Schizochytrium sp. producing ergothioneine, and application and method for producing ergothioneine

Abstract

The application discloses a schizophyllan selenium-producing schizophyllum and application and a method for producing ergothioneine, and belongs to the technical field of microbial breeding and fermentation. A wild-type schizophyllum producing ergothioneine is separated from selenium-rich soil in Akesu region in the north of Tarim Basin in southern Xinjiang, China, and is used as a starting strain. The schizophyllum ZZ is obtained through multiple rounds of ARTP mutagenesis combined with directional screening and further adaptive domestication. Through medium optimization, the shake flask yield of the strain reaches 45.8 mg / L, and the final yield of a 5L fermenter reaches 431.7 mg / L. The application provides a high-yield ergothioneine excellent strain and an efficient fermentation process, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial breeding and fermentation technology, specifically relating to a fission yeast that produces ergot selenosides, its application, and a method for producing ergot selenosides. Background Technology

[0002] Ergoselenoside is a selenium-containing ergosterol derivative with excellent antioxidant, anti-inflammatory, and immunomodulatory activities, showing broad application prospects in the pharmaceutical, health product, and functional food fields. Currently, ergoselenoside production mainly relies on chemical synthesis, but this method suffers from cumbersome procedures, low product purity, and severe environmental pollution. Microbial fermentation, due to its environmental friendliness and high product activity, has become an ideal alternative.

[0003] Schizosaccharomyces pombe, as a model eukaryotic microorganism, has advantages such as a clear genetic background, simple culture conditions, and high safety, making it a potentially excellent host for the production of ergot selenoside.

[0004] However, the existing technology faces the following prominent problems: Research on the synthesis of ergot selenoside in fissile yeast is still in its early stages. In 2014, Pluskal et al. first confirmed that the ergothioneine synthesis pathway in fissile yeast can synthesize ergot selenoside in a selenium-supplemented medium, but did not provide specific yield data for wild-type strains. In 2018, Turrini et al. constructed transgenic fissile yeast by overexpressing the egt1 gene, and obtained milligram-level ergot selenoside products after fermentation in a sodium selenite-containing medium and multi-step purification. However, the transgenic strains suffer from plasmid instability, require inducers, and the milligram-level yield is still far below the requirements for industrial production. Currently, there are no publicly reported yields of ergot selenoside in wild-type fissile yeast; existing literature only confirms its synthetic ability, but the yield is extremely low and cannot meet industrial needs. Therefore, selecting superior strains with high ergot selenoside yields has become a key technical problem that urgently needs to be solved. Summary of the Invention

[0005] To address the bottleneck problem of low yield mentioned above, this invention isolated a wild-type fissile yeast producing ergot selenoside from selenium-rich soil in Aksu region of Xinjiang. Using this wild-type fissile yeast as the starting strain, after multiple rounds of ambient pressure room temperature plasma (ARTP) mutagenesis treatment, combined with multiple rounds of directional screening (selenium tolerance screening, precursor utilization screening, and high osmotic tolerance screening) and adaptive domestication, a fissile yeast with high ergot selenoside production was successfully bred.

[0006] One objective of this invention is to provide a high-yield ergot selenozyme and its breeding method; a second objective of this invention is to provide a method for producing ergot selenozyme by shake-flask fermentation or by batch feeding in a fermenter; and a further objective of this invention is to provide the application of the high-yield ergot selenozyme in the production of ergot selenozyme.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a schizosaccharomyces pombe strain that produces ergot selenoside, characterized in that the schizosaccharomyces pombe is Schizosaccharomyces pombe ZZ, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026805.

[0009] The fission yeast (Schizosaccharomyces pombe) ZZ provided by this invention was obtained through multiple rounds of ARTP mutagenesis and selection. The specific method is as follows:

[0010] (1) Wild-type fission yeast JZ-6 was isolated from selenium-rich soil in Aksu region on the northern edge of Tarim Basin in southern Xinjiang, China, and the strain was prepared into a bacterial suspension.

[0011] (2) First round of mutagenesis: The bacterial suspension was subjected to ARTP mutagenesis treatment. The mutagenesis parameters were power 110-130W, gas flow rate 8-12 SLM, treatment distance 1-3 mm, and the mutagenesis treatment time was based on the goal of achieving a lethality rate of 85-95%. The mutagenized bacterial suspension was then spread on a screening plate with a Na2SeO3 concentration of 35-45 mg / L to obtain a selenium-resistant mutant.

[0012] (3) Second round of mutagenesis: Using the selenium-resistant mutant strain obtained in the first round as the starting strain, repeat the ARTP mutagenesis treatment in step (2). Spread the mutagenized bacterial solution on screening plates with histidine concentration of 1.5-2.5 g / L, methionine concentration of 1.5-2.5 g / L, and Na2SeO3 concentration of 35-45 mg / L to obtain a precursor utilization enhanced mutant strain.

[0013] (4) Third round of mutagenesis: Using the precursor enhanced mutant strain obtained in the second round as the starting strain, repeat the ARTP mutagenesis treatment in step (2). Spread the mutagenized bacterial solution on screening plates with glucose concentration of 55-65 g / L, histidine concentration of 1.5-2.5 g / L, methionine concentration of 1.5-2.5 g / L, and Na2SeO3 concentration of 35-45 mg / L to obtain the hyperosmolarity tolerant mutant strain.

[0014] (5) Adaptation and domestication: The high osmotic tolerance mutant obtained in step (4) was continuously domesticated by gradually increasing the concentration of ergot selenoside in a culture medium containing ergot selenoside. The domestication concentration gradient was 100-500 mg / L (including 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L). Each concentration gradient was cultured 4-6 times, and a total of 20-30 times were cultured. The fission yeast (Schizosaccharomyces pombe) ZZ was isolated and screened.

[0015] In a second aspect, the present invention provides the use of the aforementioned Schizosaccharomyces pombe ZZ in the production of ergot selenoside or ergot selenoside-containing products.

[0016] In a third aspect, the present invention provides a method for producing ergot selenium by shake-flask fermentation, characterized in that fission yeast (Schizosaccharomyces pombe) ZZ is inoculated into a fermentation medium for culture, and ergot selenium is obtained from the culture.

[0017] Culture conditions: Incubate at 30-32℃ and 200-250 rpm for 96-100 h.

[0018] The fermentation medium consists of the following components: carbon source 20-70 g / L, yeast extract 5-10 g / L, nitrogen source 5-30 g / L, histidine 0.25-2.5 g / L, methionine 0.25-2.5 g / L, cysteine ​​0-1 g / L, Na2SeO3 10-60 mg / L, KH2PO4 1-3 g / L, MgSO4·7H2O 0.5-0.8 g / L, CaCl2·2H2O 0.1-0.2 g / L, KCl 1-2 g / L, and a trace element mixture 1-1.5 mL / L, pH 5-6.

[0019] The carbon source is selected from one or a combination of two of glucose, sucrose, and glycerol; preferably, the carbon source is selected from glucose.

[0020] The nitrogen source is selected from one or a combination of two of peptone, beef extract, and ammonium sulfate; preferably, the nitrogen source is selected from peptone or ammonium sulfate; most preferably, the nitrogen source is selected from ammonium sulfate.

[0021] In this invention, unless otherwise specified, the trace element mixture is composed of the following components: FeSO4·7H2O 10 mg / L, MnSO4·H2O 5 mg / L, ZnSO4·7H2O 5 mg / L, CuSO4·5H2O 0.5 mg / L, Na2MoO4·2H2O 0.5 mg / L, and CoCl2·6H2O 0.5 mg / L.

[0022] In some embodiments of the present invention, the fermentation medium comprises the following components: glucose 20-60 g / L, yeast extract 4-6 g / L, peptone or ammonium sulfate 10-15 g / L, histidine 1-2 g / L, methionine 1-2 g / L, cysteine ​​0-1 g / L, Na2SeO3 20-40 mg / L, KH2PO4 1.5-2.5 g / L, MgSO4·7H2O 0.4-0.6 g / L, CaCl2·2H2O 0.05-0.15 g / L, KCl 0.8-1.2 g / L, and a trace element mixture 0.8-1.2 mL / L, with a pH of 5-6.

[0023] Preferably, the fermentation medium consists of the following components: glucose 40-60 g / L, yeast extract 4.5-5.5 g / L, peptone or ammonium sulfate 10-15 g / L, histidine 1-2 g / L, methionine 1-2 g / L, Na2SeO3 30-40 mg / L, KH2PO4 1.5-2.5 g / L, MgSO4·7H2O 0.4-0.6 g / L, CaCl2·2H2O 0.05-0.15 g / L, KCl 0.8-1.2 g / L, and a trace element mixture 0.8-1.2 mL / L, with a pH of 5-6.

[0024] In an optional embodiment of the present invention, the fermentation medium comprises the following components: 60 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 2 g / L histidine, 2 g / L methionine, 2 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.1 g / L CaCl2·2H2O, 1 g / L KCl, 40 mg / L Na2SeO3, 1 mL / L trace element mixture, and pH 5.5.

[0025] In another optional embodiment of the present invention, the fermentation medium comprises the following components: 60 g / L glucose, 5 g / L yeast extract, 10 g / L ammonium sulfate, 2 g / L histidine, 2 g / L methionine, 2 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.1 g / L CaCl2·2H2O, 1 g / L KCl, 40 mg / L Na2SeO3, 1 mL / L trace element mixture, and pH 5.5.

[0026] In the preferred embodiment of the present invention, the fermentation medium consists of the following components: 40 g / L glucose, 5 g / L yeast extract, 15 g / L ammonium sulfate, 1 g / L histidine, 1 g / L methionine, 30 mg / L Na2SeO3, 2 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.1 g / L CaCl2·2H2O, 1 g / L KCl, 1 mL / L trace element mixture, and pH 5.5.

[0027] In a fourth aspect, the present invention provides a method for batch-fed fermentation of ergot selenium, characterized in that fission yeast (Schizosaccharomyces pombe) ZZ is inoculated into a fermenter for fermentation, and the fermentation is carried out by feeding according to the strategies shown in 1)-3) below, thereby obtaining ergot selenium from the fermentation product:

[0028] 1) When the residual sugar concentration is below 5 g / L, add glucose solution to control the glucose concentration in the fermentation broth in the range of 1-5 g / L, wherein the glucose concentration in the glucose solution is 450-550 g / L;

[0029] 2) Start adding a mixed solution of histidine and methionine at 48h of fermentation, and control the concentration of histidine and methionine in the fermentation broth in the range of 0.5-1.5 g / L. Continue adding the solution until the end of fermentation. The concentration of histidine and methionine in the mixed solution is 15-20 g / L.

[0030] 3) Start adding Na2SeO3 solution after 48 hours of fermentation, and control the Na2SeO3 concentration in the fermentation broth to be in the range of 20-30 mg / L. The Na2SeO3 concentration in the Na2SeO3 solution is 20-25 g / L.

[0031] The residual sugar concentration mentioned in step 1) refers to the reducing sugar concentration in the fermenter, which is detected using the DNS method.

[0032] In some optional embodiments of the present invention, the culture medium in the fermenter is composed of the following components: glucose 40-60 g / L, yeast extract 4-6 g / L, peptone or ammonium sulfate 10-15 g / L, histidine 1-2 g / L, methionine 1-2 g / L, Na2SeO3 30-40 mg / L, KH2PO4 1.5-2.5 g / L, MgSO4·7H2O 0.4-0.6 g / L, CaCl2·2H2O 0.05-0.15 g / L, KCl 0.8-1.2 g / L, trace element mixture 1 mL / L, vitamin mixture 1 mL / L, pH 5-6.

[0033] The vitamin mixture consists of the following components: biotin 0.05 mg / L, calcium pantothenate 1.0 mg / L, niacin 1.0 mg / L, inositol 25 mg / L, thiamine 1.0 mg / L, and pyridoxine 1.0 mg / L.

[0034] In the preferred embodiment of the present invention, the culture medium in the fermenter is composed of the following components: 40 g / L glucose, 5 g / L yeast extract, 15 g / L ammonium sulfate, 1 g / L histidine, 1 g / L methionine, 30 mg / L Na2SeO3, 2 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.1 g / L CaCl2·2H2O, 1 g / L KCl, 1 mL / L trace element mixture, 1 mL / L vitamin mixture, and pH 5.5.

[0035] Fermentation conditions: temperature 30-32℃, stirring speed 300-700 rpm (with dissolved oxygen in conjunction, maintaining dissolved oxygen at 30% or higher), aeration rate 1-1.2 vvm, and pH controlled at 5.5±0.5 by adding ammonia or dilute hydrochloric acid.

[0036] The fermentation cycle is selected from 120-144 h, such as 120 h, 132 h, 144 h; in the preferred embodiment of the present invention, the fermentation cycle is 144 h.

[0037] The technical solution provided by this invention has the following beneficial effects:

[0038] (1) Solved the bottleneck problem of low ergot selenium production: For the first time, this invention obtained the ergot selenium-producing Schizosaccharomyces mutant strain ARTP-3X through multiple rounds of ARTP mutagenesis combined with directional screening. The yield of this mutant strain in shake flask reached 20.2 mg / L, which is 3.3 times higher than the starting strain JZ-6 (4.7 mg / L). Based on this strain, the tolerance and adaptation of ergot selenium products were domesticated to obtain a high-yielding mutant strain. This strain was preserved and named Schizosaccharomyces pombe ZZ. After domestication, the yield in shake flask increased to 31.2 mg / L, which is 54.5% higher than the ARTP-3X strain before domestication. Subsequently, after optimization of the shake flask culture medium composition, the highest yield of the ZZ strain in shake flask reached 45.8 mg / L. Further, a 5L fermenter batch feeding fermentation process was adopted, and the final yield of ergot selenium reached 431.7 mg / L, providing high-quality strain resources and mature and efficient fermentation technology for the industrial production of ergot selenium.

[0039] (2) An efficient multi-round targeted screening strategy was established: This invention adopts a multi-round targeted screening strategy combining ARTP mutagenesis with selenium tolerance screening (sodium selenite 35-45 mg / L), precursor utilization screening (histidine and methionine 1.5-2.5 g / L), high osmotic tolerance screening (glucose 55-65 g / L), and product tolerance adaptation (ergot selenoside concentration gradient 100-500 mg / L, 25 generations). The screening efficiency is high and the positive mutation rate is high, providing a technical solution that can be referenced for industrial microbial breeding.

[0040] (3) Optimized fermentation process: This invention further optimized the shake-flask fermentation medium formula for high-yield mutant strains (glucose 40 g / L, ammonium sulfate 15 g / L, histidine 1 g / L, methionine 1 g / L, sodium selenite 30 mg / L), achieving a shake-flask yield of 45.8 mg / L. Based on this, a 5L fermenter fed-batch fermentation process was developed. The initial medium used the optimized shake-flask formula and was supplemented with a vitamin mixture. Product synthesis was further enhanced through a feeding strategy (carbon source feedback feeding to maintain glucose concentration of 1-5 g / L, precursor feedback control to maintain histidine and methionine concentrations of 0.5-1.5 g / L, and selenium source dynamic control to maintain Na2SeO3 concentration of 20-30 mg / L). After 5L fermenter fed-batch fermentation, ergot selenoside yield reached 431.7 mg / L, providing reliable technical support for the industrial production of ergot selenoside.

[0041] (4) The strain has good genetic stability: After 10 consecutive generations, the yield of ergot selenium of this mutant strain still remained above 92% of the initial yield (28.9 mg / L after 10 generations, which is 92.6% of the initial yield of 31.2 mg / L), indicating that it has good genetic stability and is suitable for industrial production.

[0042] (5) Broad application prospects: The excellent strains and efficient fermentation methods provided by this invention can be used to prepare food, health products or medicines with antioxidant functions, which have good economic and social benefits.

[0043] Preservation Instructions

[0044] Latin name of Schizosaccharomyces pombe

[0045] Collection Center Registration Number: CCTCC NO: M 2026805

[0046] Preservation Institution: China Center for Type Culture Collection

[0047] Abbreviation for depository institution: CCTCC

[0048] Address of the depository: Wuhan University, Wuhan, China

[0049] Deposit date: April 24, 2026 Attached Figure Description

[0050] Figure 1 The high-performance liquid chromatogram of ergot selenine standard (detection wavelength 265 nm).

[0051] Figure 2 Ergot selenium standard quality spectrum (ESI) + (Positive ion mode).

[0052] Figure 3 Mass spectrometry image of ergot selenium fermentation product from wild-type strain JZ-6. Detailed Implementation

[0053] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0054] Example 1: Isolation and identification of fissile yeast producing ergot selenium

[0055] The target bacterial strain was isolated from soil samples rich in organic matter. The specific steps are as follows:

[0056] 1. Sample Collection and Enrichment: Samples were collected from Aksu region, located on the northern edge of the Tarim Basin in southern Xinjiang, China. A systematic study by Ma et al. on selenium-rich farmland in this region showed that the total selenium content in the soil ranged from 0.09 to 4.60 mg / kg, with an arithmetic mean of 0.356 mg / kg (Ma et al., J. Geochem. Explor., 2024, 258, 107412), confirming that the region possesses typical characteristics of selenium-rich soils. One g of soil sample collected from this region was weighed and added to a 250 mL Erlenmeyer flask containing 99 mL of sterile physiological saline (containing 0.05% Tween-80). The flask was shaken for 30 min to ensure thorough dispersion of microorganisms in the soil sample. After standing, 1 mL of the supernatant was inoculated into 50 mL of enrichment medium (20 g / L glucose, 5 g / L yeast extract, 10 g / L peptone, 10 mg / L Na2SeO3, pH 5.5) and cultured at 30 °C and 200 rpm for 48 h to selectively enrich microorganisms with selenium tolerance and selenium conversion ability.

[0057] 2. Strain Isolation: The enriched culture medium was serially diluted (10⁻⁶ ppm). -2 10 -4 10 -6 100 μL of the diluted solution was spread onto a plate (20 g / L agar was added to the enrichment medium). The plates were then incubated at 30°C for 48 h to obtain single colonies for subsequent screening.

[0058] 3. High-throughput primary screening: A total of 384 morphologically diverse single colonies were picked from the isolated plates and inoculated into 96-well plates containing 200 μL of liquid enrichment medium. The plates were incubated at 30℃ and 200 rpm for 72 h with shaking. After incubation, the 96-well plates were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected. 200 μL of methanol was added to each well, and the plates were extracted by sonication at room temperature for 30 min. After centrifugation again, the supernatant was collected, and the ergot selenium content was detected using RapidFire LC-MS. Based on the detection results, 8 strains that showed detectable ergot selenium production were selected and subjected to shake-flask secondary screening.

[0059] 4. Ergot selenium detection methods (including high-throughput primary screening and shake-flask secondary screening)

[0060] 4.1 Confirmation of Ergot Selenium Standard: The ergot selenium standard used in this invention is synthesized in-house and determined by liquid chromatography-mass spectrometry (LC-MS). Figure 1 , Figure 2 The results are as follows:

[0061] (1) Liquid chromatography analysis: Under the condition of detection wavelength of 265 nm, the standard showed a single characteristic peak at retention time of 5.050 min, with a peak area of ​​100%, indicating that the standard had high purity. Figure 1 ).

[0062] (2) Mass spectrometry analysis: The mass spectrum of the standard showed a quasi-molecular ion peak [M+H]. + The value is 278.0 m / z, consistent with the theoretical value of ergoselenosider, exhibiting typical selenium isotope distribution characteristics. Figure 2 In summary, the product is confirmed to be ergot selenium.

[0063] 4.2 High-throughput primary screening using RapidFire LC-MS: A RapidFire 400 high-throughput solid-phase extraction system was coupled with an Agilent 6470 triple quadrupole mass spectrometer. The solid-phase extraction column was an Agilent RapidFire Cartridge C18 column (5 μm); mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid methanol solution; the elution program was: loading 0.4 s, washing 5.5 s, elution 4.0 s, reequilibration 0.6 s, flow rate 3.0 mL / min. Mass spectrometry detection was performed using an AJS-ESI electrospray ionization source in positive ion mode, with data acquisition in multiple reaction monitoring (MRM) mode; the ergoselenochromic acid monitoring ion pair was m / z 278.1→234.0, collision energy 15 eV, and residence time 100 ms.

[0064] 4.3 High-performance liquid chromatography (HPLC) detection conditions for shake-flask re-screening:

[0065] 4.3.1 Chromatographic conditions: An Agilent 1260 Infinity II high-performance liquid chromatograph equipped with a VWD detector was used. The chromatographic column was a Yuexu AQ-C18 (4.6 mm × 250 mm, 5 μm). Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was methanol. The gradient elution program was as follows: 0-5 min, 100% A; 5-10 min, A decreased from 100% to 30%; 10-13 min, maintained at 30% A; 13.1-18 min, restored and maintained at 100% A. The flow rate was 1.0 mL / min, the column temperature was 30℃, the detection wavelength was 265 nm, and the injection volume was 5 μL.

[0066] 4.3.2 Preparation of Standard Solution: Accurately weigh ergoselenoside standard and prepare a 1 mg / mL stock solution with ultrapure water. Filter the solution through a 0.22 μm aqueous filter membrane. Quantification was performed using the external standard single-point method. The standard solution and the sample solution were injected and analyzed under the same chromatographic conditions. The ergoselenoside content in the sample was calculated based on the peak area ratio of the standard to the sample.

[0067] 4.3.3 Sample solution preparation: After fermentation, the fermentation broth was centrifuged at 12000 rpm for 5 min at 4℃. The supernatant was discarded and the wet cells were collected. Five times the volume of ultrapure water and an equal volume of 0.5 mm glass grinding beads were added. The mixture was vortexed at high speed for 30 s and then placed in an ice bath for 1 min. This process was repeated 8-10 times. The mixture was then centrifuged at 12000 rpm for 5 min at 4℃. The supernatant was filtered through a 0.22 μm aqueous filter membrane to obtain the ergot selenium test solution.

[0068] 5. Shake-flask re-screening: The glycerol-preserved cultures of the eight candidate strains isolated above were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of fermentation medium and cultured at 30 °C and 200 rpm for 96 h with shaking. The fermentation medium consisted of: glucose 20 g / L, yeast extract 5 g / L, peptone 10 g / L, KH₂PO₄ 2 g / L, MgSO₄·7H₂O 0.5 g / L, CaCl₂·2H₂O 0.1 g / L, KCl 1 g / L, Na₂SeO₃ 20 mg / L, and a trace element mixture of 1 mL / L, pH 5.5. The trace element mixture consisted of: FeSO4·7H2O 10 mg / L, MnSO4·H2O 5 mg / L, ZnSO4·7H2O 5 mg / L, CuSO4·5H2O 0.5 mg / L, Na2MoO4·2H2O 0.5 mg / L, and CoCl2·6H2O 0.5 mg / L. The samples were analyzed using high-performance liquid chromatography (HPLC) under shake-flask re-screening conditions.

[0069] Screening Results: The ergot selenium yields of eight candidate strains were screened through shake-flask fermentation, as shown in Table 1. Among them, strain JZ-6 showed a significantly higher yield than the other strains, reaching 4.7 mg / L. HPLC analysis revealed a characteristic absorption peak in the fermentation broth of strain JZ-6 at the same retention time as the ergot selenium standard. Simultaneously, mass spectrometry analysis of the fermentation sample of strain JZ-6 showed a quasi-molecular ion peak [M+H]. + The m / z value is 278.0, consistent with the theoretical value of ergot selenosider, exhibiting typical selenium isotope distribution characteristics, confirming that the fermentation product is ergot selenosider. Figure 3 ).

[0070] Table 1. Ergosene yield of candidate strains during shake-flask fermentation

[0071]

[0072] 6. Strain Identification: Genomic DNA was extracted from the pure culture (JZ-6), and the ITS sequence was amplified by PCR using universal fungal primers ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO:2) and ITS4: 5'-TCCTCCGCTTATTGATAT GC-3' (SEQ ID NO:3). The amplified product was sequenced to obtain the sequence shown in SEQ ID NO:1, and BLAST alignment analysis was performed in the NCBI database. The results showed that the sequence had 99.89% homology with the standard strain Schizosaccharomyces pombe (fission yeast). Based on the above molecular biological identification results, the ergot-producing strain JZ-6 of this invention was identified as Schizosaccharomyces pombe.

[0073] The sequence shown in SEQ ID NO:1 is as follows:

[0074]

[0075] Example 2: Ergot-producing fissile yeast mutant strain obtained through multiple rounds of ARTP mutagenesis screening.

[0076] The fission yeast S. pombe JZ-6, which was isolated and identified in Example 1, was used as the starting strain for ARTP mutagenesis breeding.

[0077] Sample preparation: The starting strain was inoculated into YPD liquid medium (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, pH 6.0) and cultured at 30°C with shaking at 200 rpm until the logarithmic growth phase (approximately 16-18 h, OD...). 600 ≈1.0). The bacterial culture was centrifuged at 4℃ and 6000 rpm for 10 min to collect the cells. The cells were washed twice with sterile physiological saline, and finally resuspended in sterile physiological saline to adjust the cell concentration to 102. 6 -10 7 CFU / mL.

[0078] 2.1 First round of mutagenesis (screening of selenium-tolerant mutants):

[0079] Take 10 μL of the prepared bacterial suspension and spread it evenly on a sterile slide, placing it on the operating table of the ARTP mutagenesis breeding instrument. The mutagenesis parameters were set as follows: power 120 W, gas flow rate 10 SLM, treatment distance 2 mm. Treatment time gradients were set as follows: 0 s (control), 20 s, 40 s, 60 s, 70 s, 75 s, 80 s, 85 s, 90 s, 100 s. After mutagenesis, the slide was quickly placed into a 2 mL centrifuge tube containing 1 mL of YPD liquid medium, and the cells were eluted. The eluent was appropriately diluted and spread onto YPD plates, incubated at 30℃ for 48 h, and the colony count was recorded and the lethality rate calculated.

[0080] The effects of different treatment times on the lethality of the strain are shown in Table 2. The lethality rates for treatments of 80 s, 85 s, and 90 s were 89.7%, 92.3%, and 94.1%, respectively, all within the screening range of 85%-95%. Considering that a lower lethality rate is beneficial for preserving more mutant diversity and providing richer genetic resources for subsequent rounds of mutagenesis, 80 s was selected as the treatment time for the first round of mutagenesis in this embodiment.

[0081] Table 2. Effect of ARTP treatment time on the lethality of fission yeast

[0082]

[0083] The mutagenic eluent with a lethality of 89.7% was spread onto YPD selection plates containing a high concentration of Na2SeO3 (40 mg / L) and incubated at 30°C for 3 days. Mutant colonies with larger growth were selected.

[0084] High-throughput primary screening: 270 candidate strains were inoculated into 96-well plates (each well containing 200 μL of fermentation medium, the same as the shake-flask secondary screening fermentation medium in Example 1, with Na2SeO3 at 40 mg / L). Simultaneously, three strains treated for 0s were selected from each 96-well plate as controls. The plates were cultured at 30°C and 200 rpm for 72 h with shaking. After culture, the 96-well plates were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected. 200 μL of methanol was added to each well, and the plates were ultrasonically extracted at room temperature for 30 min. After centrifugation again, the supernatant was used for RapidFire LC-MS detection (method as in Example 1, 4.2) to determine ergot selenium production. The primary screening results showed that the yield of the 270 mutant strains ranged from 0.5 to 3.2 mg / L. The top 25 strains with yields higher than the control strain (1.7 mg / L) (yield range 2.1-3.2 mg / L) were selected for shake-flask secondary screening.

[0085] Shake-flask secondary screening: 25 strains were subjected to shake-flask fermentation for secondary screening (using the same high-throughput primary screening fermentation medium as above), and cultured at 30℃ and 200 rpm for 96 h with shaking. Simultaneously, the starting strain JZ-6 was used as a control under the same conditions. The results showed that the yield of the starting strain JZ-6 under 40 mg / L selenium was 4.2 mg / L. The yield distribution of the 25 strains was as follows: 7 strains had yields higher than 10 mg / L, and 18 strains had yields between 5 and 10 mg / L. Among them, a mutant strain with the highest yield was obtained, with an ergot selenium yield of 10.79 mg / L, which was 2.6 times the yield of the starting strain under the same conditions, and was named *S. pombe* M1.

[0086] 2.2 Second round of mutagenesis (screening of mutants using precursor enhancement)

[0087] Using the high-yielding strain *S. pombe* M1 obtained in the first round of mutagenesis as the starting strain, a bacterial suspension was prepared according to the same method. ARTP treatment parameters were the same as in the first round of mutagenesis, and the lethality of strain M1 at different treatment times was determined according to the method used in the first round of mutagenesis. The results showed that the lethality at multiple time points within the treatment time range of 80-90 s was between 85% and 95%. Considering that the second round of mutagenesis aims to further break through metabolic bottlenecks and enhance precursor utilization, a treatment time slightly longer than the first round (80 s) is chosen to exert stronger selection pressure; therefore, 85 s was selected as the treatment time for the second round of mutagenesis. The mutagenesis eluent was spread on screening plates containing histidine and methionine (YPD plates containing 2 g / L histidine, 2 g / L methionine, and 40 mg / L Na2SeO3) and incubated at 30°C for 3 days. Fast-growing and larger mutant colonies were selected. Finally, a total of 260 mutant strains were selected and screened using the same high-throughput primary screening and shake-flask secondary screening as in the first round.

[0088] High-throughput primary screening: 260 candidate strains were inoculated into 96-well plates for primary screening (each well contained 200 μL of fermentation medium, the same as in the first round of high-throughput primary screening, with added histidine 2 g / L and methionine 2 g / L). The plates were cultured at 30℃ and 200 rpm for 72 h with shaking. Each 96-well plate also contained 3 starting strains of *S. pombe* M1. After fermentation, sample processing and detection methods were the same as in the first round of high-throughput primary screening. Based on the primary screening results, the yield of the 260 mutant strains ranged from 1.4 to 5.5 mg / L. The top 20 strains with yields higher than the starting strain *S. pombe* M1 (3.7 mg / L) (yield range 4.2–5.5 mg / L) were selected for shake-flask secondary screening.

[0089] Shake-flask re-screening: Twenty strains were subjected to shake-flask fermentation for re-screening (using the same high-throughput screening medium as described above), and cultured at 30℃ and 200 rpm for 96 h with shaking. Simultaneously, the starting strain *S. pombe* M1 was used as a control under the same conditions. Results showed that the yield of the starting strain *S. pombe* M1 under the condition of additional histidine and methionine supplementation was 11.9 mg / L. The yield distribution of the 20 strains was as follows: 4 strains had yields higher than 20 mg / L, and 16 strains had yields between 16 and 20 mg / L. Among them, a mutant strain with the highest yield was obtained, with an ergot selenide yield of 19.4 mg / L, which was 1.6 times the yield of the starting strain under the same conditions; this mutant was named *S. pombe* M2.

[0090] 2.3 Third round of mutagenesis (screening of high-osmotic tolerance mutants)

[0091] Using the high-yielding strain *S. pombe* M2 obtained in the second round of mutagenesis as the starting strain, a bacterial suspension was prepared according to the same method. The lethality of strain M2 at different treatment times was determined using the same method as in the first round of mutagenesis. Treatment times with a lethality between 85% and 95% were selected as the screening dose. The results showed that the lethality at multiple time points within the treatment time range of 80-95 s was consistently between 85% and 95%. Considering that the third round of mutagenesis aims to screen for mutant strains with enhanced tolerance under hyperosmolar conditions, requiring stronger selection pressure, a further increased treatment time of 90 s was selected as the screening dose for the third round of mutagenesis. The mutagenesis eluent was spread onto screening plates containing a higher concentration of glucose (same as the second round screening plates, but with the glucose concentration increased to 60 g / L) and incubated at 30°C for 3 days. Because the 60 g / L glucose concentration is much higher than the 20 g / L concentration of conventional YPD medium, it effectively applies osmotic selection pressure. At this concentration, the growth of the starting strain M2 was significantly inhibited, while the mutant strains with enhanced tolerance were able to form larger colonies. The faster-growing and larger single mutant bacteria were selected, and finally 88 mutant single colonies were selected.

[0092] High-throughput primary screening: 88 candidate strains were inoculated into 96-well plates for primary screening (each well contained 200 μL of fermentation medium, the same as in the second round of high-throughput primary screening, with the glucose concentration increased to 60 g / L), and cultured at 30℃ and 200 rpm for 72 h with shaking. Simultaneously, each 96-well plate contained 3 starting strains of *S. pombe* M2. After fermentation, sample processing and detection methods were the same as in the first round of high-throughput primary screening. Based on the primary screening results: the yield of the 88 mutant strains ranged from 1.1 to 3.9 mg / L. The top 20 strains with yields higher than the starting strain *S. pombe* M2 (2.4 mg / L) (yield range 2.9–4.3 mg / L) were selected for shake-flask secondary screening.

[0093] Shake-flask re-screening: Twenty strains were subjected to shake-flask fermentation for re-screening (using the same high-throughput primary screening medium as above), and cultured at 30℃ and 200 rpm for 96 h with shaking. Simultaneously, the starting strain *S. pombe* M2 was fermented as a control under the same conditions. Results showed that the yield of the starting strain *S. pombe* M2 under a high glucose concentration of 60 g / L was 10.4 mg / L (a decrease compared to the 20 g / L glucose condition, indicating that the high glucose environment posed a significant osmotic stress to the starting strain). The yield distribution of the 20 strains was as follows: 2 strains had yields higher than 19 mg / L, and 16 strains had yields between 10 and 20 mg / L. Among them, a mutant strain with the highest yield was obtained, with an ergot selenium yield of 20.2 mg / L, which was 1.9 times that of the starting strain under the same high glucose stress conditions. This demonstrated that the osmotic-tolerant mutant strain selected in the third round of mutagenesis screening has a significant growth and selenium production advantage under high glucose conditions. This high-yielding mutant strain was named *S. pombe* ARTP-3X.

[0094] Example 3 Adaptive domestication of ergot selenium-producing fission yeast mutants (evolution of product tolerance)

[0095] Using the high-yielding strain S. pombe ARTP-3X obtained in Example 2 as the starting strain, adaptive domestication was carried out by gradually increasing the concentration of ergot selenium in the culture environment, and evolved strains with enhanced product tolerance were screened.

[0096] 3.1 Domestication Methods

[0097] To further enhance the strain's tolerance to ergot selenoside while maintaining the original fermentation stress (selenium stress, precursor supply, hyperosmotic pressure), the basal culture medium used for acclimatization was the fermentation medium of the third round of mutagenesis screening in Example 2, and exogenous ergot selenoside was gradually added as selection stress on this basis.

[0098] The starting strain was inoculated into the above-mentioned fermentation medium containing an initial concentration of ergot selenoside and cultured at 30°C and 200 rpm for 48 h with shaking. Every 48 h, the strain was transferred to fresh medium containing a higher concentration of ergot selenoside at a 1% inoculation rate to allow the strain to gradually adapt to the high product concentration environment. The ergot selenoside concentration gradient was set at 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, with each concentration being passaged 5 times consecutively, for a total of 25 generations of adaptation and acclimatization.

[0099] During the domestication process, samples were taken every 5 generations to measure the cell density (OD). 600Simultaneously, shake-flask fermentation was performed to verify the results: the strain was cultured at 30℃ and 200 rpm for 96 h in a medium containing the corresponding concentration of exogenous ergot selenoside, and the yield of ergot selenoside was measured. The results are shown in Table 3. The results showed that with the increase of domestication generation and the increase of exogenous ergot selenoside concentration, the biosynthetic capacity of the strain under product stress gradually improved. By the 25th generation, the concentration of ergot selenoside had reached 500 mg / L, and the bacterial OD... 600 The growth was significantly inhibited when the concentration decreased from 8.56 to 6.15; at the same time, the yield increase tended to plateau, and further increasing the product concentration would easily cause excessive inhibition of cell growth and metabolism, which would be detrimental to the stability of the strain. Therefore, 25 generations was determined to be the appropriate endpoint for domestication, and further subculturing was stopped.

[0100] Table 3. Changes in strain performance during adaptive acclimatization.

[0101]

[0102] 3.2 Verification of domestication results

[0103] After domestication, the 25th generation culture was diluted and spread onto a medium plate containing 500 mg / L ergot selenoside (same as the screening plate in 2.3 of Example 2), and incubated at 30°C for 3 days. Single colonies were then picked. This strain was then subjected to shake-flask fermentation (30°C, 200 rpm shaking culture for 96 h) in the same fermentation medium with and without exogenous ergot selenoside, and the ergot selenoside yield was measured. The results showed that the yield was 27.8 mg / L with exogenous ergot selenoside added and 31.2 mg / L without, representing a 54.5% increase compared to the starting strain ARTP-3X (20.2 mg / L). This strain was named *Schizosaccharomyces pombe* ZZ.

[0104] 3.3 Genetic stability

[0105] To investigate the genetic stability of the domesticated strain *S. pombe* ZZ, the strain was passaged 10 times on basal medium slant. Samples were taken every two generations for shake-flask fermentation (using the same fermentation medium, but without exogenous ergot selenoside, cultured at 30℃ and 200 rpm for 96 h with shaking). Ergot selenoside yield was then measured. The results are shown in Table 4. The yield ranged from 28.9 to 31.2 mg / L from generation 0 to 10, and the yield at generation 10 was 28.9 mg / L, with a relative yield of 92.6%. The yield remained above 92% after 10 passages, indicating that the strain possesses good genetic stability.

[0106] Table 4. Genetic stability of domesticated strain ZZ

[0107]

[0108] 3.4 Preservation of bacterial strains

[0109] The fission yeast (Schizosaccharomyces pombe) ZZ is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026805.

[0110] Example 4: Optimization of culture medium for high-yield strains

[0111] The domesticated strain S. pombe ZZ obtained in Example 3 was used as the fermentation strain, and the culture medium was optimized and the culture was scaled up in a 5L fermenter.

[0112] 4.1 Seed liquid preparation

[0113] After activating the preserved strain by inoculating it onto YPD slant, a single colony was picked and inoculated into seed culture medium (20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract), and cultured at 30℃ and 200 rpm for 24 h to obtain the seed culture.

[0114] 4.2 Optimization of Shake Flask Culture Media

[0115] The current fermentation medium consists of: glucose 60 g / L, yeast extract 5 g / L, peptone 10 g / L, histidine 2 g / L, methionine 2 g / L, KH₂PO₄ 2 g / L, MgSO₄·7H₂O 0.5 g / L, CaCl₂·2H₂O 0.1 g / L, KCl 1 g / L, Na₂SeO₃ 40 mg / L, and a trace element mixture 1 mL / L, pH 5.5. The trace element mixture contains: FeSO₄·7H₂O 10 mg / L, MnSO₄·H₂O 5 mg / L, ZnSO₄·7H₂O 5 mg / L, CuSO₄·5H₂O 0.5 mg / L, Na₂MoO₄·2H₂O 0.5 mg / L, and CoCl₂·6H₂O 0.5 mg / L. This medium will be used as the basal medium for further optimization.

[0116] (1) Single-factor experiment

[0117] The effects of different carbon sources, nitrogen sources and precursors on ergot selenogen production were investigated.

[0118] Carbon source experiment: Different carbon sources (glucose, sucrose, and glycerol) were added at 60 g / L to replace glucose in the basal medium. The results showed that glucose was the optimal carbon source, with a yield of 31.2 mg / L, which was significantly higher than that of sucrose (21.5 mg / L) and glycerol (23.6 mg / L).

[0119] Nitrogen source experiment: Different nitrogen sources (peptone, beef extract, and ammonium sulfate) were added at 10 g / L to replace peptone in the basal medium. The results showed that ammonium sulfate was the most suitable nitrogen source with a yield of 34.5 mg / L; peptone (31.2 mg / L) and beef extract (28.6 mg / L) were the next most effective.

[0120] Precursor assay: Based on the basal fermentation medium (containing 2 g / L each of histidine and methionine), 1 g / L of cysteine ​​was added as an additional precursor. The results showed that the yield after adding cysteine ​​was 30.9 mg / L, which was not significantly higher than the control (31.2 mg / L). This indicates that cysteine ​​is not a limiting factor under the existing medium conditions; therefore, further optimization will continue to focus on histidine and methionine as the main precursors.

[0121] (2) Results of multi-factor stepwise optimization experiment

[0122] Single-factor experiments showed that glucose was the optimal carbon source and ammonium sulfate was the optimal nitrogen source for ergot selenoside synthesis. However, these experiments only examined the effect of individual factors on product synthesis without considering the interactions between carbon sources, nitrogen sources, precursor amino acids, and selenium sources, making it difficult to obtain the optimal ratio under the synergistic effect of all factors. Therefore, a multi-factor stepwise optimization experiment was conducted, simultaneously controlling the concentration levels of glucose, ammonium sulfate, histidine, methionine, and Na₂SeO₃ to explore the optimal range for multi-factor combinations. The results are shown in Table 5. As the levels of each factor increased synchronously, the yield of ergot selenoside showed a trend of first increasing and then decreasing. Among them, the yield of group 3 (glucose 40 g / L, ammonium sulfate 15 g / L, histidine 1 g / L, methionine 1 g / L, Na₂SeO₃ 30 mg / L) reached the maximum value of 45.8 mg / L, which was 46.8% higher than the yield of the basal medium (31.2 mg / L). As the levels of various factors continue to increase, yield begins to decline. This is because excessively high concentrations of nutrients and precursors can cause osmotic inhibition or selenium source toxicity inhibition, which is detrimental to ergot selenosyn synthesis, rather than due to insufficient substrate supply.

[0123] Table 5. Experimental Design and Results of Multi-Factor Stepwise Incremental Optimization

[0124]

[0125] (3) Optimization results

[0126] The results of the multi-factor stepwise optimization experiment showed that after culturing for 96 h at 30℃ and 200 rpm in a shaker, the ergoselenoside yield of the third formulation reached 45.8 mg / L, the highest value among all experimental groups. Therefore, the optimized culture medium formulation was determined to be: glucose 40 g / L, yeast extract 5 g / L, ammonium sulfate 15 g / L, histidine 1 g / L, methionine 1 g / L, Na2SeO3 30 mg / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, CaCl2·2H2O 0.1 g / L, KCl 1 g / L, and trace element mixture 1 mL / L (specific components as above), pH 5.5.

[0127] Example 5: Feed-in fermentation of high-yield strains in a 5L fermenter

[0128] (1) Fermentation conditions

[0129] Based on the optimization results of the shake flask culture medium, the basic culture medium formula for the fermenter was determined to be: the optimized culture medium formula determined above.

[0130] Considering the long fermentation cycle and expected high cell density, an additional 1 mL / L of vitamin mixture was added to the basal culture medium to meet the vitamin nutrition requirements for high-density growth. For ease of preparation, storage, and aseptic operation, all added solutions were first prepared as high-concentration stock solutions, filtered through a 0.22 μm sterile membrane, and stored at 4°C for later use. After autoclaving the fermentation medium at 121°C for 20 min and cooling, the calculated amounts of the sterile stock solutions were added to the medium in a laminar flow hood and diluted to the final working concentration. The final working concentrations of each component are as follows: Vitamin mixture: Biotin 0.05 mg / L, Calcium pantothenate 1.0 mg / L, Nicotinic acid 1.0 mg / L, Inositol 25 mg / L, Thiamine 1.0 mg / L, Pyridoxine 1.0 mg / L.

[0131] Seed culture preparation: After activating the preserved strain S. pombe ZZ by inoculating it onto YPD slant, a single colony was picked and inoculated into seed culture medium (20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract). The culture was carried out at 30℃ and 200 rpm for 24 h with shaking to obtain the seed culture.

[0132] Inoculation and fermentation conditions: The seed culture was inoculated at a rate of 5% (v / v) into a 5 L fermenter containing 2 L of the optimized vitamin-added medium. The temperature was 30℃, the stirring speed was 300-700 rpm (with dissolved oxygen controlled to maintain dissolved oxygen at 30% or higher), the aeration rate was 1 vvm, and the pH was kept constant at 5.5±0.5 by adding ammonia or dilute hydrochloric acid.

[0133] (2) Feeding strategy

[0134] Carbon source feeding: Feeding is based on residual sugar concentration feedback. When the residual sugar concentration is below 5 g / L, fed medium (500 g / L glucose) is started. By adjusting the feeding rate, the glucose concentration in the fermentation broth is maintained at 1-5 g / L to avoid carbon source limitation.

[0135] Precursor feeding: The initial culture medium contained 1 g / L each of histidine and methionine, which was sufficient for the initial synthesis. After 48 h, the product entered a rapid synthesis phase, and the precursor consumption rate increased significantly. To avoid a decrease in precursor concentration that would limit synthesis, a mixed solution of histidine and methionine (20 g / L histidine and 20 g / L methionine) was fed into the fermentation broth starting at 48 h. The precursor concentration in the fermentation broth was maintained at 0.5-1.5 g / L through feedback control, and the feeding continued until the end of fermentation (144 h) to ensure a continuous and sufficient supply of precursors.

[0136] Na₂SeO₃ Control: Based on the results of multi-factor optimization in shake flasks, the optimal Na₂SeO₃ concentration for the fermentation system was determined to be 30 mg / L. Considering selenium oxidation loss, non-specific cell binding, and the need for synthetic redundancy, a "sufficient initial addition + subsequent continuous addition" strategy was adopted: 30 mg / L of Na₂SeO₃ was initially added to ensure that cell growth was not significantly inhibited while fully meeting the selenium source requirements for product synthesis in the early stages of fermentation. After 48 h of fermentation, the rapid product synthesis phase began, and selenium source consumption increased significantly. At this point, a continuous addition of Na₂SeO₃ solution (20 g / L) was initiated. Through online monitoring and control, the Na₂SeO₃ concentration in the fermentation broth was dynamically maintained within the range of 20-30 mg / L, ensuring a continuous selenium source supply while avoiding excessive concentrations that could cause toxicity. This resulted in efficient and stable synthesis over a fermentation period of 144 h.

[0137] (3) Sampling and testing

[0138] Sampling and testing: Sampling was conducted according to the time points shown in Table 6 to determine cell concentration (dry weight method), ergot selenoside yield (HPLC method), and residual sugar concentration (DNS method). Intensive sampling and testing were performed before and after key feeding points. The ergot selenoside extraction and determination methods were the same as in Example 1.

[0139] (4) Fermentation results

[0140] The parameter changes during the batch-feed fermentation process in the 5L fermenter are shown in Table 6. After 144 h of fermentation, the biomass reached 34.5 g / L, and the ergot selenium yield reached 431.7 mg / L.

[0141] Table 6. Parameter changes during batch-feed fermentation in a 5L fermenter

[0142]

[0143] Fermentation process analysis: The rapid synthesis period of ergot selenosiders occurred from 72 to 86 hours, with a yield reaching 8.57 mg / (L·h), the highest value of the entire fermentation process. This indicates that the supply of carbon, precursor, and selenium sources was sufficient during this stage, and the cell metabolic activity was at its strongest. After 86 hours, residual sugar dropped to 1.1 g / L, triggering fed-batch feeding. By 96 hours, residual sugar had rebounded to 3.8 g / L, and remained within the 1-5 g / L range throughout the subsequent process, providing a stable carbon source supply for the continuous and efficient synthesis of the product. After 132 hours, as fermentation entered the later stages, cell growth tended to stagnate, and the ergot selenosider synthesis rate decreased significantly (the yield dropped to 0.56 mg / (L·h) from 132 to 144 hours). This may be related to the consumption of nutrients, the accumulation of metabolic byproducts, and the decrease in cell activity during the later stages of fermentation.

[0144] After optimization of the shake-flask culture medium, the yield of ergot selenosider reached 45.8 mg / L (96 h). Based on this, a dissolved oxygen-coupled residual sugar feedback feeding strategy and a dynamic feed-on-feed strategy for precursors and selenium sources were adopted in a 5 L fermenter, and the fermentation period was extended to 144 h, ultimately increasing the yield to 431.7 mg / L, which is 9.4 times the yield after shake-flask optimization. These results indicate that, based on the shake-flask optimized culture medium, combined with a feed-on-feed strategy in the fermenter, the synthesis efficiency of ergot selenosider can be significantly improved. This invention significantly improves the fermentation yield of ergot selenosider, providing high-quality strain resources and an efficient fermentation process for the industrial production of ergot selenosider.

[0145] The above specific embodiments are merely illustrative of the invention and do not represent a limitation thereof. Those skilled in the art will recognize that other variations of the specific structure of this invention are possible.

Claims

1. A fissile yeast producing ergot selenium, characterized in that, The fissorimycosis yeast is *Schizosaccharomyces pombe* ZZ, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026805.

2. The use of the fission yeast (Schizosaccharomyces pombe) ZZ as described in claim 1 in the production of ergot selenoside or products containing ergot selenoside.

3. A method for producing ergot selenium by shake-flask fermentation, characterized in that, The fission yeast (Schizosaccharomyces pombe) ZZ described in claim 1 was inoculated into a fermentation medium and cultured to obtain ergot selenium from the culture.

4. The method according to claim 3, characterized in that, The fermentation medium comprises the following components: carbon source 20-70 g / L, yeast extract 5-10 g / L, nitrogen source 5-30 g / L, histidine 0.25-2.5 g / L, methionine 0.25-2.5 g / L, cysteine ​​0-1 g / L, Na₂SeO₃ 10-60 mg / L, KH₂PO₄ 1-3 g / L, MgSO₄·7H₂O 0.5-0.8 g / L, CaCl₂·2H₂O 0.1-0.2 g / L, KCl 1-2 g / L, and a trace element mixture 1-1.5 mL / L, pH 5-6; preferably, the trace element mixture comprises the following components: FeSO₄·7H₂O 10 mg / L, MnSO₄·H₂O 5 mg / L, ZnSO₄·7H₂O 5 mg / L, CuSO₄·5H₂O 0.5 mL / L. mg / L, Na2MoO4·2H2O 0.5 mg / L, CoCl2·6H2O 0.5 mg / L; The carbon source is selected from one or a combination of two of glucose, sucrose, and glycerol, and the nitrogen source is selected from one or a combination of two of peptone, beef extract, and ammonium sulfate.

5. The method according to claim 4, characterized in that, The fermentation medium consists of the following components: glucose 20-60 g / L, yeast extract 4-6 g / L, peptone or ammonium sulfate 10-15 g / L, histidine 1-2 g / L, methionine 1-2 g / L, cysteine ​​0-1 g / L, Na2SeO3 20-40 mg / L, KH2PO4 1.5-2.5 g / L, MgSO4·7H2O 0.4-0.6 g / L, CaCl2·2H2O 0.05-0.15 g / L, KCl 0.8-1.2 g / L, and a trace element mixture of 0.8-1.2 mL / L, pH 5-6.

6. The method according to claim 5, characterized in that, The fermentation medium consists of the following components: glucose 40-60 g / L, yeast extract 4.5-5.5 g / L, peptone or ammonium sulfate 10-15 g / L, histidine 1-2 g / L, methionine 1-2 g / L, Na2SeO3 30-40 mg / L, KH2PO4 1.5-2.5 g / L, MgSO4·7H2O 0.4-0.6 g / L, CaCl2·2H2O 0.05-0.15 g / L, KCl 0.8-1.2 g / L, and a trace element mixture 0.8-1.2 mL / L, pH 5-6.

7. The method according to claim 5, characterized in that, The fermentation medium consists of the following components: glucose 60 g / L, yeast extract 5 g / L, peptone 10 g / L, histidine 2 g / L, methionine 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, CaCl2·2H2O 0.1 g / L, KCl 1 g / L, Na2SeO3 40 mg / L, and a trace element mixture 1 mL / L, pH 5.5; or The fermentation medium consists of the following components: glucose 60 g / L, yeast extract 5 g / L, ammonium sulfate 10 g / L, histidine 2 g / L, methionine 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, CaCl2·2H2O 0.1 g / L, KCl 1 g / L, Na2SeO3 40 mg / L, and a trace element mixture 1 mL / L, pH 5.5; or The fermentation medium consists of the following components: 40 g / L glucose, 5 g / L yeast extract, 15 g / L ammonium sulfate, 1 g / L histidine, 1 g / L methionine, 30 mg / L Na2SeO3, 2 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.1 g / L CaCl2·2H2O, 1 g / L KCl, and 1 mL / L of trace element mixture, pH 5.

5.

8. A method for producing ergot selenium in a fermenter using a batch feeding method, characterized in that, The fissor yeast (Schizosaccharomyces pombe) ZZ described in claim 1 was inoculated into a fermenter for fermentation, and fed according to the strategies shown in 1)-3) below to obtain ergot selenium from the fermentation product: 1) When the residual sugar concentration is below 5 g / L, add glucose solution to control the glucose concentration in the fermentation broth in the range of 1-5 g / L, wherein the glucose concentration in the glucose solution is 450-550 g / L; 2) Start adding a mixed solution of histidine and methionine at 45-50 h of fermentation, and control the concentration of histidine and methionine in the fermentation broth in the range of 0.5-1.5 g / L. Continue adding the solution until the end of fermentation. The concentration of histidine and methionine in the mixed solution is 15-20 g / L. 3) Start adding Na2SeO3 solution after 45-50 hours of fermentation, and control the Na2SeO3 concentration in the fermentation broth to be in the range of 20-30 mg / L. The Na2SeO3 concentration in the Na2SeO3 solution is 20-25 g / L.

9. The method according to claim 8, characterized in that, The culture medium in the fermenter consists of the following components: glucose 40-60 g / L, yeast extract 4-6 g / L, peptone or ammonium sulfate 10-15 g / L, histidine 1-2 g / L, methionine 1-2 g / L, Na2SeO3 30-40 mg / L, KH2PO4 1.5-2.5 g / L, MgSO4·7H2O 0.4-0.6 g / L, CaCl2·2H2O 0.05-0.15 g / L, KCl 0.8-1.2 g / L, trace element mixture 1 mL / L, vitamin mixture 1 mL / L, pH 5-6; More preferably, the vitamin mixture is composed of the following components: biotin 0.05 mg / L, calcium pantothenate 1.0 mg / L, niacin 1.0 mg / L, inositol 25 mg / L, thiamine 1.0 mg / L, and pyridoxine 1.0 mg / L.

10. The method according to claim 8, characterized in that, Fermentation conditions are: temperature 30-32℃, stirring speed 300-700 rpm, dissolved oxygen linked to maintain dissolved oxygen above 30%, aeration rate of 1-1.2 vvm, pH controlled at 5.5±0.5 by adding ammonia or dilute hydrochloric acid; fermentation cycle is 120-144 h.

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