CN109913386B - Tea tree endophyte CsE7 capable of producing theanine at high yield and application thereof - Google Patents
Tea tree endophyte CsE7 capable of producing theanine at high yield and application thereof Download PDFInfo
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- DATAGRPVKZEWHA-YFKPBYRVSA-N N(5)-ethyl-L-glutamine Chemical compound CCNC(=O)CC[C@H]([NH3+])C([O-])=O DATAGRPVKZEWHA-YFKPBYRVSA-N 0.000 title claims abstract description 91
- 229940026510 theanine Drugs 0.000 title claims abstract description 35
- 235000009024 Ceanothus sanguineus Nutrition 0.000 title claims abstract description 17
- 235000015459 Lycium barbarum Nutrition 0.000 title claims abstract description 17
- 241000366182 Melaleuca alternifolia Species 0.000 title 1
- 240000003553 Leptospermum scoparium Species 0.000 claims abstract description 16
- DATAGRPVKZEWHA-UHFFFAOYSA-N L-gamma-glutamyl-n-ethylamine Natural products CCNC(=O)CCC(N)C(O)=O DATAGRPVKZEWHA-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 241000191025 Rhodobacter Species 0.000 claims abstract description 6
- 241000506875 Luteibacter sp. Species 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- 241000546193 Clusiaceae Species 0.000 claims description 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 abstract description 10
- 241000593508 Garcinia Species 0.000 abstract description 6
- 235000000885 Garcinia xanthochymus Nutrition 0.000 abstract description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 abstract description 5
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 abstract description 5
- 239000001963 growth medium Substances 0.000 abstract description 4
- 230000028327 secretion Effects 0.000 abstract description 4
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- 241001122767 Theaceae Species 0.000 description 5
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- 241001276011 Rhodanobacter Species 0.000 description 2
- 241001430267 Rhodobacteraceae Species 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
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- 230000000877 morphologic effect Effects 0.000 description 2
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- DATAGRPVKZEWHA-RXMQYKEDSA-N (2r)-2-azaniumyl-5-(ethylamino)-5-oxopentanoate Chemical compound CCNC(=O)CC[C@@H](N)C(O)=O DATAGRPVKZEWHA-RXMQYKEDSA-N 0.000 description 1
- 241001112741 Bacillaceae Species 0.000 description 1
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- 102000004190 Enzymes Human genes 0.000 description 1
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- 206010028980 Neoplasm Diseases 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses tea tree endophyte with high theanine yield and application thereof, the tea tree endophyte is classified and named as rhodobacter (Rhodanobacteriaceae Luteibacter sp.) CsE7, and is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2018816, the preservation address is Wuhan university in Wuhan city, Hubei province, China. The L-theanine-producing strain is a theanine-producing endophyte CsE7 of the genus Garcinia of the family of rhodobacter, which is separated from tea trees for the first time. The strain CsE7 can synthesize L-theanine on an LB culture medium added with glutamine and ethylamine, the synthesized theanine can be quickly secreted to the outside of cells, and the secretion efficiency reaches more than 90%.
Description
Technical Field
The invention belongs to the technical field of preparation of theanine, and particularly relates to a tea tree endophyte CsE7 capable of producing theanine at a high yield and application thereof.
Background
Theanine (L-Theanine) is N-ethyl-gamma-glutamine, accounts for more than 70% of the total amount of amino acids in tea, is a special non-protein free amino acid in tea trees, is also a main substance for endowing tea with special flavor, and the content of Theanine directly determines the quality of tea. Theanine has the medicinal effects of relaxing nerves, resisting tumor and depression, protecting blood vessel, lowering blood pressure, inhibiting obesity, improving immunity, etc., and can be used as functional food additive in food field. Therefore, theanine is more and more favored and is in great demand by the international market.
At present, there are three main methods for producing theanine. One is extracted from tea. The theanine content in the tea is low, and the raw material source is limited, so that the production cost is high. The second method is a chemical synthesis method. The method can not only cause toxic substance residue, but also produce theanine which is a mixture of D-theanine and L-theanine isomers, and the resolution and refining process is complicated. The third method is to utilize the microorganism and the enzyme thereof to ferment and produce the theanine. Microorganisms capable of synthesizing theanine, which have been reported so far, include Escherichia coli, Bacillus subtilis, etc., but these are free microorganisms in nature and are not isolated from the body of tea tree.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects in the prior art, the invention aims to provide a tea tree endophyte with high theanine yield, which is an endophyte separated from tea trees for the first time and meets the use requirement. The invention also aims to provide application of the tea tree endophyte with high theanine yield in the production of L-theanine.
The technical scheme is as follows: in order to achieve the purpose of the invention, the invention adopts the technical scheme that:
an endophyte of tea trees with high theanine yield is classified and named as Rhodobacter luteobacter sp 7 of the family of rhodamine, and is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2018816, the preservation date is 11 months and 23 days in 2018; the preservation address is Wuhan university in Wuhan city of Hubei province in China.
The application of the CsE7 of the genus Garcinia of the family of the rhodamine norobacteriaceae in the preparation of L-theanine.
The application is as follows: the CsE7 strain was shake-cultured at 28 ℃ to OD600When the concentration is 1.0, simultaneously adding glutamine and ethylamine into the culture solution, culturing for 1-15 d, taking the culture solution, carrying out water bath pyrolysis at 100 ℃ for 30min, repeatedly freezing and thawing at-20 ℃ for 2-3 times, centrifuging at 12000rpm for 15min, taking the supernatant, filtering by a 0.22 mu m water-phase filter membrane, and carrying out liquid-phase mass spectrometry detection; obtaining the supernatant containing L-theanine.
The application is characterized in that the final concentration of the added glutamine is 20 mM. Ethylamine was added to a final concentration of 20 mM.
The application is preferably culture for 7 d.
The L-theanine-producing strain CsE7 of the invention is a theanine-producing endophyte which is firstly separated from tea trees, and 16SrDNA is identified as Rhodobacteraceae Guttiferae (Rhodanobacter Luteibacter sp.). The CsE7 strain grows well on LB culture medium, and after being cultured for 2-3 days at 28 ℃, a single strain falls into bright yellow. After amplification, sequencing and comparison, the 16S rDNA has high homology with Rhodanobacter luteobacter sp (GenBank access No. KM187159) in the family of rhodamine Bacillaceae in NCBI database, and reaches over 99 percent. The growth conditions were determined to be of the genus Garcinia of the family of rhodamine, classified and named as Rhodobacteraceae Garcinia (Rhodanobacteriaceae Luteibacter sp.) CsE7, in combination with morphological characteristics of the bacteria.
Has the advantages that: compared with the prior art, the L-theanine-producing strain is the theanine-producing endophyte CsE7 of the genus Garcinia of the family of the rhodobacter in the family of the rhodobacter, which is separated from tea trees for the first time. The strain CsE7 can synthesize L-theanine on an LB culture medium added with glutamine and ethylamine, the synthesized theanine can be quickly secreted to the outside of cells, the secretion efficiency reaches more than 90%, and the strain has good practicability.
Drawings
FIG. 1 is a photographic image of a colony of CsE 7;
FIG. 2 is a gram-stained microscopic image of a CsE7 colony;
FIG. 3 is a graph showing the results of mass spectrometric detection of theanine, a synthetic product of CsE 7.
FIG. 4 is a graph showing the change in the amount of theanine synthesized by CsE7 cultured for various periods of time.
FIG. 5 is a graph showing the results of the efficiency of theanine secretion by CsE7 in cultures of 5d and 7 d.
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1
1) Isolation of CsE7 Strain
And (3) selecting mixed endophytes secreted by the tea tree tissue culture seedlings, separating the monoclonals on a solid LB (lysogeny broth) plate, numbering the separated monoclonals, detecting the theanine synthesis capacity, and finally selecting the CsE7 as a target strain. Then, extracting genomic DNA of a target strain, identifying the 16S rDNA (the sequence is shown as SEQ ID NO. 1) of the strain by using a molecular cloning technology, and comparing the strain with an NCBI database to determine that the strain belongs to the genus Garcinia of the family of the rhodamine anobacteriaceae.
2) Gram staining and morphological characterization of CsE7 strain
The bacterial strain has a round bacterial plaque, a smooth, moist and convex surface, is opaque and yellow, and has neat edges (as shown in figure 1). The strain is red and rod-shaped after gram staining, and is identified as gram-negative bacteria (shown in figure 2).
3) Sample preparation and detection process for CsE7 synthetic theanine
The strain was shake-cultured at 28 ℃ to OD600When the concentration is 1.0, simultaneously adding 20mM glutamine and 20mM ethylamine into LB culture solution, simultaneously taking a bacterium solution with the same concentration without any substrate and a blank culture medium with the same concentration as a control test, continuously culturing for 1d, 3d, 5d, 7d, 10d and 15d under the same shaking culture condition at 28 ℃, respectively cracking the culture solution obtained at different time points in a water bath at 100 ℃ for 30min, repeatedly freezing and thawing for 2-3 times at-20 ℃, then carrying out 12000rpm centrifugation for 15min, taking supernatant, filtering the supernatant through a 0.22 mu m water-phase filter membrane, and carrying out ultra-high performance liquid mass spectrometry (UPLC-MS/MS) detection.
The secondary mass spectrum identification result shows that: the culture solutions all stably contained theanine at different concentrations (see FIG. 3 for mass spectrometry results), and the theanine content was about 3.8mg/L on average at day 7 after the substrate addition (see FIG. 4), whereas in the control group, only a very small amount of theanine was detected in the same concentration of the culture solution without any substrate addition, and the presence of theanine was not detected in the blank medium with the substrate addition. Thus, the strain has the capability of efficiently synthesizing theanine.
And theanine synthesized by the strain can be quickly secreted to the outside of cells, and the secretion efficiency reaches about 90 percent (see figure 5).
Sequence listing
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<120> tea tree endophyte CsE7 with high theanine yield and application thereof
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gggataacgt agggaaactt acgctaatac cgcatacgtc ctacgggaga aagcggggga 60
tcgcaagacc tcgcgcggtt ggatggaccg atgtgcgatt agctagttgg tgaggtaacg 120
gctcaccaag gcgacgatcg ctagctggtc tgagaggatg atcagccaca ctgggactga 180
gacacggccc agactcctac gggaggcagc agtggggaat attggacaat gggcgcaagc 240
ctgatccagc aatgccgcgt gtgtgaagaa ggccctcggg ttgtaaagca cttttatcag 300
gagcgaaatc tgcaaggtta atacctttgc agtctgacgg tacctgagga ataagcaccg 360
gctaactccg tgccagcagc cgcggtaata cggagggtgc aagcgttaat cggaattact 420
gggcgtaaag cgtgcgtagg cggttcgtta agtctgttgt gaaagccccg ggctcaacct 480
gggaatggca atggatactg gcgagctaga gtgtgtcaga ggatggtgga attcccggtg 540
tagcggtgaa atgcgtagag atcgggagga acatcagtgg cgaaggcggc catctgggac 600
aacactgacg ctgaggcacg aaagcgtggg gagcaaacag gattagatac cctggtagtc 660
cacgccctaa acgatgcgaa ctggatgttg gtctcaactc ggagatcagt gtcgaagcta 720
acgcgttaag ttcgccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 780
gggggcccgc acaagcggtg gagtatgtgg tttaattcga tgcaacgcga agaaccttac 840
ctggccttga catgtccgga atccagcaga gatgcaggag tgccttcggg aatcggaaca 900
caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 960
agcgcaaccc ttgtccttag ttgccagcga gtaatgtcgg gaactctaag gagactgccg 1020
gtgacaaacc ggaggaaggt ggggatgacg tcaagtcatc atggccctta cggccagggc 1080
tacacacgta ctacaatggt cggtacagag ggttgcgata ccgcgaggtg gagctaatcc 1140
cagaaagccg atcccagtcc ggattggagt ctgcaactcg actccatgaa gtcggaatcg 1200
ctagtaatcg cagatcagct atgctgcggt gaatacgttc ccgggccttg tacacaccgc 1260
ccgtcacacc atgggagtga gctgctccag aagccgttag tctaaccgca agggggacga 1320
cgaccacgga gtggttcatg actggg 1346
Claims (2)
1. An endophyte of tea trees with high theanine yield is classified and named as Rhodobacter luteobacter (Rhodanobacteriaceae Luteibacter sp.) CsE7, is deposited in China center for type culture collection with the collection number of CCTCC NO: m2018816, the preservation date is 11 months and 23 days in 2018; the preservation address is Wuhan university in Wuhan city of Hubei province in China.
2. The use of the CsE7 belonging to the genus Guttiferae of the family Rhodobacteriaceae in the preparation of L-theanine as claimed in claim 1.
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| CN2018115458909 | 2018-12-17 | ||
| CN201811545890.9A CN109402028A (en) | 2018-12-17 | 2018-12-17 | One plant height produces tea tree endophyte CsE7 and its application of theanine |
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| CN109913386A CN109913386A (en) | 2019-06-21 |
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| CN109402028A (en) * | 2018-12-17 | 2019-03-01 | 安徽农业大学 | One plant height produces tea tree endophyte CsE7 and its application of theanine |
| CN111073830B (en) * | 2019-11-29 | 2021-07-23 | 河南巨龙生物工程股份有限公司 | Lactobacillus casei with high yield of gamma-glutamyltranspeptidase and application thereof in production of L-theanine |
| CN114507623B (en) * | 2022-03-04 | 2023-03-10 | 安徽农业大学 | Bacillus and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402028A (en) * | 2018-12-17 | 2019-03-01 | 安徽农业大学 | One plant height produces tea tree endophyte CsE7 and its application of theanine |
| WO2019046439A1 (en) * | 2017-08-30 | 2019-03-07 | Monsanto Technology Llc | Methods for detecting microbes |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019046439A1 (en) * | 2017-08-30 | 2019-03-07 | Monsanto Technology Llc | Methods for detecting microbes |
| CN109402028A (en) * | 2018-12-17 | 2019-03-01 | 安徽农业大学 | One plant height produces tea tree endophyte CsE7 and its application of theanine |
Non-Patent Citations (3)
| Title |
|---|
| Endophytic Bacteria as Contributors to Theanine Production in Camellia sinensis;Jun Sun等;《Journal of Agricultural and Food Chemistry》;20190903;第67卷(第38期);第10685-10693页 * |
| Isolation, Diversity, and Growth-Promoting Activities of Endophytic Bacteria From Tea Cultivars of Zijuan and Yunkang-10;Xiaomei Yan等;《Frontiers in Microbiology》;20180821;第9卷(第1848期);第1-11页 * |
| 茶树茶氨酸代谢相关基因表达组织特异性分析;陈琪等;《核农学报》;20150731;第29卷(第7期);第1285-1291页 * |
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| CN109402028A (en) | 2019-03-01 |
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