Marine vibrio m0101 and application thereof

Abstract

This invention provides a strain of Vibrio marineis M0101 and its applications. Vibrio marineis M0101 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 26, 2017, with accession number CGMCC 14026. The Vibrio marineis M0101 provided by this invention possesses many excellent properties, including the ability to efficiently synthesize tetrahydropyrimidine using alginate as a carbon source. This solves the problem in existing technologies where tetrahydropyrimidine cannot be effectively synthesized using non-grain biomass raw materials, and thus has significant application prospects and commercial value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a marine Vibrio M0101 strain and its applications. Background Technology

[0002] Tetrahydropyrimidine is an important secondary metabolite of various halophilic / halophilic microorganisms. It is a physiologically inert compound that can stabilize the structure of biomolecules and mitigate the toxic effects of extreme conditions such as hyperosmolarity, high temperature, freeze-thaw cycles, drying, radiation, and chemical reagents on cells, biofilms, proteins, enzymes, and nucleic acids. Therefore, tetrahydropyrimidine has broad application prospects in biotechnology, cosmetics, and pharmaceuticals, and has developed into a product with high commercial value. The increasing market demand has prompted the search for and development of cost-effective, sustainable microbial sources and processes with the potential for large-scale production of tetrahydropyrimidine.

[0003] Currently, tetrahydropyrimidine is mainly obtained through biosynthesis. This is achieved using fermentation methods such as batch fermentation, fed-batch fermentation, bacterial milking / lactation, and combined growth and resting cell preparation using wild-type strains screened in high-salt environments. There are also methods using synthetic gene cluster recombination and gene knockout to construct engineered bacteria using *E. coli* or *Corynebacterium glutamicum* as host bacteria to produce tetrahydropyrimidine in a heterologous low-salt environment, achieving significant breakthroughs in yield. Currently, commonly used fermentation methods for the biosynthesis of tetrahydropyrimidine primarily use glucose as the carbon source, although glycerol, sucrose, yeast extract, aspartate, glutamate, methane, and molasses are also used. Glucose mainly comes from dietary starch; large-scale tetrahydropyrimidine production using glucose as the carbon source may exacerbate the food supply and demand imbalance. Furthermore, glutamate, glycerol, and L-aspartate are expensive, resulting in high production costs for tetrahydropyrimidine using these as carbon sources.

[0004] Marine Vibrio are a group of short, straight or curved, facultative anaerobic, heterotrophic, Gram-negative bacteria widely found in aquatic environments such as estuaries, coastlines, sediments, and aquaculture areas. They are halophilic, motile, and oxidase-positive. Marine Vibrio are among the most common bacterial groups in the marine environment and hold a dominant position in marine ecosystems. Some marine Vibrio species can produce tetrahydropyrimidine, but only a few species have been isolated and screened to date. Therefore, further research on marine Vibrio microorganisms is still needed. Summary of the Invention

[0005] This invention provides a marine Vibrio M0101 strain that can effectively synthesize tetrahydropyrimidine using alginate as a carbon source, and has great application prospects and commercial value.

[0006] According to a first aspect of the present invention, a strain of Vibrio marineis M0101 is provided, which was deposited at the China General Microbiological Culture Collection Center on April 26, 2017, with the accession number CGMCC 14026. The Vibrio marineis M0101 provided by the present invention possesses many excellent properties, enabling the efficient synthesis of tetrahydropyrimidine using alginate as a carbon source. This solves the problem in the prior art of effectively utilizing non-grain biomass raw materials for the synthesis of tetrahydropyrimidine, and has enormous application prospects and commercial value.

[0007] Preferably, the 16S rDNA nucleotide sequence of Vibrio marineis M0101 is shown in SEQ ID NO: 1.

[0008] According to another aspect of the invention, the use of the above-mentioned marine Vibrio M0101 in the synthesis of tetrahydropyrimidine is provided.

[0009] According to another aspect of the present invention, a method for synthesizing tetrahydropyrimidine using Vibrio marineis MO101 includes the following steps: activating Vibrio marineis MO101 to obtain a seed culture; then inoculating the seed culture into a fermentation medium for fermentation to synthesize tetrahydropyrimidine, wherein the fermentation medium uses alginate as a carbon source. The method for synthesizing tetrahydropyrimidine using Vibrio marineis MO101 provided by the present invention, using alginate as a carbon source, can effectively synthesize tetrahydropyrimidine, and is low in cost, high in yield, simple to operate, suitable for large-scale production, and has broad application prospects and commercial value.

[0010] Preferably, the inoculation amount of seed liquid is 5-10% (v / v).

[0011] Preferably, the concentration of alginate in the fermentation medium is 10–70 g / L. On the one hand, alginate at the above concentration can promote the growth of Vibrio marineis MO101 as a carbon source; on the other hand, alginate at the above concentration can improve the carbon source conversion rate and the carbon source utilization rate of Vibrio marineis MO101, that is, promote the synthesis of tetrahydropyrimidine by Vibrio marineis MO101 and reduce the generation and accumulation of by-products.

[0012] Preferably, the concentration of alginate in the fermentation medium is 30 g / L.

[0013] Preferably, the fermentation medium also includes a nitrogen source, which includes organic nitrogen and / or inorganic nitrogen; wherein the organic nitrogen includes at least one of peptone and yeast extract, and the inorganic nitrogen includes at least one of ammonium nitrate, ammonium chloride, and ammonium sulfate.

[0014] Preferably, the nitrogen source is ammonium nitrate. Since Vibrio marineis MO101 can synthesize succinic acid through the metabolism of nitrate, which then enters the tricarboxylic acid cycle to be converted into oxaloacetic acid, and finally synthesizes tetrahydropyrimidine, introducing nitrate as the nitrogen source in the fermentation medium can further promote the synthesis of tetrahydropyrimidine by Vibrio marineis MO101 and increase the yield of tetrahydropyrimidine.

[0015] Preferably, the concentration of ammonium nitrate in the fermentation medium is 15–50 g / L. Fermentation media with ammonium nitrate at this concentration as a nitrogen source can promote thorough fermentation, increase the yield of tetrahydropyrimidine, and is beneficial for industrial production.

[0016] Preferably, the concentration of ammonium nitrate in the fermentation medium is 15 g / L.

[0017] Preferably, the fermentation medium also includes NaCl, wherein the concentration of NaCl in the fermentation medium is 50–110 g / L. The fermentation medium with the above-mentioned NaCl concentration can promote the synthesis of tetrahydropyrimidine by Vibrio marineis MO101 using alginate, thereby increasing the yield of tetrahydropyrimidine.

[0018] Preferably, the concentration of NaCl in the fermentation medium is 100 g / L.

[0019] Preferably, the fermentation temperature is 28–35°C, and the fermentation time is 18–72 hours. These fermentation conditions facilitate the full utilization of alginate by Vibrio marineis MO101 during fermentation, reduce the production of byproducts, and increase the yield of tetrahydropyrimidine.

[0020] Preferably, the fermentation temperature is 30°C and the time is 24 hours.

[0021] Preferably, the specific operation for strain activation is as follows: Vibrio marineis M0101 is inoculated into LB solid medium and cultured, and then a single colony in the LB solid medium is picked and inoculated into LB liquid medium and cultured to obtain seed culture. The culture temperature is 28-35℃ and the time is 18-36 hours.

[0022] Preferably, the culture temperature is 30°C and the time is 24 hours.

[0023] According to another aspect of the present invention, a microbial agent is provided, the active ingredient of which comprises the above-mentioned Vibrio marineis MO101. Attached Figure Description

[0024] Figure 1 This is a colony morphology diagram of strain M0101 from Example 1.

[0025] Figure 2 This is the phylogenetic tree of strain M0101 from Example 1.

[0026] Figure 3 The image shows the high-performance liquid chromatography (HPLC) peaks of the synthesis of tetrahydropyrimidine by strain M0101 in Example 4 using different concentrations of alginate.

[0027] Figure 4 Example 5 illustrates the metabolic pathway by which strain M0101 synthesizes tetrahydropyrimidine using alginate. Detailed Implementation

[0028] The technical features of the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Isolation and Identification of Vibrio marineis M0101

[0030] 1. Separation

[0031] In this embodiment, Sargassum fusiforme collected from the coast of Weizhou Island, Guangxi Province was used. 0.2g of Sargassum fusiforme was weighed and placed in a 1.5mL sterile centrifuge tube. 0.8mL of sterile water was added, and the mixture was ground into a homogenate using an electric grinder. The homogenate was then serially diluted 10-fold. 0.1mL of the diluted solution was spread onto a separation medium. The plates were inverted and incubated at 30℃ for 2–5 days. Colonies with good growth and different morphologies and colors were selected for purification culture until a pure culture was obtained. The separation medium was sodium alginate medium, and the formulation of sodium alginate medium (1L) was as follows: (NH4)2SO4 5g, K2HPO4 1g, NaCl 15g, MgSO4 1g, H2SO4·7H2O 0.01g, sodium alginate 6g, agar 17g, pH 7.5.

[0032] 2. Identification

[0033] Single colonies were picked from the plate and transferred to liquid separation medium. The culture was carried out at 30°C and 200 rpm for 24 h. The bacterial solution was diluted with sterile physiological saline to an appropriate concentration. 100 μL of the solution was spread on the primary screening medium and incubated upside down at 30°C for 3 days. The morphology of the strain was observed and identified according to the "Handbook of Systematic Identification of Common Bacteria". The bacterial cells were sent to the Institute of Microbiology, Chinese Academy of Sciences, for physiological and biochemical characterization and determination of cellular fatty acid composition.

[0034] (1) Morphological characteristics of strain M0101

[0035] Strain M0101 was cultured on selection medium at 30°C for 3 days. The colony morphology is shown in the figure below. Figure 1 As shown. By Figure 1 It can be seen that the colonies are round, with neat edges, pale yellow color, and smooth surface.

[0036] (2) Physiological and biochemical characteristics of strain M0101

[0037] This strain can grow in glucose, mannitol, mannose, glycerol, acetylglucosamine, and maltose, and its (G+C) mol% content is 41.1%. The main fatty acid components and contents of strain M0101 are C12:0 (9.38%), C14:0 (4.24%), C16:0 (15.61%), C18:1ω7c (14.69%), C18:1ω9c (1.95%), C14:0 3OH / C16:1iso I (9.09%), and C16:1w7c / C16:1w6c (45.06%). After testing and identification by the Institute of Microbiology, Chinese Academy of Sciences, strain M0101 is identified as Vibrio sp.

[0038] (3) 16S rDNA identification

[0039] DNA extraction: DNA from the strain was extracted using the Chelex-100 method. A small amount of bacterial cells was picked up with a sterile toothpick and placed into a 1.5 mL centrifuge tube. 50 μL of 10% Chelex-100 solution was added, the mixture was shaken to mix, heated at 100 °C for 10 min, cooled to room temperature, and centrifuged at 12000 r / min for 10 min. The supernatant was then used for subsequent DNA amplification.

[0040] 16S rDNA sequencing: PCR amplification was performed using universal 16S rRNA primers 27F (5'GAGTTTGATCCTGGCTCAG 3') and 1492R (5'GGTTACCTTGTTACGACTT 3'). The amplification products were detected by gel electrophoresis and sent to Shanghai Sangon Biotech Co., Ltd. for 16S rDNA sequencing. The 16S rDNA sequence of strain M0101 is shown in SEQ ID NO:1.

[0041] Phylogenetic tree construction: Sequencing results were analyzed by BLAST in the NCBI (National Center for Biotechnology Information) nucleotide database. Highly homologous 16S rRNA sequences were selected, and a phylogenetic tree of the strains was constructed using MEGA 7.0 (neighbor-joining). The results are as follows: Figure 2 As shown. By Figure 2 It can be seen that strain M0101 is 100% homologous to Vibrio gangliei.

[0042] Based on the above morphological, physiological and biochemical characteristics and 16S rDNA sequence homology analysis results, the isolated and purified strain M0101 was identified as a Vibrio and named Vibrio gangliei M0101. Marine Vibrio M0101 was deposited on April 26, 2017, at the China General Microbiological Culture Collection Center, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC 14026.

[0043] Example 2: Synthesis of Tetrahydropyrimidine using strain M0101

[0044] 1. Synthesis of tetrahydropyrimidine

[0045] S1. Activation of microorganisms and preparation of seed culture:

[0046] Vibrio marineis M0101 preserved in glycerol tubes was streaked onto LB solid medium and incubated at 30°C for 48 hours. Single colonies were picked and inoculated into 50 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and incubated overnight on a shaker at 30°C and 200 rpm to obtain the seed culture.

[0047] S2. The seed culture was inoculated into the fermentation medium at an inoculation rate of 5% (v / v) and fermented on a shaker at 30℃ and 200 rpm / min for 24 h to obtain the fermentation broth. The fermentation medium formula is as follows: alginate (purchased from Aladdin, product number: S278630) 30 g / L, NaCl 100 g / L, (NH4)2SO4 5 g / L, K2HPO4 1 g / L, MgSO4·7H2O 9.7 g / L, KCl 2 g / L, anhydrous CaCl2 0.2 g / L.

[0048] 2. Detection of bacterial biomass

[0049] Take 1 mL of fermentation broth, dilute it 3 times, and use a TU-1900 double-beam ultraviolet spectrophotometer to measure the absorbance at 600 nm.

[0050] 3. Detection of tetrahydropyrimidine content

[0051] S1. Collect tetrahydropyrimidine released by cells: Take 1 mL of fermentation broth, centrifuge, and collect the cells. Resuspend the cells in a hypotonic solution (10 g / L NaCl solution), and shake at 200 rpm for 20 min on a shaker at 30℃. Centrifuge at 12000 rpm for 2 min and collect the supernatant. The supernatant is the sample to be tested for tetrahydropyrimidine released by the cells into the extracellular space.

[0052] S2. Determination of tetrahydropyrimidine content using high performance liquid chromatography (HPLC): A Waters e2695 HPLC system equipped with a 2998 PDA detector was used for detection. The chromatographic conditions were as follows:

[0053] a) Liquid chromatography column: Waters C18 column (250 mm in length, 4.6 mm in inner diameter, 5 μm in particle size); b) Mobile phase: 10% acetonitrile: 90% water;

[0054] c) Flow rate: 0.5 mL / min;

[0055] d) Column temperature: 30℃;

[0056] e) Injection volume: 10 μL;

[0057] f) Detection wavelength: 210nm.

[0058] Tetrahydropyrimidine standard (purchased from Aladdin, catalog number: E292674) was tested under the chromatographic conditions described above, and a standard curve was plotted. Then, the sample to be tested was tested under the same chromatographic conditions, and the content of tetrahydropyrimidine in the sample was calculated based on the peak area and the standard curve.

[0059] 4. Test Results

[0060] The yield of tetrahydropyrimidine was 0.19 g / L, which indicates that the marine Vibrio M0101 provided by the present invention has the ability to synthesize tetrahydropyrimidine and the yield of synthesized tetrahydropyrimidine is high.

[0061] Example 3: Effect of NaCl concentration in fermentation medium on the synthesis of tetrahydropyrimidine by strain MO101

[0062] 1. Synthesis of tetrahydropyrimidine

[0063] This embodiment synthesizes tetrahydropyrimidine according to Example 2. The difference from Example 2 is that the concentration of NaCl in the fermentation medium is 50 g / L, 80 g / L, 110 g / L, and 120 g / L, respectively. Apart from the above differences, the culture medium formulation and preparation procedures used in this embodiment are strictly consistent with those in Example 2.

[0064] 2. Detection of bacterial biomass

[0065] The test method for detecting bacterial biomass is the same as the test method performed in Example 2.

[0066] 3. Detection of tetrahydropyrimidine content

[0067] The test method for detecting the content of tetrahydropyrimidine is the same as the test method performed in Example 2.

[0068] 4. Test Results

[0069] Table 1. Effects of NaCl concentration on the growth and tetrahydropyrimidine synthesis of strain M0101

[0070]

[0071]

[0072] The test results are shown in Table 1. Table 1 shows that low salt concentration is beneficial to the growth of Vibrio marineis M0101, but detrimental to the accumulation of intracellular tetrahydropyrimidine. With increasing NaCl concentration in the fermentation medium, the growth rate (OD) of strain M0101 decreased. 600 The concentration of NaCl in the fermentation medium gradually decreased, but the ability of strain M0101 to synthesize tetrahydropyrimidine showed a trend of first increasing and then decreasing. When the NaCl concentration in the fermentation medium reached 100 g / L, the yield of tetrahydropyrimidine was the highest, reaching 0.19 g / L. Further increasing the NaCl concentration to 110 g / L caused the growth of strain M0101 to continue to decrease, and the yield of tetrahydropyrimidine also decreased significantly, reaching only 0.09 g / L. When the NaCl concentration in the medium was 120 g / L, it was unfavorable for the growth of strain M0101, with an OD600 of 0.106, indicating almost no release of tetrahydropyrimidine. Therefore, when the NaCl concentration in the fermentation medium was 100 g / L, the yield of tetrahydropyrimidine synthesized using strain M0101 was optimal.

[0073] Example 4: Effect of alginate concentration in fermentation medium on tetrahydropyrimidine synthesis by strain M0101

[0074] 1. Synthesis of tetrahydropyrimidine

[0075] This embodiment synthesizes tetrahydropyrimidine according to Example 2. The difference from Example 2 is that the concentration of alginate in the fermentation medium is 10 g / L, 50 g / L, and 70 g / L, respectively. Apart from the above differences, the fermentation medium formulation and preparation procedures used in this embodiment are strictly consistent with those in Example 1.

[0076] 2. Detection of bacterial biomass

[0077] Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 2 min, collect the precipitate, resuspend in ultrapure water, and disrupt the cells using an ultrasonic disruptor. Centrifuge at 12000 rpm for 2 min, collect the supernatant, and use it to determine the total bacterial protein. The modified Bradford protein assay kit from Shanghai Sangon Biotech Co., Ltd. was used to determine the total bacterial protein content, following the instructions. The total bacterial protein content reflects the growth status of strain M0101.

[0078] 3. Detection of tetrahydropyrimidine content

[0079] The test method for detecting the content of tetrahydropyrimidine is the same as the test method performed in Example 2.

[0080] 4. Test Results

[0081] Table 2 Effects of alginate concentration on the growth and tetrahydropyrimidine synthesis of strain M0101

[0082]

[0083]

[0084] The test results are shown in Table 2 and Figure 3 As shown in Table 2, an appropriate concentration of alginate is beneficial to the growth of strain M0101. When the alginate concentration is between 10 and 30 g / L, the cell growth increases with increasing alginate concentration; however, when the alginate concentration is between 30 and 70 g / L, the cell growth decreases with increasing alginate concentration. This indicates that excessively high alginate concentration in the fermentation medium leads to increased viscosity and inhibits the growth of strain M0101. Furthermore, Table 2 also shows that the alginate concentration in the fermentation medium has a significant impact on the synthesis of tetrahydropyrimidine by strain M0101. When the alginate concentration was between 10 and 50 g / L, the amount of tetrahydropyrimidine released by the cells increased with increasing alginate concentration. When the alginate concentration in the fermentation medium was between 30 and 70 g / L, the amount of tetrahydropyrimidine synthesized by strain M0101 was 0.32 g / L, 0.39 g / L, and 0.37 g / L, respectively, with little difference in yield. The amount of tetrahydropyrimidine synthesized was the largest at an alginate concentration of 50 g / L, reaching 0.39 g / L. When the alginate concentration in the culture medium was 70 g / L, the amount of tetrahydropyrimidine synthesized decreased to 0.37 g / L, indicating that an excessively high alginate concentration in the fermentation medium was not conducive to the accumulation of tetrahydropyrimidine.

[0085] Depend on Figure 3 It was found that when the alginate concentration exceeded 50 g / L, the byproducts synthesized by strain M0101 increased. This indicates that excessively high alginate concentrations in the fermentation medium are detrimental to the growth of strain M0101 and the synthesis of tetrahydropyrimidine, easily leading to byproduct accumulation and reduced effective carbon source conversion. Therefore, for the synthesis of tetrahydropyrimidine using Vibrio marineis M0101, the optimal alginate concentration in the fermentation medium is 30 g / L.

[0086] Example 5: Effect of nitrogen source type in fermentation medium on tetrahydropyrimidine synthesis by strain M0101

[0087] 1. Synthesis of tetrahydropyrimidine

[0088] This embodiment synthesizes tetrahydropyrimidine according to Example 2. The difference in composition compared to Example 2 is that the nitrogen sources in the fermentation medium are 5 g / L yeast extract, 5 g / L peptone, 5 g / L ammonium nitrate, and 5 g / L ammonium chloride, respectively. Apart from the above differences, the fermentation medium formulation and preparation procedures used in this embodiment are strictly consistent with those in Example 2.

[0089] 2. Detection of bacterial biomass

[0090] The test method for detecting bacterial biomass is the same as the test method performed in Example 4.

[0091] 3. Detection of tetrahydropyrimidine content

[0092] The test method for detecting the content of tetrahydropyrimidine is the same as the test method performed in Example 2.

[0093] 4. Test Results

[0094] Table 3. Effects of different nitrogen sources on the growth and tetrahydropyrimidine synthesis of strain M0101

[0095] nitrogen source Total bacterial protein (μg / mL) Tetrahydropyrimidine (g / L) yeast powder 396.56 0.31 peptone 332.69 0.25 ammonium sulfate 313.06 0.37 ammonium nitrate 315.04 1.79 ammonium chloride 275.66 0.44

[0096] The test results are shown in Table 3. Table 3 shows that yeast extract was beneficial to the growth of strain M0101, resulting in the largest cell growth and a total protein content of 396.56 ug / mL. Peptone, ammonium sulfate, and ammonium nitrate were the next most effective nitrogen sources, while ammonium chloride resulted in the smallest cell growth. However, when ammonium nitrate was used as the nitrogen source, strain M0101 produced the highest yield of tetrahydropyrimidine, reaching 1.79 g / L, which is 4.8 times that of ammonium sulfate. Ammonium chloride produced the second highest yield at 0.44 g / L, ammonium sulfate at 0.37 g / L, yeast extract at 0.31 g / L, and peptone at the lowest yield at 0.25 g / L.

[0097] The reason is that, for example Figure 4 As shown, the tetrahydropyrimidine metabolic pathway includes a nitrate metabolic pathway. Nitrate is converted to succinate through a series of enzymatic reactions, then enters the tricarboxylic acid cycle to be converted to oxaloacetate. Oxaloacetate is catalyzed by aspartate transaminase to be converted to aspartic acid, which then enters the tetrahydropyrimidine synthesis pathway and is finally converted to tetrahydropyrimidine. It is speculated that strain M0101 also has such a metabolic pathway (see appendix). Figure 1 The use of alginate promotes the synthesis of tetrahydropyrimidine in strain M0101. Therefore, when ammonium nitrate is used as the nitrogen source, the yield of tetrahydropyrimidine increases by 4.8 times. In conclusion, ammonium nitrate is the preferred nitrogen source for the production of tetrahydropyrimidine by Vibrio marineis M0101 using alginate.

[0098] Example 6: Effect of ammonium nitrate concentration in fermentation medium on tetrahydropyrimidine synthesis by strain M0101

[0099] 1. Synthesis of tetrahydropyrimidine

[0100] This embodiment synthesizes tetrahydropyrimidine according to Example 2. The difference in composition compared to Example 2 is that the nitrogen sources in the fermentation medium are 1 g / L NH4NO3, 3 g / L NH4NO3, 5 g / L NH4NO3, 7 g / L NH4NO3, 9 g / L NH4NO3, 11 g / L NH4NO3, 13 g / L NH4NO3, 15 g / L NH4NO3, 30 g / L NH4NO3, and 50 g / L NH4NO3, respectively. Apart from the above differences, the fermentation medium formulation and preparation procedures used in this embodiment are strictly consistent with those in Example 2.

[0101] 2. Detection of bacterial biomass

[0102] The test method for detecting bacterial biomass is the same as the test method performed in Example 4.

[0103] 3. Detection of tetrahydropyrimidine content

[0104] The test method for detecting the content of tetrahydropyrimidine is the same as the test method performed in Example 2.

[0105] Table 4 Effects of NH4NO3 concentration on the growth and tetrahydropyrimidine synthesis of strain M0101

[0106] <![CDATA[NH4NO3 concentration (g / L)]]> Tetrahydropyrimidine (g / L) 1 0.58 3 1.14 5 1.79 7 2.33 9 2.80 11 2.66 13 3.20 15 5.80 30 5.25 50 5.18

[0107] Table 4 shows that an appropriate amount of NH4NO3 is beneficial for strain M0101 to synthesize tetrahydropyrimidine using alginate. When the NH4NO3 concentration is 1–15 g / L, the amount of tetrahydropyrimidine produced by strain M0101 using alginate increases with increasing NH4NO3 concentration. The highest tetrahydropyrimidine yield (5.8 g / L) is achieved when the NH4NO3 concentration in the culture medium is 15 g / L. When the NH4NO3 concentration is 15–50 g / L, the amount of tetrahydropyrimidine produced by strain M0101 using alginate decreases with increasing NH4NO3 concentration. When the NH4NO3 concentration is 50 g / L, the tetrahydropyrimidine yield drops to 5.18 g / L, indicating that further increasing the NH4NO3 concentration in the fermentation medium does not promote the synthesis of tetrahydropyrimidine by strain M0101 using alginate; on the contrary, it reduces the yield. Therefore, when the NH4NO3 concentration in the fermentation medium is 15 g / L, the yield of tetrahydropyrimidine synthesized by Vibrio marineis MO101 is better.

[0108] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A strain of Vibrio vulnificus ( Vibrio gangliei M0101 was deposited on April 26, 2017, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 14026.

2. The application of Vibrio marineis MO101 as described in claim 1 in the synthesis of tetrahydropyrimidine.

3. A method for synthesizing tetrahydropyrimidine using Vibrio marineis MO101 as described in claim 1, characterized in that, The process includes the following steps: activating the Vibrio marineis M0101 strain to obtain a seed culture; then inoculating the seed culture into a fermentation medium for fermentation, thereby synthesizing tetrahydropyrimidine; The fermentation medium comprises the following components: 10~70 g / L carbon source, 15~50 g / L nitrogen source, and 50~110 g / L sodium chloride; The carbon source is alginate, and the nitrogen source is ammonium nitrate.

4. The method for synthesizing tetrahydropyrimidine using Vibrio marineis MO101 as described in claim 3, characterized in that, The fermentation temperature is 28~35℃, and the time is 18~72 hours.

5. The method for synthesizing tetrahydropyrimidine using Vibrio marineis MO101 as described in claim 3, characterized in that, The specific operation for activating the strain is as follows: Vibrio marineis M0101 is inoculated into LB solid medium and cultured, and then a single colony in the LB solid medium is picked and inoculated into LB liquid medium and cultured to obtain seed culture. The culture temperature is 28~35℃ and the time is 18~36 hours.

6. A microbial agent, characterized in that, The active ingredient of the bacterial agent includes Vibrio marineis MO101 as described in claim 1.

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