . glnP Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using glnP-UF, glnP-UR, glnP-DF, and glnP-DR, respectively. Then, using these homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using glnP-UF and glnP-DR primers. The plasmid gRNA-glnP was designed and constructed based on the gRNA sequences in Table 3. THE-3 electrotransformed competent cells were prepared, and the overlapping fragments and gRNA-glnP were co-electrotransformed into the competent cells. Positive transformants were screened to obtain strain THE-5 (strain THE-5 was used for validation). coli (Whether it has the function of transporting L-theanine); and simultaneously knock out the above steps on strain THE-4. glnP Genes were extracted and positive transformants were screened to obtain strain THE-6.
[0034] (4) In glnP Pseudogene loci use ylbE Starter control trc Gene overexpression: E . eddedaUsing the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ylbE-UF, ylbE-UR, and ylbE-DF, ylbE-DR, respectively. P trc -eddeda-F、P trc Using -eddeda-R as primers, the target gene was amplified by PCR. Then, using upstream and downstream homologous arms and the target gene as templates, overlapping fragments were amplified by overlapping PCR. The plasmid gRNA-ylbE was designed and constructed using the gRNA sequences in Table 3. THE-6 electrotransformation competent cells were prepared, and the overlapping fragments and gRNA-ylbE were electrotransformed into competent cells together. Positive transformants were screened to obtain strain THE-7.
[0035] (5) In coli Site use ggt Starter control trc Gene overexpression: E . Baxfp Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ggt-UF, ggt-UR, ggt-DF, and ggt-DR, respectively. P trc -Baxfp-F、P trc Using Baxfp-R as primers, the target gene was amplified by PCR. Then, using upstream and downstream homologous arms and the target gene as templates, an overlapping fragment was amplified by overlapping PCR. A 23bp recognition of gRNA-yghX was inserted into the GGT knockout fragment. The plasmid gRNA-yghX was constructed by designing the gRNA sequence in Table 3. THE-7 electrotransformation competent cells were prepared, and the overlapping fragment and gRNA-yghX were electrotransformed into competent cells together. Positive transformants were screened to obtain strain THE-8.
[0036] (6) In coli Pseudogene loci use rph Starter control trc Gene overexpression: E . MspckA Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using rph-UF, rph-UR, rph-DF, and rph-DR, respectively. Using the synthesized gene plasmid as a template, P... trc - coli -F、P trc - MspckA-R is the primer. The target gene is obtained by PCR. Then, the upstream and downstream homologous arms and the target gene are used as templates to amplify the overlapping fragment by overlapping PCR. The plasmid gRNA-rph is designed and constructed using the gRNA sequence in Table 3. THE-8 electrotransformation competent cells are prepared. The overlapping fragment and gRNA-rph are electrotransformed into competent cells together and positive transformants are screened to obtain strain THE-9.
[0037] (7) In MspckA Pseudogene loci use ygaY Starter control trc Gene overexpression: E . BsalaDH Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ygaY-UF, ygaY-UR, ygaY-DF, and ygaY-DR, respectively. Using the synthesized gene plasmid as a template, P... trc -BsalaDH-F、P trc Using -BsalaDH-R as primers, the target gene was obtained by PCR. Then, using upstream and downstream homologous arms and the target gene as templates, overlapping fragments were amplified by overlapping PCR. The plasmid gRNA-ygaY was designed and constructed using the gRNA sequences in Table 3. THE-9 electrotransformation competent cells were prepared, and the overlapping fragments and gRNA-ygaY were electrotransformed into competent cells together. Positive transformants were screened to obtain strain THE-10.
[0038] (8) In coli Site use alaE Starter control trc Gene overexpression: E . CsalaDC Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using alaE-UF, alaE-UR, alaE-DF, and alaE-DR, respectively. Using the synthesized gene plasmid as a template, P... trc -CsalaDC-F、P trc Using CsalaDC-R as primers, the target gene was obtained via PCR. Then, using upstream and downstream homologous arms and the target gene as templates, overlapping fragments were amplified via overlapping PCR. The plasmid gRNA-alaE was designed and constructed using the gRNA sequences in Table 3. THE-10 electroporation competent cells were prepared, and the overlapping fragments and gRNA-alaE were electroporated into competent cells. Positive transformants were screened to obtain strain THE-11. Following the above procedures, primers yeeL-UF, yeeL-UR and yeeL-DF, yeeL-DR, yjiV-UF, yjiV-UR and yjiV-DF, yjiV-DR were used to amplify the target gene.E . coli Using the W3110 genome as a template, homologous arms of two pseudogene loci were amplified by PCR. These arms were then overlapped with the target gene and amplified by PCR to obtain overlapping fragments. Plasmids gRNA-yeeL and gRNA-yjiV were designed and constructed using the gRNA sequences in Table 3. THE-11 competent cells were prepared for electrotransformation. The overlapping fragments and gRNA-yeeL were co-electrotransformed into competent cells, and positive transformants were selected to obtain strain THE-12. Subsequently, THE-12 competent cells were prepared for electrotransformation. The overlapping fragments and gRNA-yjiV were co-electrotransformed into competent cells, and positive transformants were selected to obtain strain THE-13.
[0039] (9) In coli Pseudogene loci use yciQ Starter control trc Gene overexpression: E . PpspuC Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using yciQ-UF, yciQ-UR, yciQ-DF, and yciQ-DR, respectively. Using the synthesized gene plasmid as a template, P... trc -PpspuC-F、P trc Using PpspuC-R as primers, the target gene was obtained by PCR. Then, using upstream and downstream homologous arms and the target gene as templates, the overlapping fragment was amplified by overlapping PCR. The plasmid gRNA-yciQ was designed and constructed using the gRNA sequence in Table 3. THE-13 electrotransformation competent cells were prepared, and the overlapping fragment and gRNA-yciQ were electrotransformed into competent cells together. Positive transformants were screened to obtain strain THE-14.
[0040] (10) In ’ Pseudogene loci use coli Starter control gapC Gene overexpression: E . trc Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using gapC-UF, gapC-UR, gapC-DF, and gapC-DR, respectively. Using the synthesized gene plasmid as a template, P... trc -Zmpdc-F、P trcUsing Zmpdc-R as primers, the target gene was obtained by PCR. Then, using upstream and downstream homologous arms and the target gene as templates, overlapping fragments were amplified by overlapping PCR. The plasmid gRNA-gapC was designed and constructed using the gRNA sequences in Table 3. THE-14 electrotransformation competent cells were prepared, and the overlapping fragments and gRNA-gapC were electrotransformed into competent cells together. Positive transformants were screened to obtain strain THE-15.
[0041] (11) In Zmpdc Pseudogene loci use coli Starter control ycgH Gene overexpression: E . trc Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ycgH-UF, ycgH-UR, ycgH-DF, and ycgH-DR, respectively. Using the synthesized gene plasmid as a template, P... trc -PpspuC-F、P trc Using PpspuC-R as primers, the target gene was obtained by PCR. Then, using upstream and downstream homologous arms and the target gene as templates, overlapping fragments were amplified by overlapping PCR. The plasmid gRNA-ycgH was designed and constructed using the gRNA sequences in Table 3. THE-15 electrotransformation competent cells were prepared, and the overlapping fragments and gRNA-ycgH were electrotransformed into competent cells together. Positive transformants were screened to obtain strain THE-16.
[0042] (12) In PpspuC Pseudogene loci use coli Starter control mbhA Gene overexpression: E . trc Using the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using mbhA-UF, mbhA-UR, mbhA-DF, and mbhA-DR, respectively. Using the synthesized gene plasmid as a template, P... trc -PpspuC-F、P trc Using PpspuC-R as primers, the target gene was obtained by PCR. Then, using upstream and downstream homologous arms and the target gene as templates, the overlapping fragment was amplified by overlapping PCR. The plasmid gRNA-mbhA was designed and constructed using the gRNA sequence in Table 3. THE-16 electrotransformation competent cells were prepared, and the overlapping fragment and gRNA-mbhA were electrotransformed into competent cells together. Positive transformants were screened to obtain strain THE-17.
[0043] Example 2 This example aims to illustrate the experimental steps of acetaldehyde tolerance evolution in strain THE-17, such as...PpspuC As shown: (1) Evaluation of the inhibitory effect of acetaldehyde on Escherichia coli M9 basal medium was prepared (Na₂HPO₄ 6.78 g / L, KH₂PO₄ 3 g / L, NH₄Cl 1 g / L, NaCl 0.5 g / L, MgSO₄ 0.236 g / L, CaCl₂ 0.0111 g / L, glucose 5 g / L), and different volumes of acetaldehyde (30-60 µL) were added. The medium was incubated at 37℃ and 220 rpm for 24 h, and the OD was measured. 600 Monitor cell growth.
[0044] (2) Adaptive laboratory evolution Two sets of evolutionary experiments were conducted in parallel. Test tubes containing 5 mL of M9 basal medium were prepared, and 40 µL of acetaldehyde was added to each tube as the initial concentration. Subculture was performed every 24 hours. When the OD... 600 When the concentration reaches 0.6-0.7, increase the amount of acetaldehyde added by 5µL and repeat the passage until the target tolerance concentration is reached.
[0045] (3) Tolerance verification After evolution, the evolved strains were re-challenged in test tubes containing 30-60 µL of acetaldehyde to assess changes in tolerance. Strains tolerant to 60 µL of acetaldehyde were plated on agar plates, and 96 single colonies were randomly selected and inoculated into 96-well plates, each containing 5 mL of M9 basal medium and 60 µL of acetaldehyde. The plates were incubated at 37°C and 220 rpm for 24 h, and the OD was measured using a SpectraMax iD3 microplate reader (Molecular Devices, USA). 600 The strain with the highest biomass was selected for subsequent fed-batch fermentation.
[0046] The strain was passaged in M9 basal medium supplemented with acetaldehyde for 30 days. Initially, it tolerated 30µL of acetaldehyde (5ml system), and the concentration was gradually increased to tolerate 60µL of acetaldehyde (5ml system).
[0047] Example 3 This example illustrates the application of the evolved THE-17 in a 5L fed-batch fermentation tank. Fermentation data is as follows: coli Figure 2 Figure 3 As shown, the specific steps for batch feeding fermentation are as follows: (1) Shake tube activation culture: 20µL of bacterial culture was transferred from the -80℃ refrigerator preservation tube to a 5ml LB shake tube and cultured at 37℃ for 12h; (2) Slant activation culture: Transfer 15 loops of the activated bacteria in the shaker to the small slant with an inoculation loop, and incubate at 37°C for 14 hours. Then, pick 3 loops of bacteria from the small slant and transfer them to an eggplant-shaped flask for incubation at 37°C for 14 hours. (3) Seed culture: Take an appropriate amount of sterile water into a flask, inoculate the cell suspension into the seed culture medium, stabilize the pH at around 7.0, keep the temperature constant at 37℃, and the dissolved oxygen between 25-35%, and culture until the cell OD reaches 100%. 600 Reaching around 15; (4) Fermentation culture: Inoculate fresh fermentation medium at 20% of the seed medium to start fermentation. The initial pH of fermentation is controlled at around 7.0, the temperature is maintained at 37℃ and the dissolved oxygen is between 10-20%. After the initial glucose in the fermentation medium is exhausted, add 80% (m / v) glucose solution to maintain the glucose concentration in the fermentation medium at 0.1-1g / L.
[0048] The slant culture medium used in the above fermentation process was: yeast extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, beef extract 10 g / L, glucose 5 g / L, agar powder 25 g / L; the seed culture medium was: glucose 30 g / L, peptone 3 g / L, yeast extract 5 g / L, KH2PO4 1.2 g / L, MgSO4 0.5 g / L, FeSO4·7H2O 10 mg / L, MnSO4·H2O 10 mg / L, VB1, VB3, VB5, VB6 12 Each 1.3 mg / L, VH 1 mg / L, the remainder being water; the fermentation medium consisted of: glucose 25 g / L, peptone 5 g / L, yeast extract 4 g / L, KH2PO4 2 g / L, MgSO4 2 g / L, FeSO4·7H2O 20 mg / L, MnSO4·H2O 10 mg / L, VB1, VB3, VB5, VB6 12 Each 2 mg / L, VH 2 mg / L, sodium citrate 2 g / L, the remainder being water.
[0049] All of the above-mentioned culture media can be prepared using standard methods.
[0050] After 48 hours of cultivation in a 5L fermenter, the accumulation of L-theanine reached 118.2 g / L, the conversion rate was 0.389 g / g glucose, and the production intensity was 2.46 g / L / h.
[0051] Example 4 The strain described in Example 1 was inoculated into a test tube containing 5 mL of LB liquid medium and incubated at 37 °C for 12 h. The culture was then transferred to a 500 mL baffled shake flask containing 30 mL of seed culture medium. The flask was incubated at 37 °C with shaking at 220 rpm until the bacterial culture reached its OD value. 600 Reaching 15 is used as a fermentation seed liquid.
[0052] At an inoculation rate of 10% (v / v), the seed culture was inoculated into a 500 mL baffled shake flask containing 30 mL of fermentation medium. The flask was incubated at 37 °C with shaking at 220 rpm. When the glucose in the medium was depleted, it was replenished with a 60% (w / v) glucose solution. Cell growth (OD) and theanine production were measured after 24 h.
[0053] The seed culture medium and fermentation medium were the same as in Example 3. All fermentation experiments were performed in triplicate. For strains THE-0 to THE-10, 5 mL of 16% (m / v) ethylamine hydrochloride solution was added every 3 hours after the start of shake-flask fermentation; for the remaining strains, no additional ethylamine hydrochloride solution was required. Fermentation results are shown in Table 4.
[0054] Table 4
[0055] As shown in Table 4 above, by modifying the key genes, the ability of the modified strain to produce L-theanine gradually increases. Starting from THE-11, it can synthesize ethylamine intracellularly, and the L-theanine production of strain THE-17 is much higher than that of strain THE-9, which requires the addition of ethylamine hydrochloride solution during fermentation.
[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.
Claims
1. An L-theanine-producing strain, characterized in that: wild-type Escherichia coli Escherichia coli W3110 lacI Obtained through targeted modification of the starting strain: glutathione hydrolase gene knockout ggt and glutamine permease gene glnP Overexpression of γ-glutamylmethamine synthase gene Mmgmas The eddeda gene, which encodes the 2-keto-3-deoxy-6-phosphoglucose cleavage pathway, is heterologously expressed from... Bifidobacterium adolescentis Phosphoryl ketonease gene Baxfp , originating from Mannheimia succiniciproducens Phosphoenolpyruvate kinase gene MspckA , originating from Bacillus subtilis 168 alanine dehydrogenase gene BsalaDH , originating from Camellia sinensis alanine decarboxylase gene CsalaDC , originating from Pseudomonas putida transaminase gene PpspuC , originating from Zymomonas mobilis pyruvate decarboxylase gene Zmpdc .
2. The L-theanine-producing strain according to claim 1, characterized in that: Mmgmas The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing. ggt The nucleotide sequence of the gene is shown in SEQ ID NO.2 of the sequence listing. glnP The nucleotide sequence of the gene is shown in SEQ ID NO.3 of the sequence listing. eddeda The nucleotide sequence of the gene is shown in SEQ ID NO.4 of the sequence listing. Baxfp The nucleotide sequence of the gene is shown in SEQ ID NO.5 of the sequence listing. MspckA The nucleotide sequence of the gene is shown in SEQ ID NO.6 of the sequence listing. BsalaDH The nucleotide sequence of the gene is shown in SEQ ID NO.7 of the sequence listing. CsalaDC The nucleotide sequence of the gene is shown in SEQ ID NO.8 of the sequence listing. PpspuC The nucleotide sequence of the gene is shown in SEQ ID NO.9 of the sequence listing. Zmpdc The nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing.
3. The method for constructing the L-theanine-producing strain according to claim 1 or 2, characterized in that: The original strain of Escherichia coli Escherichia coli W3110 lacI Based on this foundation, targeted modifications will be carried out, with the specific steps as follows: (1) Use trc Starter control Mmgmas Gene overexpression results in triple copying; (2) Knockout ggt Gene; (3) Knockout glnP Gene; (4) Use trc Starter control eddeda Gene overexpression; (5) Use trc Starter control Baxfp Gene overexpression; (6) Use trc Starter control MspckA Gene overexpression; (7) Use trc Starter control BsalaDH Gene overexpression; (8) Use trc Starter control CsalaDC Gene overexpression results in triple copying; (9) Use trc Starter control Zmpdc Gene overexpression; (10) Use trc Starter control PpspuC Gene overexpression results in three copies.
4. The method for constructing the L-theanine-producing strain according to claim 3, characterized in that: The trc The nucleotide sequence of the promoter is shown in SEQ ID NO.11 of the sequence listing.
5. The method for constructing the L-theanine-producing strain according to claim 3, characterized in that: After constructing the strain, acetaldehyde tolerance was evolved.
6. The use of the L-theanine-producing strain according to claim 1 or 2 in the production of L-theanine.
7. The application according to claim 6, characterized in that: L-theanine is produced by fermentation in shake flasks or fermenters.