A method for increasing the yield of emestrin, a polythiodiketopiperazine compound.

Abstract

This invention discloses a method for increasing the yield of emestrin, a polythiodiketopiperazine compound, comprising: adding glutathione to a fermentation medium, sterilizing the fermentation medium, inoculating with a seed culture of microorganisms, and fermenting to obtain emestrin; wherein 0.001–99.999 g of glutathione is added per 100 g of fermentation medium; and the mass-to-volume ratio of the fermentation medium to the seed culture is 1:0.001–1:99.999 g / mL. This method significantly increases emestrin yield, saves fermentation time, improves the efficiency of emestrin production, and reduces production costs, making it suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to a method for increasing the yield of emestrin, a polythiodikepiperazine compound, a secondary metabolite of microorganisms. Background Technology

[0002] Epipolythiodioxopiperazines (ETPs) are mainly derived from fungal secondary metabolites. Their structural feature is a cyclic dipeptide structure formed by the condensation of two amino acids through peptide bonds. The stable six-membered ring skeleton structure makes diketopiperazines an important pharmacophore in medicinal chemistry, exhibiting a variety of biological and pharmacological activities, which has attracted great attention. Currently, research has found that polythiodiketopiperazines possess a variety of biological activities, such as antitumor, immunosuppression, and antiviral activity. Li Liyuan et al. classified this class of compounds into 15 structural types based on the different types and modifications of the amino acids that make up their parent nucleus (diketopiperazines containing sulfur bridges) and the different connection modes of each segment. These include: gliotoxins, sirodesmins, sporidesmins, chetomins, chaetocins, verticillins, leptosins, emestrins, scabrosins, epicorazines, aronatins, emethallicins, gliocladine C, hyalodendrin, and dithiosilvatin.

[0003] Emestrins are polythiodiketopiperazine compounds with unique structural fragments, primarily derived from fungal secondary metabolism. These compounds contain a distinctive 4,5-dihydro-oxepin fragment, and most also exhibit a 15-membered lactone ring. Emestrins possess antibacterial, antitumor, and CCR2 antagonistic activities.

[0004] In 1985, Hideyuki Seya et al. first isolated emestrin, a polythionid dionepiperazine compound, from *Emerricella striata*, and determined its precise structure using single-crystal X-ray diffraction in 1986. This compound exhibits good antifungal activity, with MICs of 10 and 2.5 μg·mL against *Fusarium graminearum* and *Penicillium graminearum*, respectively. -1 The inhibitory activity against Escherichia coli and Bacillus subtilis was 10 μg·disc -1 2.5μg·disc -1Furthermore, it exhibits cytotoxicity against multiple tumor cell lines, including T47D, HepG2, and HeLa. Based on cytotoxicity assays, cell cycle analysis, and apoptosis analysis, emestrin can be inferred to have potential antitumor activity. In addition, some researchers have found that this compound can act on target organs such as the heart, liver, and thymus in mice, leading to acute poisoning. The LD50 at 24h and 48h after intraperitoneal injection was [not specified]. 50 The concentrations were 17.7 and 13.0 mg·kg, respectively. -1 In 1987, Nozawa et al. obtained emestrin B from *Emericella striata*, a compound with antibacterial activity. In 1997, Ooike M et al. isolated emestrin, emestrin B, and secoemestrin C from *Emericella foveolata*. In 2004, Tsumagari et al. isolated the antitumor compound MPC1001 from the fungus *Cladorrhinum sp.*, and Onodera H et al. isolated its homologues MPC1001s B, C, D, and E. Their IC50 values ​​for human prostate cancer DU145 cell lines... 50 The values ​​were 9.3, 39, 12, 16, and 83 nmol / L, respectively. In 2005, Herath et al. discovered four novel ETP-like chemokine receptor (CCR2) antagonists, emestrin C, emestrin D, emestrin E, and secoemestrin C1, from a fungus collected from Alaska. Most emestrin compounds exhibit strong antitumor activity and hold promise for development into new antitumor drugs.

[0005] Methods to improve microbial secondary metabolism typically include: 1) altering culture conditions, such as changing the nutrient conditions of the culture medium; changing the culture time; and altering oxygen, light, and water levels; 2) physical mutagenesis, such as ultraviolet mutagenesis and plasma mutagenesis; and chemical mutagenesis, such as adding chemical mutagens like diethyl sulfate (DES) and guanidine nitrite; 3) synthetic biology methods, which involve analyzing biosynthetic pathways and precursors to regulate microbial biosynthesis by adding relevant substances as needed, thereby increasing yield; and 4) overexpressing relevant genes through genetic engineering. However, there are currently no reports on improving emestrin yield. Summary of the Invention

[0006] The purpose of this invention is to provide a method for increasing the yield of emestrin, a microbial secondary metabolite, by adding a biosynthetic precursor to the culture medium.

[0007] The structure of Emestrin is shown in Figure I:

[0008]

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A method for increasing the yield of the polythiodiketopiperazine compound emestrin includes: adding glutathione to a fermentation medium, sterilizing the fermentation medium, inoculating a seed culture of microorganisms, fermenting, and obtaining the compound emestrin.

[0011] The fermentation medium includes, but is not limited to, natural media such as rice medium, wheat bran medium, flour medium, and corn flour medium, as well as artificial synthetic media such as PDA medium and PDB medium; rice medium is preferred.

[0012] The rice culture medium is prepared by mixing rice and distilled water at a mass ratio of 1:1 and sterilizing at 121°C for 15 minutes.

[0013] The carbon source of the fermentation medium is selected from one or a combination of glucose, sucrose, lactose, maltose, maltodextrin, starch, sorbitol, and glycerol. The nitrogen source of the fermentation medium is selected from one or a combination of soybean meal, yeast powder, rice protein powder, corn protein powder, cottonseed meal, peanut meal, soybean flour, corn steep liquor powder, yeast extract, and peptone.

[0014] Add 0.001 to 99.999 g of glutathione per 100 g of fermentation medium; preferably, add 0.25 to 20 g of glutathione per 100 g of fermentation medium.

[0015] Specifically, when the culture medium is selected from rice culture medium, 0.25 to 20 g of glutathione is added to every 100 g of rice culture medium.

[0016] The microorganisms mentioned are any microorganisms that can produce emestrin, including but not limited to fungi of the genus Emericella.

[0017] Specifically, the microorganism is the fungus Emericella sp. 1454, classified as a member of the genus Emericella sp., and was deposited on May 30, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 17765. Emericella sp. 1454 is not a new biological material; for details, please refer to the applicant's invention patent application filed on November 23, 2019 (publication number CN 110818728A).

[0018] The method for preparing the seed culture of fungus Emericella sp. 1454 is as follows: Take the activated fungal strain and inoculate it on a PDA slant, and incubate it in a constant temperature incubator at 25℃ for one week; cut off a piece of the seed slant, crush it, and inoculate it into a container of 100mL PDB medium, and incubate it with shaking at 25℃ for 5 days to obtain the seed culture.

[0019] The mass-to-volume ratio of the fermentation medium to the seed liquid is 1:0.001 to 1:99.999 g / mL, preferably 100:5 g / mL.

[0020] The fermentation time is 1 to 180 days, preferably 6 to 30 days, and more preferably 6 to 10 days.

[0021] The fermentation temperature is 18–45°C, preferably 25°C.

[0022] The beneficial effects of this invention are:

[0023] The method of this invention significantly increases emestrin yield, saves fermentation time, improves the efficiency of emestrin production, reduces production costs, and is suitable for large-scale industrial production. Attached Figure Description

[0024] Figure 1 The bar chart shows the effect of glutathione on emestrin production.

[0025] Figure 2 The figures show a comparison of HPLC analysis of rice fermentation by strain Emericella sp. 1454 and rice fermentation with glutathione; where A represents rice fermentation by Emericella sp. 1454 for 6 days; B represents rice fermentation by Emericella sp. 1454 with glutathione for 6 days; C represents rice fermentation by Emericella sp. 1454 for 10 days; and D represents rice fermentation by Emericella sp. 1454 with glutathione for 10 days. Detailed Implementation

[0026] The embodiments of the present invention are applicable to increasing the yield of emestrin produced by fungal fermentation from any microorganism, and are not limited to it. The following examples are provided to help those skilled in the art better understand the technical solutions of the present invention, but are not intended to limit the invention in any way.

[0027] Example 1

[0028] Activated fungal strain Emericella sp. 1454 (CGMCC No. 17765) was inoculated onto a PDA slant and cultured at 25°C for one week. The seed slant was then cut into pieces, crushed, and inoculated into three 500mL Erlenmeyer flasks containing 100mL PDB medium. The flasks were then shaken and cultured at 25°C for 5 days to obtain the seed culture.

[0029] Treatment group: 50g of rice, 0.5g of reduced glutathione and 50mL of distilled water were added to each plastic bag. After sealing, the bags were sterilized at 121℃ for 15min. After cooling, 5mL of the above seed culture was added to each bag, and the bags were cultured at 25℃ for 6 days (two bags) and 10 days (two bags). After fermentation, the fermentation broth was extracted twice with ethyl acetate, using 300mL of ethyl acetate each time. The extracts were combined, concentrated under reduced pressure to remove ethyl acetate, dissolved in 1mL of methanol, filtered through a 0.22μm filter membrane, and analyzed by HPLC.

[0030] Blank control group: No reduced glutathione was added, and all other aspects were the same as the treatment group; after fermentation for 6 days (two bags) and 10 days (two bags) respectively, the fermentation broth was treated as in the treatment group and analyzed by HPLC.

[0031] HPLC conditions: Column: COSMOSIL C18-PAQ analytical column (5μm, 4.6×150mm), mobile phase: acetonitrile: 0.1% trifluoroacetic acid water = 5:95~100:0V / V, linear elution; detection wavelength 220nm.

[0032] See results Figure 1 and Figure 2 By comparing the peak areas, it can be seen that, under the same culture time, the addition of the precursor compound glutathione during fermentation can significantly increase the yield of emestrin compared with the blank control group.

[0033] Further purification yielded the compound emestrin. Preparation and purification conditions: The rice culture medium obtained after Emericella sp. 1454 fermentation was extracted twice with 95% ethanol and once with 50% ethanol using ultrasound, each extraction lasting 30 min. The extracts were combined, filtered, and concentrated under reduced pressure to obtain a crude extract aqueous solution. This solution was extracted three times with an equal volume of ethyl acetate, and the ethyl acetate layer was concentrated under reduced pressure to obtain a crude extract ER. The crude extract ER was dissolved in appropriate amounts of methanol and dichloromethane, mixed with silica gel (100-200 mesh), and the sample solution was added to the silica gel in small amounts several times, ground evenly, and allowed to stand until the solvent evaporated. Select an appropriate chromatography column, pack it dry with silica gel (300-400 mesh), and load the sample dry. Use a dichloromethane-methanol system with gradient elution at volume ratios of 50:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 1:1, and 1:0, eluting three column volumes per gradient, with each 300 mL fraction representing one fraction. Analyze each fraction by TLC, combine similar fractions, and evaporate to dryness under reduced pressure to obtain 16 fractions Fr.1-Fr.16. Dissolve Fr.9 (1.5 g) in methanol, and perform isocratic elution on a Sephadex LH-20 gel column with methanol as the mobile phase, eluting in 20 mL fractions. Based on the TLC development results, combine similar fractions to obtain 5 subfractions (Fr.9.1-Fr.9.5). Fr.9.3 Semi-preparative RP-HPLC (Capcell Pak MGⅡC18; 5 μm; 4.6 × 250 mm; 43% ACN-H2O containing 0.1% TFA, isocratic elution; 1.5 mL·min) was used. -1 The compound emestrin was prepared.

[0034] Both the treatment group and the blank control group were fermented using a total of 100g of rice (prepared into a 200g rice culture medium with distilled water). After 6 days of culture, the blank control group yielded 4.96mg of emestrin, while the treatment group (with added glutathione) yielded 48.89mg after 6 days. After 10 days of culture, the blank control group yielded 10.06mg of emestrin, while the treatment group (with added glutathione) yielded 63.88mg after 10 days. That is, adding glutathione increased the emestrin yield by 9.85 times after 6 days of fermentation, and by 6.35 times after 10 days of fermentation.

Claims

1. A method of increasing the production of a polysulfide dione piperazine compound emestrin, characterized by: The application relates to a preparation method of emestrin. The emestrin is obtained by adding glutathione into a fermentation medium, sterilizing the fermentation medium, inoculating a seed liquid of microorganisms, and fermenting; wherein 0.001-99.999 g of glutathione is added into 100 g of the fermentation medium; the mass-volume ratio of the fermentation medium and the seed liquid is 1:0.001-1:99.999 (g / mL); the microorganisms are fungi Emericella sp. 1454, and the preservation number is CGMCC No. 17765; The structure of the emestrin is shown in I. ； Ⅰ。 2. The method of increasing the production of emestrin, a polysulfidodiketopiperazine compound, according to claim 1, characterized by: The fermentation medium is rice medium, wheat bran medium, flour medium, corn flour medium, PDA medium or PDB medium.

3. The method of increasing the production of emestrin, a polysulfidodiketopiperazine compound, according to claim 2, characterized by: The fermentation medium is rice medium.

4. The method of increasing the production of emestrin, a polysulfidodiketopiperazine compound, according to claim 2 or 3, characterized by: The rice medium is prepared by mixing rice and distilled water at a mass ratio of 1:1, sterilizing at 121 DEG C for 15 min.

5. The method of increasing the production of emestrin, a polysulfidodiketopiperazine compound, according to claim 1, characterized by: 0.5-20 g of glutathione is added into 100 g of the fermentation medium.

6. The method of increasing emestrin production of a polysulfurated dione piperazine compound according to claim 1, characterized in that: The seed liquid of the fungi Emericella sp. 1454 is prepared by inoculating activated bacteria on a PDA slant, culturing in a thermostat at 25 DEG C for one week, crushing the seed slant, inoculating into 100 mL of PDB medium, and culturing at 25 DEG C for 5 days.

7. The method of increasing emestrin production of a polysulfurated dione piperazine compound according to claim 1, characterized in that: The mass-volume ratio of the fermentation medium and the seed liquid is 100:5 (g / mL).

8. The method of increasing emestrin production of a polysulfurated dione piperazine compound according to claim 1, characterized by: The fermentation time is 1-180 days.

9. The method of increasing the production of emestrin, a polysulfidodiketopiperazine compound, according to claim 8, characterized by: The fermentation time is 6-30 days.

10. The method of increasing the production of emestrin, a polysulfidodiketopiperazine compound, according to claim 9, characterized by: The fermentation time is 6-10 days.

11. The method of increasing emestrin production of a polysulfurated dione piperazine compound according to claim 1, characterized in that: The fermentation temperature is 18-45 DEG C.

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