A Sphingomonas strain and its application

Abstract

The present invention discloses a sphingomonas and its application, belonging to the field of ergothioneine biosynthesis. The bacterial name is sphingomonas sp.HBJ-193, and the deposit number is CGMCC NO.24613. The sphingomonas HBJ-193 of the present invention has the characteristics of efficient production of ergothioneine, and has a more efficient production of ergothioneine ability under the fermentation conditions in the present invention, which is embodied in that the EGT synthesized by HBJ-193 is mainly distributed outside the cell and is easy to extract; The best fermentation conditions for synthesizing EGT by sphingomonas HBJ-193 are obtained by single factor and response surface experiments, and its yield can be as high as 100.04mg / L. The sphingomonas HBJ-193 is a newly discovered EGT synthetic strain, and has the characteristics of high yield, short fermentation time, and easy extraction of fermentation products, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of ergothioneine biosynthesis, and particularly relates to a ergothioneine-producing sphingomonas strain and a method for culturing the high-yield ergothioneine. Background Art

[0002] Ergothioneine (EGT), also known as 2-sulfhydryl-L-histidine trimethylolate, is a natural antioxidant that is abundant in many animals and plants. It is synthesized only in some microorganisms (actinomycetes, streptomyces), mushrooms, and certain cyanobacteria, and cannot be synthesized by the animal body itself. Pure EGT is a white crystal, water-soluble, and will not self-oxidize at physiological pH and in strong alkaline solutions.

[0003] Many studies have shown that EGT has many important physiological functions, such as anti-inflammatory function, cell protection function, anti-depression function, eye protection function, cardiovascular and cerebrovascular protection function, prevention and treatment of neurodegenerative diseases, etc. In addition, the stability of EGT aqueous solution and its heavy metal ion chelation properties make it an ideal protective solution for transplanted organs; EGT can scavenge free radicals to the greatest extent, which can protect cells from radiation damage. It has excellent stability and biosafety, so it has broad application prospects in the fields of medicine, food and cosmetics.

[0004] Due to the high cost of chemically synthesizing EGT, the current industrial EGT production method is mainly deep fermentation of edible mushroom mycelium. This method has low EGT yield, long fermentation cycle and complex extraction process. Therefore, it is necessary to explore new EGT synthetic strains. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a Sphingomonas strain.

[0006] Another object of the present invention is to provide the application of the above-mentioned Sphingomonas.

[0007] In order to overcome the shortcomings and deficiencies of the existing EGT production source strains, low yield, and complex extraction process, the present invention provides a high-EGT-producing sphingomonas sp. HBJ-193 and a high-EGT-producing cultivation method thereof. The method is used to ferment sphingomonas sp. HBJ-193, and its EGT yield is increased from 1.58 mg / L to 100.04 mg / L.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The invention provides a sphingomonas strain named as Sphingomonas sp. HBJ-193, which is separated from distiller's yeast.

[0010] The preservation information of the Sphingomonas sp. HBJ-193 is as follows: the preservation unit is the General Microbiology Center (CGMCC) of China Microbiological Culture Collection Administration, the preservation date is March 29, 2022, the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.24613.

[0011] The Sphingomonas sp. HBJ-193 has the following morphological characteristics: the colonies are regular circles on the LB plate, the color is between golden yellow and orange yellow, the surface of the colonies is smooth and easy to pick up.

[0012] The present invention also provides an application of the sphingomonas in producing ergothioneine by liquid fermentation.

[0013] The formula of the modified LB liquid culture medium used for liquid fermentation is: carbon source 2.5-15 g / L, nitrogen source 2.5-17.5 g / L, NaCl 10-15 g / L, precursor 0-64 mM, pH 4.4-9.4.

[0014] Furthermore, the formula of the improved LB liquid culture medium is: carbon source 2.5-10 g / L, nitrogen source 7.5-17.5 g / L, NaCl 10-15 g / L, precursor 0-64 mM, pH 5.4-8.4.

[0015] Furthermore, the formula of the improved LB liquid culture medium is: carbon source 2.5-7.5 g / L, nitrogen source 10-15 g / L, NaCl 10-15 g / L, precursor 0-64 mM, pH 5.4-8.4.

[0016] The carbon source is at least one of yeast extract powder, starch, sucrose, lactose, glucose, maltose, sorbitol, galactose, glycerol and mannitol; preferably glycerol.

[0017] The nitrogen source is at least one of ammonium sulfate, ammonium chloride, diammonium phosphate, sodium nitrate, gelatin and trypsin; preferably trypsin.

[0018] The precursor is at least one of cysteine, histidine, glutamic acid and methionine, preferably cysteine.

[0019] When the precursor is cysteine, its content is 0-64 mM; further 8-64 mM; further 32-48 mM; further 40 mM.

[0020] When the precursor is histidine, its content is 0 to 32 mM; further 0 to 8 mM; further 8 mM.

[0021] When the precursor is glutamate, its content is 0-8mM;

[0022] When the precursor is methionine, its content is 0-24mM;

[0023] The fermentation temperature used in liquid fermentation is 25°C to 37°C; further 25°C to 30°C; further 28°C.

[0024] The fermentation time used for liquid fermentation is 96 to 192 hours; further, 120 to 168 hours; further, 132 to 156 hours; further, 144 hours.

[0025] The liquid volume used for liquid fermentation is 25-150mL / 250mL; further, 25-100mL / 250mL; further, 25-50mL / 250mL; further, 25mL / 250mL.

[0026] The present invention also provides a method for high-yield ergothioneine by utilizing the above-mentioned sphingomonas, comprising the following steps:

[0027] The seed liquid of Sphingomonas sp. HBJ-193 is inoculated into a modified LB liquid culture medium, and the culture is shaken and fermented to obtain a fermentation liquid, thereby achieving the purpose of high-yield ergothioneine.

[0028] The rotation speed of the shaking fermentation culture is 150-200 rpm; further 150 rpm.

[0029] Compared with the prior art, the present invention has the following advantages and effects:

[0030] (1) The present invention not only provides a high-yield EGT-producing Sphingomonas sp. HBJ-193, but also provides a method for high-yield EGT of Sphingomonas sp. HBJ-193 through optimization experiments, thus providing a new idea for the industrial production of EGT.

[0031] (2) The sphingomonas sp. HBJ-193 provided by the present invention has the characteristics of efficient production of ergothioneine, and the strain has a more efficient ability to produce ergothioneine under the fermentation conditions of the present invention, which is specifically embodied in:

[0032] 1) EGT synthesized by Sphingomonas sp. HBJ-193 is mainly distributed outside the cell and is easy to extract;

[0033] 2) The optimal fermentation conditions for the synthesis of EGT by Sphingomonas sp. HBJ-193 were obtained through single factor and response surface experiments, and the yield was as high as 100.04 mg / L. The Sphingomonas sp. HBJ-193 provided by the present invention is a newly discovered EGT synthesis strain, and the provided strain has the characteristics of high yield, short fermentation time, and easy extraction of fermentation products, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the standard curve of ergothioneine HPLC detection.

[0035] Figure 2 The strain morphology (left) and optical microscopy results (right) of Sphingomonas HBJ-193.

[0036] Figure 3 This is the phylogenetic tree of Sphingomonas HBJ-193.

[0037] Figure 4 It is the detection curve of ergothioneine chromatography fluid.

[0038] Figure 5 It is the electrospray ionization mass spectrum of Sphingomonas HBJ-193EGT.

[0039] Figure 6 This is the result of determining the optimal carbon source for high-yield EGT of Sphingomonas HBJ-193.

[0040] Figure 7 This is the result of determining the optimal nitrogen source for high-yield EGT of Sphingomonas HBJ-193.

[0041] Figure 8 This is the result of determining the optimal carbon source addition amount for high-yield EGT of Sphingomonas HBJ-193.

[0042] Fig. 9 This is the result of determining the optimal nitrogen source addition amount for high-yield EGT of Sphingomonas HBJ-193.

[0043] Fig.10 This is the initial pH determination result of the optimal culture medium for high EGT production of Sphingomonas HBJ-193.

[0044] Fig.11 This is the result of determining the optimal culture medium volume for high-yield EGT of Sphingomonas HBJ-193.

[0045] Fig.12 This is the result of determining the optimal culture temperature for high-yield EGT of Sphingomonas HBJ-193.

[0046] Fig.13 It is a three-dimensional surface diagram and contour diagram of the response surface of culture temperature-initial pH to the high production of EGT by Sphingomonas HBJ-193.

[0047] Fig.14 It is a three-dimensional surface diagram and contour diagram of the response surface of culture temperature-nitrogen source addition to the high production of EGT by Sphingomonas HBJ-193.

[0048] Fig.15 It is a three-dimensional surface diagram and contour diagram of the response surface of nitrogen source addition amount-initial pH to the high production of EGT by Sphingomonas HBJ-193.

[0049] Fig.16 These are the results of the determination of the effect of glutamate addition on the high production of EGT by Sphingomonas HBJ-193.

[0050] Fig.17 These are the results of the determination of the effect of histidine addition on the high production of EGT by Sphingomonas HBJ-193.

[0051] Fig.18 These are the results of the determination of the effect of methionine addition on the high production of EGT by Sphingomonas HBJ-193.

[0052] Fig.19 These are the results of the determination of the effect of cysteine ​​addition on the high production of EGT by Sphingomonas HBJ-193.

[0053] Fig. 20 This is the result of determining the optimal culture time for high-yield EGT of Sphingomonas HBJ-193. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0055] The experimental methods in the following examples without specific experimental conditions are usually carried out under conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used, unless otherwise specified, are all obtained from commercial sources.

[0056] LB medium used in the examples: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, pH 7.4; 20 g / L agar powder was added (solid medium only).

[0057] Example 1 Construction of EGT synthetic strain library

[0058] Add the koji to the sterile water, collect the soaking liquid and dilute it, then spread it on the plate and culture it at a constant temperature. Pick a single colony and streak it on the plate for culture, and purify it repeatedly; after obtaining a single colony, inoculate it into the corresponding liquid culture medium, culture it overnight to obtain the seed liquid, and inoculate it into the corresponding liquid culture medium with an inoculum of 2% (v / v). Ferment the bacteria at 37°C and the fungi at 25°C. Take the fermentation liquid after 7 days, centrifuge it at 10000r / min for 10min to get the bacterial sediment, resuspend and wash it three times with ddH2O, collect the bacterial cells, and freeze-dry them in a vacuum freeze dryer. Weigh 0.1g of freeze-dried bacterial cells, grind them into powder in a mortar, resuspend them with ddH2O to 1mL, add 4mL of ethanol extract (70% ethanol, 0.17% SDS), let it stand at 4°C for 12h, centrifuge it at high speed to get the supernatant, take 1mL and pass it through a 0.22μm organic microporous filter membrane for high performance liquid chromatography (HPLC).

[0059] HPLC detection conditions: Shimadzu LC 2030 CN instrument was used for ergothioneine high performance liquid chromatography detection. The chromatographic column used was Welch Ultimate HILIC AmphionⅡ chromatographic column, the detection wavelength was 257nm, the mobile phase was acetonitrile: water = 80:20, the flow rate was 1mL / min, the injection volume was 20μL, and the column temperature was 30℃.

[0060] Ergothioneine standard curve drawing: weigh ergothioneine standard, dissolve it with a small amount of ultrapure water, and then use ultrapure water to make up to the volume, and configure it into a gradient concentration of ergothioneine standard solution: 20, 10, 5, 2.5, 1.25 mg / L. Take about 1mL of ergothioneine standard solution of each concentration through a 0.22μm water system microporous filter membrane, and then perform HPLC detection under the above conditions. After HPLC detection, record the peak area corresponding to the standard solution of different concentrations, use the peak area as the horizontal coordinate, and use the standard solution concentration (mg / L) as the vertical coordinate to make a linear regression equation. Ergothioneine standard curve is shown in Figure 1 After measuring the sample, the concentration of ergothioneine in the sample solution can be calculated using this equation.

[0061] Under the above measurement method, the strain numbered HBJ-193 was detected to have an EGT production of 0.6 mg / gDW.

[0062] Example 2 Identification of HBJ-193 strain

[0063] (1) Morphological identification: Strain HBJ-193 was spread on LB plates and cultured at 30°C for 48 h. The characteristics of the colonies were observed. Figure 2As shown: The colonies are regular circles, with colors ranging from golden yellow to orange-yellow. The surface of the colonies is smooth and easy to pick up. Under optical microscopy, the bacteria have an elongated spindle-shaped structure.

[0064] (2) 16S rDNA molecular identification:

[0065] To confirm the species of HBJ-193, 16S rDNA sequencing was used to identify it. The primers used were 16S universal primers 27F and 1492R, and the primer sequences were:

[0066] 27F: 5′-TACGGYTACCTTGTTACGACTT-3′;

[0067] 1492R: 5′-AGAGTTTGATCMTGGCTCAG-3′;

[0068] The template preparation method is alkaline lysis. The specific implementation method is: take 50 μL of pure strain culture solution under a sterile environment, add 50 μL of bacterial lysis solution and mix well, place in a 98℃ metal bath for 30 minutes, and then add 25 μL of bacterial neutralization solution and mix well to obtain a molecular amplification template for molecular biological identification. The preparation method of bacterial lysis solution and bacterial neutralization solution is:

[0069] Bacterial lysis solution (50 mM NaOH solution): weigh 2 g NaOH and dissolve it in 80 mL ultrapure water. After it is completely dissolved, use ultrapure water to make up to 100 mL and sterilize it at 121°C for 30 min.

[0070] Bacteria neutralization solution (1M Tris-HCl solution, pH=8.0): weigh 15.76 g Tris-HCl, dissolve in 80 mL ultrapure water, then use ultrapure water to make up to 100 mL, use NaOH to adjust the pH to 8.0, and sterilize at 121°C for 30 min.

[0071] The PCR amplification system was: 2× PrimeSTAR Max Premix 10 μL, upstream primer 1 μL, downstream primer 1 μL, molecular amplification template 2 μL, and ddH2O 6 μL.

[0072] The PCR amplification program was: 98°C for 2 min; 94°C for 30 sec, 55°C for 45 sec, 72°C for 8 sec, 30 cycles; 72°C for 5 min.

[0073] After obtaining the rDNA amplification product of the strain, the corresponding fragments were recovered by gel excision and Sanger sequencing was performed. After obtaining the data off the machine, the software Chromas was used to interpret the peak graph to remove connector contamination and misreading sites, and the software seqMan was used to splice the bidirectional sequencing data to obtain the 16S rDNA nucleic acid sequence of HBJ-193 for comparison. After obtaining the 16S rDNA sequence of the strain, it was compared with the rDNA nucleic acid library on the NCBI website. The comparison results showed that the homology between the 16S rDNA gene sequence of strain HBJ-193 and the reported Sphingomonas paucimobilis strain H4 reached 100%. The sequences were sorted by maximum score, and the top 50 sequences in the comparison results were taken. The neighbor joining method was used with MEGA v7.0.2 for phylogenetic evolution analysis, and the results close to the HBJ-193 branch were shown as follows. Figure 3 As shown, HBJ-193 and Sphingomonas yabuuchia are in the same branch of the phylogenetic tree, with a confidence level of 73. Combined with the morphological characteristics of strain HBJ-193, HBJ-193 was identified as belonging to the genus Sphingomonas sp.

[0074] Therefore, the strain was named Sphingomonas sp. HBJ-193, and its preservation information is as follows: Preservation unit: China General Microbiology Center (CGMCC), the preservation date is March 29, 2022, the preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC NO.24613.

[0075] The 16S rDNA gene sequence of Sphingomonas sp. HBJ-193 is shown in SEQ ID NO: 1.

[0076] Example 3 Purification of EGT from Sphingomonas sp. HBJ-193 Fermentation Broth

[0077] (1) Sample preparation: Sphingomonas sp. HBJ-193 was fermented in LB liquid medium with a liquid volume of 100 mL / 250 mL at 30°C and 150 rpm for 6 days, and the inoculum size was 2% (v / v) to obtain a fermentation broth; 200 mL of the fermentation broth was added with 800 mL of ethanol extract (70% ethanol, 0.17% SDS), and the mixture was allowed to stand at 4°C for 12 h. The supernatant was centrifuged at 8000 rpm for 20 min, and concentrated by rotary evaporation at 60°C and 80 rpm to less than 20 mL. The concentrate was collected and centrifuged at 8000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter membrane for chromatographic separation.

[0078] (2) Pretreatment of dextran gel: Weigh 25g of dextran gel Sephadex G-10 and place it in a beaker. Add an appropriate amount of deionized water to soak and swell for 24 hours, then let it stand. After the gel is completely settled, pour off the water on the top of the gel. Then slowly pour 2 times the volume of deionized water into the beaker, gently stir the gel with a glass rod while pouring to avoid particle breakage, let it stand for 5 minutes to allow the gel to settle, and pour off the suspended and floating fine particles with the upper water. Float 5 times until there are no fine particles in the upper water; then seal the beaker of Sephadex G-10 aqueous solution with plastic wrap, poke several small holes in the plastic wrap, and place it in an ultrasonic cleaner for 10 minutes (high temperatures generated by long-term ultrasonic oscillation should be avoided) to remove bubbles in the gel solution. Take out the beaker and let it stand for 5 minutes, and pour off part of the upper water so that the volume of the gel particles is roughly equivalent to the volume of the upper water.

[0079] (3) Column loading: Clamp the 1.6 cm × 60 cm chromatography column rinsed with deionized water vertically on the iron frame, open the column inlet, and use a constant flow pump to inject deionized water from the column outlet (avoid generating bubbles) so that the final water height in the tube is about 3 cm; gently stir (to prevent air from escaping) the pre-treated Sephadex G-10 solution to form a uniform slurry, and then quickly use a glass rod to drain it along the wall and pour it into the chromatography column. After the slurry is naturally precipitated to form a column bed about 2 cm high, open the column outlet to allow the water in the column to flow out continuously, and at the same time, pour the slurry from the column inlet continuously to allow the gel particles in the column to continuously settle. The final column height is 40 cm. If there is no continuous gel particle sedimentation on the surface of the column bed, use a glass rod to gently stir the surface of the column bed to resuspend it and then add the slurry, otherwise it will form a fracture and affect the chromatography results. After the column loading is completed, check whether there are fractures or bubbles in the gel in the column. If there are, the column should be reloaded. After checking, allow the column bed to stabilize for 15 to 20 minutes, then connect the elution bottle and the column inlet, use a constant flow pump to equilibrate the column with 3 to 5 times the column volume of deionized water at a constant pressure, and maintain a flow rate of 1 mL / min.

[0080] (4) Loading and eluting: Before loading, check whether the surface of the column bed is flat. If it is not flat, use a glass rod to gently stir the column bed, and then allow the gel to naturally settle into a flat column bed. Turn off the constant flow pump, open the column inlet to allow the water in the column to flow out naturally, and close the column outlet after the liquid level drops to almost expose the surface of the column bed. Use a pipette to slowly add 2mL of sample to the chromatography column (avoid impacting the column bed surface), then open the chromatography column outlet and allow water to flow out slowly to ensure that the sample is replaced as narrowly as possible. After all the samples have entered the gel, gently add deionized water (i.e., eluent) to the place where the sample is added. When the liquid level is about 3cm higher than the column bed, close the column inlet and connect the constant flow pump. Elute with eluent (deionized water), control the outlet flow rate to maintain at 1mL / min, and collect the eluent in 15mL glass test tubes, 3mL / tube, collect 40 tubes, and use HPLC method to test according to the above conditions. The test results are as follows Figure 4 As shown, the peaks of tubes 23 to 30 were at the EGT retention time, but the ergothioneine content of tubes 23, 24, 29, and 30 was low, and the peak area accounted for a small proportion, and there were many impurities. Therefore, the collected liquids of tubes 25 to 28 were concentrated to obtain the EGT purified extract.

[0081] Example 4 Mass Spectrometry Confirmation of EGT Synthesis by HBJ-193

[0082] The purified extract of the HBJ-193 fermentation broth described in Example 3 was taken for LC-MS qualitative detection. The detection was entrusted to the Testing Center of Sun Yat-sen University, and the instrument used was UPLC: Ultimate3000 (Thermo Fisher, Germany), MS: timsTOF (Bruker, Germany), Mass parameters were: Source type: ESI, Ion Polarity: positive, Capillary: 4500V, Nebulizer: 1.5Bar, Dry gas: 8.0L / min, Dry temperature: 220°C, and the chromatographic conditions were: the chromatographic column used was Waters ACQUITY UPLC BEH C18, the column temperature was 40°C, the mobile phase A was 0.1% FA, B was CH3CN, and the ratio used a multi-step gradient, as shown in Table 1.

[0083] Table 1 LC-MS mobile phase multi-stage gradient ratio

[0084]

[0085] ESI-MS results are as follows Figure 5As shown in the figure, there is a main molecular ion [M+H]+ in the m / z, which is consistent with the calculated accurate molecular ion mass 230.0963. The identification results of the ion fragments are as follows: m / z 186 is 2-(2-mercapto-imidazol-5-yl) N,N,N-trimethyl-1-amino (C8H 16 N3S + ), which is an EGT molecule lacking a carboxyl group. It can be confirmed that the synthetic product of Sphingomonas HBJ-193 detected is ergothioneine.

[0086] Example 5 Optimization of the fermentation system of Sphingomonas HBJ-193 for high EGT production

[0087] (i) The inoculation amount of Sphingomonas HBJ-193 was 2% (v / v), and the culture medium was fermented at 37° C. and 150 rpm for 168 h in LB liquid culture medium with a liquid volume of 150 mL / 250 mL to obtain a fermentation broth; the EGT yield in the fermentation broth was 1.58 mg / L.

[0088] LB liquid culture medium: tryptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, pH 7.4.

[0089] (ii) The optimization of the fermentation system of Sphingomonas HBJ-193 for high-yield EGT was implemented by the following steps:

[0090] (1) Different carbon sources: The formula of the fermentation medium was improved on the basis of LB liquid medium. The strain HBJ-193 was shaken and fermented in the improved LB liquid medium. When the carbon sources of the medium were yeast extract powder, starch, sucrose, lactose, glucose, maltose, sorbitol, galactose, glycerol, and mannitol, the inoculation amount was 2% (v / v), and the liquid volume was 100 mL / 250 mL. The LB liquid medium was shaken and fermented at 30°C and 150 rpm for 168 h to obtain the fermentation broth. The fermentation broth with glycerol as the carbon source had the highest EGT yield after extraction and purification and detection by HPLC ( Figure 6 ).

[0091] (2) Different nitrogen sources: The formula of the fermentation medium was improved on the basis of LB liquid medium. The strain HBJ-193 was fermented in the improved LB liquid medium by shaking. When the nitrogen sources of the medium were ammonium sulfate, ammonium chloride, diammonium phosphate, sodium nitrate, gelatin, and trypsin, the other fermentation conditions were referred to the above step (1). The fermentation broth with trypsin as the nitrogen source had the highest EGT yield after extraction and purification and was detected by HPLC ( Figure 7 ).

[0092] (3) Different amounts of glycerol: The strain HBJ-193 was fermented in a modified LB liquid medium containing 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, and 15 g / L of carbon source glycerol, respectively. Other fermentation conditions were referred to the above step (1). When the glycerol content in the medium was 7.5 g / L, the fermentation broth had the highest EGT yield after extraction, purification, and HPLC detection ( Figure 8 ).

[0093] (4) Different amounts of tryptone: The strain HBJ-193 was fermented by shaking in modified LB liquid medium with nitrogen source tryptone contents of 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, 15 g / L, and 17.5 g / L, respectively. The other fermentation conditions were referred to the above step (1). When the tryptone content in the medium was 12.5 g / L, the fermentation broth had the highest EGT yield after extraction, purification, and HPLC detection ( Fig. 9 ).

[0094] (5) Different initial pH: The fermentation medium was improved on the basis of LB liquid medium with an initial pH of 7.4. The strain HBJ-193 was fermented at initial pHs of 3.4, 4.4, 5.4, 6.4, 7.4, 8.4, and 9.4, respectively. The other fermentation conditions were referred to the above step (1). When the fermentation broth with an initial pH of 5.4 was extracted and purified and detected by HPLC, the EGT yield was the highest ( Fig.10 ).

[0095] (6) Different liquid volumes: The liquid volume of the fermentation medium was improved on the basis of 100 mL / 250 mL of LB liquid medium. The strain HBJ-193 was subjected to oscillation fermentation at liquid volumes of 25 mL / 250 mL, 50 mL / 250 mL, 75 mL / 250 mL, 100 mL / 250 mL, 150 mL / 250 mL, and 200 mL / 250 mL, respectively. Other fermentation conditions were referred to the above step (1). When the liquid volume was 25 mL / 250 mL, the fermentation broth had the highest EGT yield after extraction and purification and detection by HPLC ( Fig.11 ).

[0096] (7) Different culture temperatures: When strain HBJ-193 was fermented under shaking conditions at 25°C, 28°C, 30°C, 34°C, and 37°C, except for the culture temperature, other fermentation conditions were the same as those in step (1) above. When the fermentation broth was extracted and purified at 28°C and tested by HPLC, the EGT yield was the highest ( Fig.12 ).

[0097] According to the above-mentioned optimization experiment, nitrogen source (tryptone) addition, initial pH, culture temperature are optimized by three-factor three-level response surface, wherein, in the improved LB liquid medium, except nitrogen source (tryptone), the remaining components and content are: glycerol 7.5g / L, NaCl 10g / L; Inoculum size is 2% (v / v), and liquid volume is 100mL / 250mL, 150rpm shaking fermentation 6d. Software Design-Expert.V8.0.6.1 software is used to carry out Box-Behnken experimental design, and the obtained factor level table is as shown in Table 2, and experimental result is as shown in Table 3. Regression fitting is carried out according to the data in the table, and the quadratic multiple regression equation with thioneine output as response value is obtained:

[0098] P=+23.29+0.87A-0.21B+0.65C+1.26AB+1.73AC+0.69BC-3.70A 2 -1.42B 2 -1.30C 2

[0099] (where P: EGT yield; A: pH; B: temperature; C: nitrogen source addition amount)

[0100] Table 2 Box-Behnken design factors and levels

[0101]

[0102] Table 3 Box-Behnken design experimental results

[0103]

[0104] In the above equation, the absolute value of the coefficient can indicate the influence of the factor on the yield, and the positive and negative values ​​of the coefficient can reflect the direction of the influence. Therefore, it can be concluded that in this experiment, the influencing factors of the response value ergothioneine are from large to small: pH> nitrogen source addition> temperature, and the coefficients of the quadratic terms in the equation are all negative. It can be seen that the parabolic graph of the response value opens downward and has a maximum value within the experimental range, which can be used to optimize the experiment.

[0105] The software Design-Expert.V8.0.6.1 was used for multivariate quadratic fitting to obtain the three-dimensional surface plot and contour plot of the response surface, as shown in Fig.13 , 14 、15.

[0106] The increase of any of the three factors will lead to the EGT production showing a trend of first increasing and then decreasing. In the three interactive combinations, the three-dimensional surfaces all open downward and have a maximum value. Through contour map analysis, it can be inferred that the culture temperature and initial pH have a strong influence on the EGT production of strain HBJ-193, which can provide guidance for the industrial fermentation of EGT production by strain HBJ-193.

[0107] Under the optimized culture medium and culture conditions, the precursors of different working concentrations were added by single factor experiment: histidine, cysteine, methionine, glutamate, among which the modified LB liquid medium: glycerol 7.5g / L, tryptone 12.5g / L, NaCl 10g / L, pH5.4; the culture conditions were: inoculation volume 2%, temperature 28℃, liquid volume 50mL / 250mL, 150rpm, and culture for 6 days. When the working concentration of cysteine ​​was 40mM, the fermentation broth was extracted and purified and the EGT yield detected by HPLC was the highest (69.28mg / L) ( Fig.16 , 17 , 18, 19).

[0108] In summary, based on the above optimization experiments, the EGT yield at different fermentation times was explored:

[0109] The strain HBJ-193 was cultured in an optimized LB medium (i.e., improved LB liquid medium: glycerol 7.5 g / L, tryptone 12.5 g / L, NaCl 10 g / L, pH 5.4), with an inoculation volume of 2%, a liquid volume of 25 mL / 250 mL, a culture temperature of 28°C, a rotation speed of 150 rpm, and a cysteine ​​addition amount of 40 mM. The fermentation broth was taken every 12 hours, and the bacterial precipitate and supernatant were collected by centrifugation at 8000 rpm, and 4 times the volume of ethanol extract was added to the supernatant, and the supernatant was kept at 4°C overnight. The bacterial precipitate was washed 3 times with sterile water, and then 1 mL of sterile water was added to resuspend it, and then 4 mL of ethanol extract was added to keep it at 4°C overnight. The overnight extracts were all centrifuged at high speed to obtain the supernatant, and HPLC was used to detect after passing through a 0.22 μm filter membrane. The results are as follows Fig. 20 As shown, the EGT production of strain HBJ-193 reached the highest when the culture time was 144 h, and the total intracellular and extracellular EGT production reached 100.04 mg / L.

[0110] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. Sequence Listing <110> South China Agricultural University <120> A Sphingomonas strain and its application <160> 3 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 1330 <212> DNA <213> Artificial Sequence <220> <223> 16S rDNA of Sphingomonas sp. HBJ-193 <400> 1 tattgctagg ttggcgcaac gccttcgggt gaatccaact cccatggtgt gacgggcggt 60 gtgtacaagg cctgggaacg tattcaccgc ggcatgctga tccgcgatta ctagcgattc 120 cgccttcatg ctctcgagtt gcagagaaca atccgaactg agacaacttt tggggattag 180 ctcgccctcg cagggtcgct gcccactgta gttgccattg tagcacgtgt gtagcccagc 240 gcgtaagggc catgaggact tgacgtcatc cccaccttcc tccggcttat caccggcggt 300 tcctttagag tacccaacca aatgatggta actaaaggcg agggttgcgc tcgttgcggg 360 acttaaccca acatctcacg acacgagctg acgacagcca tgcagcacct gtgtgtaggt 420 ccccgaaggg aagaaatcca tctctggaag tcgtcctacc atgtcaaacg ctggtaaggt 480 tctgcgcgtt gcttcgaatt aaaccacatg ctccaccgct tgtgcaggcc cccgtcaatt 540 cctttgagtt ttaaccttgc ggccgtactc cccaggcgga taacttaatg cgttagctgc 600 gccacccaag caccaagtgc ccggacagct agttatcatc gtttacggcg tggactacca 660 gggtatctaa tcctgtttgc tccccacgct ttcgcacctc agcgtcaata ccagtccagt 720 cagccgcctt cgccactggt gttcttccga atatctacga atttcacctc tacactcgga 780 attccactga cctctcctgg attcaagcga tgcagtctca aaggcagttc tggagttgag 840 ctccaggctt tcacctctga cttacaaagc cgcctacgtg cgctttacgc ccagtaattc 900 cgaacaacgc tagccccctc cgtattaccg cggctgctgg cacggagtta gccggggctt 960 attctcccgg tacagtcatt atcttcccgg gtaaaagagc tttacaaccc tagggccttc 1020 atcactcacg cggcattgct ggatcaggct ttcgcccatt gtccaatatt ccccactgct 1080 gcctcccgta ggagtctggg ccgtgtctca gtcccagtgt ggctgatcat cctctcagac 1140 cagctatgga tcgtcggctt ggtaggcctt taccccacca actacctaat ccaacgcggg 1200 ctcatcctca ggcgataaat ctttgatctc gcgatatcat ccggtattag cagccgtttc 1260 cagctgttat tccgaaccta agggcagatt cccacgcgtt acgcacccgt gcgccactaa 1320 ggccgaagcc 1330 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> 27F <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1492R <400> 3 agagtttgat cmtggctcag 20

Claims

1. A strain of Sphingomonas ( Sphingomonas sp.) HBJ-193, characterized in that: The Sphingomonas HBJ-193 was deposited on March 29, 2022 at the General Microbiology Center of the China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC NO.24613.

2. the application of Sphingomonas described in claim 1 in producing thioneine by liquid fermentation.

3. The use according to claim 2, characterized in that: The formula of the modified LB liquid culture medium used for liquid fermentation is: carbon source 2.5-15 g / L, nitrogen source 2.5-17.5 g / L, NaCl 10-15 g / L, precursor 0-64 mM, pH 4.4-9.

4.

4. The use according to claim 3, characterized in that: The formula of the improved LB liquid culture medium is: carbon source 2.5-10 g / L, nitrogen source 7.5-17.5 g / L, NaCl 10-15 g / L, precursor 0-64 mM, pH 5.4-8.

4.

5. The use according to claim 4, characterized in that: The formula of the improved LB liquid culture medium is: carbon source 2.5-7.5 g / L, nitrogen source 10-15 g / L, NaCl 10-15 g / L, precursor 0-64 mM, pH 5.4-8.

4.

6. The use according to claim 3, characterized in that: The carbon source is at least one of yeast extract powder, starch, sucrose, lactose, glucose, maltose, sorbitol, galactose, glycerol and mannitol; The nitrogen source is at least one of ammonium sulfate, ammonium chloride, diammonium phosphate, sodium nitrate, gelatin, and trypsin; The precursor is at least one of cysteine, histidine, glutamic acid and methionine.

7. The use according to claim 6, characterized in that: When the precursor is cysteine, its content is 0-64 mM; When the precursor is histidine, its content is 0-32 mM; When the precursor is glutamate, its content is 0 to 8 mM; When the precursor is methionine, its content is 0-24 mM.

8. The use according to claim 7, characterized in that: When the precursor is cysteine, its content is 8 to 64 mM; When the precursor is histidine, its content is 0-8 mM.

9. The use according to any one of claims 2 to 8, characterized in that: The fermentation temperature used for liquid fermentation is 25℃~37℃.

10. The use according to claim 9, characterized in that: The fermentation temperature is 25℃~30℃.

11. The use according to claim 10, characterized in that: The fermentation temperature was 28°C.

12. The use according to any one of claims 2 to 8, characterized in that: The fermentation time used for liquid fermentation is 96 to 192 h.

13. The use according to claim 12, characterized in that: The fermentation time is 120 to 168 hours.

14. The use according to claim 13, characterized in that: The fermentation time is 132 to 156 hours.

15. The use according to claim 14, characterized in that: The fermentation time was 144 h.

16. The use according to any one of claims 2 to 8, characterized in that: The liquid volume used for liquid fermentation is 25-150 mL / 250 mL.

17. The use according to claim 16, characterized in that: The liquid volume is 25~100 mL / 250mL.

18. The use according to claim 17, characterized in that: The liquid volume is 25-50 mL / 250 mL.

19. The use according to claim 18, characterized in that: The liquid volume is 25 mL / 250 mL.

20. A method for high production of ergothioneine using the sphingomonas according to claim 1, characterized in that, The steps include: The sphingomonas HBJ-193 seed liquid described in claim 1 is inoculated into the improved LB liquid culture medium described in any one of claims 3 to 8, and the culture is shaken and fermented to obtain a fermentation liquid, thereby achieving the purpose of high-yield ergothioneine.

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