Curvularia sturtii producing brefeldin A and fermentation method thereof

Abstract

The present invention provides a strain of Curvularia pseudorobusta HDCC00041, which was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on August 22, 2022, with a deposit number of CGMCC No. 40275. The strain belongs to Curvularia pseudorobusta, and can be fermented to produce Brefeldin A (BFA for short); the strain has few by-products in the fermentation broth, and the BFA purity is high, which is more conducive to subsequent purification; the strain can tolerate higher temperature fermentation, such as 33°C, thereby reducing the energy consumption of chilled water; the strain has a short fermentation cycle, and can improve the production efficiency of BFA compared to the prior art; due to the use of the strain for fermentation, the advantages in energy consumption, subsequent purification process, short fermentation cycle, etc., make the cost of industrial production lower, more conducive to industrial scale-up production.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Curvularia pseudorobustica producing brefeldin A and a fermentation method thereof. Background Art

[0002] Brefeldin A (BFA) was first discovered in 1958 from the fermentation broth of Penicillium decumbens. It can induce the decomposition of the Golgi apparatus and inhibit the transfer of proteins from the endoplasmic reticulum to the Golgi apparatus. It is a macrolide antibiotic with biological activities such as antifungal, antiviral, antimitotic, and antitumor. The American Cancer Society found that BFA can induce tumor cell differentiation and has great application prospects as a reagent for hemotherapy for tumor treatment. At present, BFA has been widely used in the pharmaceutical field as an important raw material. Since the synthesis method for preparing BFA is cumbersome, low in yield, high in cost, and not environmentally friendly, the synthesis of BFA by microorganisms is low in cost, so it is the main synthetic preparation method.

[0003] Currently, there are many strains of BFA that can be synthesized. "Development of macrolide lactoneantibiotic brefeldin A fermentation process with Eupenicillium brefeldianum ZJB082702" disclosed that it was obtained by fermentation of Eupenicillium brefeldianum, with a titer of 1304.7 mg / L. US3896002A disclosed that BFA was obtained by fermentation of fungi of the genus Phyllosticta, with a yield of about 3.0 grams per 10 liters of culture medium. In addition, the production of BFA by strains of the genus Curvularia is also a research direction. "Constituents of some Curvulariaspecies" first disclosed that BFA was obtained in the fermentation broth of strains of the genus Curvularia. However, the document found that only C.lunata strains and C.subulata could be fermented, among which C.lunata had the highest titer of 250 mg / L. Later, "Antiviral activity metabolites isolated from Curvularia pallescens" and "Brefeldin-A: an Antiproliferative Metabolite of the Fungus Curvularia trifolii Collected from the Veracruz Coral Reef System, Mexico" also disclosed that BFA was found in the fermentation broth of Curvularia pallescens and Curvularia trifolii, but the yields obtained from these fermentations were generally low. In order to find a strain suitable for industrial production of BFA, the applicant screened and obtained a strain of Curvularia pseudorobusta, which can be fermented to obtain BFA with high titer. At present, there is no literature publicly reporting that this strain can obtain BFA. Summary of the invention

[0004] In order to obtain a strain with high fermentation titer of brefeldin A, the present invention provides a strain of Curvularia pseudorobusta HDCC00041, which was deposited in the General Microbiology Center (CGMCC) of China Microorganism Culture Collection Administration on August 22, 2022, with a deposit number of CGMCC No.40275.

[0005] The strain CGMCC No.40275 was obtained after screening and mutagenesis. The strain can be fermented at 25°C and can even tolerate a higher temperature of 33°C. Generally speaking, species of the genus Curvularia or Curvularia robusta cannot tolerate fermentation at 33°C, or their activity is greatly reduced at this temperature, while the strain CGMCC No.40275 can tolerate fermentation at 33°C. Compared with fermentation at 25°C, at 33°C, the strain CGMCC No.40275 can shorten the total production cycle from 6d (40h+4d) to 4d (22h+3d). On the one hand, by increasing the suitable temperature for fermentation of the strain, the power cost consumption of the cooling system can be reduced. On the other hand, the total production cycle can be shortened.

[0006] In addition, the CGMCC No.40275 strain was cultured at 33°C for 3 days, and the BFA yield was as high as 8.9 g / L, with an HPLC purity of 71.9%, which is more conducive to subsequent purification and increasing the output per unit fermentation volume.

[0007] The present invention also provides a use of the Curvularia pseudorobust for preparing brefeldin A;

[0008] Alternatively, it is used for preparing one or more of medicines, feeds, additives, research reagents, and antibacterial agents containing Brefeldin A.

[0009] The invention also provides a fermentation product of Curvularia pseudorobust.

[0010] The fermentation product referred to in this application is: a product containing active ingredients formed after microorganisms are cultured in a culture medium.

[0011] Preferably, the fermentation product is fermentation liquid, seed liquid, culture liquid or bacterial suspension.

[0012] The present invention also provides a use of the fermentation broth for preparing brefeldin A;

[0013] Alternatively, it is used for preparing one or more of medicines, feeds, additives, research reagents, and antibacterial agents containing Brefeldin A.

[0014] The present invention also provides a fermentation method for producing brefeldin A by using Curvularia pseudorobust.

[0015] Preferably, the fermentation is carried out in a fermentation medium containing an assimilable carbon source and / or nitrogen source.

[0016] Preferably, the assimilable carbon source is selected from glucose, sucrose, maltose, lactose, fructose, glycerol, soybean oil, maltodextrin, starch; or the assimilable carbon source is a combination of glucose and starch.

[0017] Preferably, the assimilable nitrogen source is selected from yeast extract powder, yeast powder, peptone, amino acid, casein, soybean cake powder, peanut cake powder, cottonseed cake powder, gluten powder, yeast extract, beef extract; or the assimilable nitrogen source is a combination of yeast extract powder, peptone and gluten powder.

[0018] Preferably, the fermentation medium also includes inorganic salts.

[0019] Preferably, the fermentation temperature of Curvularia pseudorobust is 20-35°C; or 22-35°C; or 25-35°C; or 25-33°C; or 32-34°C; or 33°C.

[0020] Preferably, the total fermentation time of Curvularia spp. is no more than 6 days; or no more than 5 days; or no more than 4 days.

[0021] Beneficial effects:

[0022] 1. The CGMCC No.40275 strain provided by the present invention belongs to Curvularia spp., which can produce BFA by fermentation, but the prior art does not report that Curvularia spp. can produce BFA;

[0023] 2. The CGMCC No.40275 strain provided by the present invention has high yield, few by-products in the fermentation broth, high BFA purity, which is more conducive to subsequent purification and improves the output per unit fermentation volume;

[0024] 3. The CGMCC No.40275 strain provided by the present invention can be fermented at a higher temperature, such as 33°C, thereby reducing the power cost consumption of the cooling system caused by cooling;

[0025] 4. The CGMCC No.40275 strain provided by the present invention has a short fermentation cycle and can obtain the expected amount of BFA in a shorter time;

[0026] 5. Due to the advantages of using the CGMCC No.40275 strain provided by the present invention for fermentation in terms of energy consumption, subsequent purification process, short fermentation cycle, etc., the cost in industrial production is lower and it is more conducive to industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the HPLC spectrum of Brefeldin A reference substance.

[0028] Figure 2 This is the HPLC chart of the final fermentation broth of Example 5.

[0029] Figure 3The ion current scanning spectrum of LCMS in positive ion mode, m / z = 281.2 [M+H] + .

[0030] Figure 4 The ion current scanning spectrum of LCMS in negative ion mode, m / z = 279.3 [MH] - . DETAILED DESCRIPTION

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples are all common commercial products and can be purchased on the market.

[0033] The present invention will be further described below by way of examples, which are not intended to further limit the present invention. It should be understood by those skilled in the art that equivalent replacements or corresponding improvements made to the present invention still fall within the scope of protection of the present invention.

[0034] Example 1

[0035] Isolation and purification of original strains

[0036] Wild roots, stems and leaves of Dendrobium officinale were obtained from Tianmu Mountain, Lin'an, Hangzhou. They were rinsed with tap water and dried with absorbent paper. Then they were cut into small pieces, rinsed repeatedly with sterile water for 5 times, and finally dried with sterile filter paper. The processed materials were cut into pieces in a clean bench, sterile water was added, mixed, and the clear water was absorbed into a PDA plate (containing bacterial antibiotics). Two pieces were placed on each plate. The fifth washing water was diluted and applied to the PDA plate. The above plates were cultured at a constant temperature of 25°C for 10 days. Different colony digging pieces were transferred to the PDA plate culture medium coated with Candida albicans using a puncher, and cultured at 25°C for 7 days. The formation of the inhibition zone was observed and recorded, and the strain HZ-TM182 with the most obvious inhibition zone was screened.

[0037] The colony of strain HZ-TM182 was broken up and inoculated into PDB medium, cultured at 25°C for 3 days, and then inoculated into 1.5 times the concentration of PDB medium at a ratio of 5%, cultured at 25°C for 10 days, 100 ml of the fermentation broth was taken, soaked in 4 times the volume of anhydrous ethanol for 30 min, concentrated to 178 ml by vacuum rotary evaporation, added with 3% macroporous adsorption resin, adsorbed for 24 hours, separated and collected the resin, soaked and eluted with 2 times the volume of anhydrous ethanol for 4 hours, separated the resin, and continued to soak and elute with 2 times the volume of anhydrous ethanol for 4 hours, combined the eluate, concentrated in vacuum until all the ethanol was volatilized, and then added with 2 ml of anhydrous ethanol to dissolve, filtered with a 0.45 μm membrane and detected, and the molecular weight of the obtained compound was determined to be 280 by negative ion mode LCMS ion current scanning spectrum and positive ion mode LCMS ion current scanning spectrum, which was consistent with Brefeldin A (hereinafter referred to as BFA).

[0038] Example 2

[0039] The strain HZ-TM182 was used as the test object, and the morphological and physiological and biochemical properties were studied with reference to the "Handbook of Fungal Identification", "Chinese Fungi Collection", "Chinese Fungi" and "Berger's Bacteria Identification Manual". The results showed that the colony of the strain was grayish white, fluffy, the spores were curved, septate, branched, smooth, the conidial cells were cylindrical, the conidia were curved, elliptical, with 3 septa, and dark crystal-like dots on the longitudinal surface of the septa. The more suitable growth temperature was 22-27℃, and the tolerable growth temperature was 20-35℃; the more suitable growth pH was 5-6, and the tolerable growth pH range was 4-7; it can grow using nutrients such as glycerol, glucose, sorbitol, starch, ammonium sulfate, potassium nitrate, and urea.

[0040] Fresh cells were collected and the Institute of Bioengineering of Zhejiang University of Technology was commissioned to conduct 18S rDNA sequencing and identification. After proofreading, the 18S rDNA sequence of the strain was compared with the sequences of related species and genera in the GenBank database by homologous sequence BLAST, and it was found that it had a high homology with Curvularia pseudorobusta, with the highest homology reaching 98.09%. At the same time, combined with the morphological characteristics of the strain, the strain was identified as Curvularia pseudorobusta.

[0041] Example 3

[0042] The spores of HZ-TM182 strain were used as experimental objects, and ethyl methanesulfonate was used for mutagenesis. After activation growth and spot purification, mutant colonies were obtained. After the mutant colonies were cultured by seeds and fermentation, the strain with the highest BFA content in the fermentation broth was screened, and the strain HDCC00041 was obtained. The strain was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on August 22, 2022, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No.40275.

[0043] Example 4

[0044] Take a glycerol tube of CGMCC No.40275 strain, thaw it at 40℃, take 0.5ml and inoculate the PDA slant, spread it evenly and incubate it at 25℃ for 7 days to obtain mature slant spores. Then scrape 1cm 2 The spores were inoculated into a 500 ml triangular flask containing 100 ml seed culture medium, wrapped and placed on a shaker at 25°C and 250 rpm for 40 hours, and the pH value of the seed solution was controlled at 5.0-6.0. The qualified seed solution was inoculated into 500 ml fermentation medium at a ratio of 2% (V / V), wrapped and placed at 25°C for 4 days.

[0045] After the fermentation, 1 ml of the fermentation liquid was taken, soaked in 4 times volume of anhydrous ethanol for 30 min, mixed and diluted 4 times with anhydrous ethanol, and the supernatant was taken after high-speed centrifugation and tested by HPLC. The results showed that the BFA content in the fermentation liquid was 8.7 g / L and the HPLC purity was 70.3%.

[0046] The seed culture medium formula consists of: 1.5% glucose, 1.5% corn starch, 0.5% yeast extract powder, 0.5% peptone, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, and 0.2% calcium carbonate. The pH is adjusted to 6.0 before disinfection, and the disinfection conditions are 121-123°C for 30 minutes.

[0047] The fermentation medium formula consists of 2% glucose, 4% corn starch, 4% soluble starch, 2% yeast extract powder, 1% peptone, and 1% gluten powder. The pH is adjusted to 5.5 before disinfection, and the disinfection conditions are 121-123° C. and 30 min.

[0048] Example 5

[0049] The same as Example 4, except that the seed solution of CGMCC No.40275 strain was shaken and cultured on a shaker at 33°C for 22 hours, and then the seed solution was inoculated into a shake flask containing 25 ml of fermentation medium at a ratio of 5% (V / V). The fermentation was terminated after culturing at 33°C for 3 days. The fermentation solution was detected by HPLC as follows: Figure 2 According to the spectrum shown, the BFA content in the fermentation broth was calculated to be 8.9 g / L and the HPLC purity was 71.9%.

[0050] Comparative Example 1

[0051] Same as Example 4, except that the cultured strain is HZ-TM182 strain, which is cultured in 500 ml of liquid fermentation medium for 6 days. After the fermentation, the BFA content in the fermentation broth was measured by HPLC to be 0.027 g / L, and the HPLC purity was 20.3%.

[0052] Comparative Example 2

[0053] Same as Example 4, except that the cultured strain is HZ-TM182 strain, the fermentation temperature is 33° C., and the strain is cultured in 500 ml of liquid fermentation medium for 6 days. After the fermentation is completed, the BFA content in the fermentation broth is measured by HPLC to be 0.019 g / L, and the HPLC purity is 16.5%.

Claims

1. A strain of Curvularia pseudorobusta HDCC00041 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on August 22, 2022, with the deposit number CGMCC No.40275.

2. A use of Curvularia spp. as claimed in claim 1, characterized in that: For the preparation of Brefeldin A; Alternatively, the invention can be used for preparing a bacteriostatic agent containing brefeldin A.

3. A fermentation product containing Curvularia pseudorobust as claimed in claim 1.

4. A fermentation product according to claim 3, characterized in that: The fermentation product is fermentation liquid, seed liquid, culture liquid or bacterial suspension.

5. A use of the fermentation liquid as claimed in claim 4, characterized in that: For the preparation of Brefeldin A; Alternatively, the invention can be used for preparing a bacteriostatic agent containing brefeldin A.

6. A fermentation method for producing brefeldin A using Curvularia pseudorobust as claimed in claim 1.

7. The fermentation method according to claim 6, characterized in that: It includes fermenting in a fermentation medium containing an assimilable carbon source and / or nitrogen source.

8. The fermentation method according to claim 7, characterized in that: The assimilable carbon source is selected from glucose, sucrose, maltose, lactose, fructose, glycerol, soybean oil, maltodextrin, and starch; or the assimilable carbon source is a combination of glucose and starch.

9. The fermentation method according to claim 7, characterized in that: The assimilable nitrogen source is selected from yeast extract powder, yeast powder, peptone, amino acid, casein, soybean cake powder, peanut cake powder, cottonseed cake powder, gluten powder, yeast extract, beef extract; or the assimilable nitrogen source is a combination of yeast extract powder, peptone and gluten powder.

10. The fermentation method according to claim 7, characterized in that: The fermentation temperature of Curvularia spp. is 20-35°C.

11. The fermentation method according to claim 7, characterized in that: The fermentation temperature of Curvularia spp. is 22-35°C.

12. The fermentation method according to claim 7, characterized in that: The fermentation temperature of Curvularia spp. is 25-35°C.

13. The fermentation method according to claim 7, characterized in that: The fermentation temperature of Curvularia spp. is 25-33°C.

14. The fermentation method according to claim 7, characterized in that: The fermentation temperature of Curvularia spp. is 32-34°C.

15. The fermentation method according to claim 7, characterized in that: The fermentation temperature of Curvularia pseudorobust was 33°C.

16. The fermentation method according to claim 7, characterized in that: The total fermentation time of Curvularia spp. is no more than 6 days.

17. The fermentation method according to claim 7, characterized in that: The total fermentation time of Curvularia spp. is no more than 5 days.

18. The fermentation method according to claim 7, characterized in that: The total fermentation time of Curvularia spp. is no more than 4 days.

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