Fusarium r1-uv-4-3a with high yield of cyclophellolactone and its application

Abstract

The application discloses a fusarium R1-UV-4-3a capable of producing cyclophellitol compounds and an application thereof. The fusarium strain R1-UV-4-3a has good genetic stability, and the level of producing cyclophellitol compounds in the sixth generation is equivalent to that in the first generation. Meanwhile, the titer of cyclophellitol compounds in the fermentation product of the strain is increased by about 48% compared with the titer of cyclophellitol compounds in the fermentation product of the original strain, and the strain can be applied to the industrial production of cyclophellitol compounds.

Description

(I) TECHNICAL FIELD

[0001] The present application relates to a fusarium R1-UV-4-3a with high yield of cyclophellolone compound and its application. (II) BACKGROUND

[0002] SA is a cyclophellolone compound, which was first obtained from the liquid fermentation products (mycelium and fermentation broth) of fusarium sp. CNL-619 by Cueto et al. in 2000, with a yield of about 3 mg / L; in 2023, Park et al. found that fusarium solani f. also produced SA in agar medium, but no yield was reported.

[0003]

[0004] Modern pharmacological studies have shown that SA has significant anti-tumor activity. SA and its analogues can effectively inhibit the growth of pancreatic cancer cell lines and can inhibit the proliferation of bladder cancer cells and colon cancer cells. SA shows potential anti-tumor activity in the treatment of different types of cancer, providing a promising direction for the development of new anti-tumor drugs. Patent application CN115505033A discloses a preparation method of SA, which is: centrifuging the fermentation mixture of fusarium CGMCC NO. 17763, adding methanol to the precipitate after centrifugation and performing ultrasonic assisted extraction. The organic layer is concentrated under reduced pressure to obtain a crude extract, which is dissolved with chromatographic methanol, filtered and then prepared by high performance liquid chromatography to obtain SA. Although the SA yield of fusarium CGMCC NO. 17763 fermentation conditions has been improved to a certain extent (Fermentation. 2024;10(3): 140), it is still difficult to realize large-scale fermentation preparation.

[0005] Therefore, it is necessary to screen an excellent strain capable of high-yield SA. (III) SUMMARY

[0006] The present application aims to provide fusarium R1-UV-4-3a with high yield of SA and its application, which solves the problem of low fermentation titer of SA obtained by using traditional fermentation methods of the original strain.

[0007] The technical solution adopted by the present application is:

[0008] The application provides a high SA-yield Fusarium sp. R1-UV-4-3a, which is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC NO: M 20241573, a preservation date of July 15, 2024, and an address of Wuhan University, Wuhan, China, and a postal code of 430072. The Fusarium sp. R1-UV-4-3a is obtained by screening and mutagenizing the Fusarium sp. CGMCC NO. 17763.

[0009] The application also provides an application of the Fusarium sp. R1-UV-4-3a in fermentation to produce SA.

[0010] Further, the application comprises the following steps: (1) inoculating the Fusarium sp. R1-UV-4-3a into PDA culture medium, activating the culture at 30-37 DEG C for 3-5 days, then inoculating into PDB liquid culture medium, and culturing at 30-37 DEG C and 150-200 rpm for 60-80 hours to obtain a seed liquid; (2) inoculating the seed liquid into fermentation culture medium at a volume concentration of 1-10%, and statically fermenting at 20-30 DEG C for 10-15 days to obtain a fermentation liquid containing SA, and separating and purifying to obtain SA; the fermentation culture medium comprises 20-30 g / L sucrose, 10-20 g / L tryptone, 20-40 mg / L yeast extract and 10-30 g / L sea salt, has a pH of 6.5, and uses water as a solvent; the liquid loading amount of the fermentation culture medium is 50% of the container capacity, and the fermentation culture medium is sterilized at 121 DEG C for 20 minutes.

[0011] Further, the PDA culture medium comprises 200.0 g / L potatoes, 20.0 g / L glucose and 15.0-20.0 g / L agar, has a natural pH, and uses water as a solvent; the PDB liquid culture medium comprises 200.0 g / L potatoes and 20.0 g / L glucose, has a natural pH, and uses water as a solvent.

[0012] Further, the temperature of the seed culture in step (1) is 35 DEG C, the rotating speed is 180 rpm, and the time is 72 hours.

[0013] Further, the volume inoculation amount of the seed liquid in step (2) is 5%.

[0014] Further, the fermentation culture medium comprises 22.5 g / L sucrose, 16.5 g / L tryptone, 24 mg / L yeast extract and 20.0 g / L sea salt, has a pH of 6.5, and uses water as a solvent.

[0015] Further, the fermentation culture temperature in step (2) is 25 DEG C, and the static culture is performed for 13 days.

[0016] Compared with the prior art, the beneficial effects of the present application mainly embody that the present application provides a fusarium strain R1-UV-4-3a with high SA yield, which has good genetic stability, and the yield of the compound SA in the sixth generation is equivalent to that in the first generation; meanwhile, the titer of SA in the fermentation product of the strain is increased by about 48% compared with that in the fermentation product of the original strain, and the strain can be applied to the industrial production of the compound SA. (V) Brief Description of the Drawings

[0017] Figure 1 Figure 4 is a curve of the lethal rate of the original strain with respect to the ultraviolet irradiation time.

[0018] Figure 2 Figure 5 is a morphological difference diagram of the original strain (a) and the mutant strain (b).

[0019] Figure 3 Figure 6 is an HPLC analysis spectrum of the fermentation product of the original strain.

[0020] Figure 4 Figure 7 is an HPLC analysis spectrum of the fermentation product of the fusarium R1-UV-4-3a. (V) Brief Description of the Drawings

[0021] The present application will be further described below in combination with specific examples, but the protection scope of the present application is not limited to this:

[0022] In the embodiment of the present application, the PDA culture medium is composed of 200.0 g / L potatoes, 20.0 g / L glucose, 15.0-20.0 g / L agar, pH natural, solvent is water, and sterilized at 121℃ for 20 min; the PDB liquid culture medium is composed of 200.0 g / L potatoes, 20.0 g / L glucose, pH natural, solvent is water, and sterilized at 121℃ for 20 min.

[0023] The fermentation culture medium is composed of 22.5 g / L sucrose, 16.5 g / L tryptone, 0.024 g / L yeast extract, 20.0 g / L sea salt, pH 6.5, solvent is water, the liquid volume of the fermentation liquid is 50% of the container capacity, and sterilized at 121℃ for 20 min.

[0024] Example 1, mutation and screening of the fusarium strain R1-UV-4-3a

[0025] The fusarium strain R1-UV-4-3a of the present application is obtained by ultraviolet irradiation mutagenesis screening based on the fusarium CGMCC NO.17763 (which has been disclosed in the patent application CN110563740A) as the original strain, and includes the following steps:

[0026] (1) The original strain was inoculated in PDA medium and incubated at 30°C for 3-5 days; distilled water was added, and spores were scraped off with an inoculation loop, and the concentration of the spore suspension was adjusted to 10 5 CFU / mL with a hemocytometer.

[0027] (2) The spore suspension obtained in step (1) was irradiated with ultraviolet light at a power of 8 W and a distance of 10 cm for 0 s, 60 s, 120 s, 180 s, 240 s, and 300 s, respectively, and then coated on PDA medium and incubated at 30°C for 3-5 days. According to the growth of single colonies on the plates with different irradiation times, the effect of different ultraviolet irradiation times on the lethality of the strain was calculated with the blank as a control. Figure 1 The colony morphology of the mutant strain after mutagenesis was almost the same as that of the original strain Figure 2 , so the colonies on the plates with a lethality of more than 80% were selected for subsequent fermentation and yield detection.

[0028] The lethality calculation formula is:

[0029] Lethality = [(1- number of colonies after mutagenesis) / number of colonies before mutagenesis] x 100%

[0030] (3) The colonies on the plates with a lethality of more than 80% in step (2) were inoculated into PDB liquid medium for seed culture, and the culture conditions were 35°C, 180 rpm, and 3 days.

[0031] (4) The seed liquid obtained in step (3) was inoculated into fermentation medium at a volume concentration of 5% for fermentation culture, and the culture temperature was 25°C with static culture for 13 days.

[0032] (5) The fermentation liquid in step (4) was filtered with gauze, and about 25 g of the bacterial cells were ultrasonically extracted with 300 mL of methanol at 480 W for 30 min, then subjected to suction filtration, the filtrate was concentrated to a liquid-free state by rotary evaporation, dissolved in 15 mL of chromatographic methanol, filtered with a 0.22 μm organic filter, and 1 mL of the liquid was detected by HPLC to detect the yield change of SA. Strains with a significant change in SA yield compared with the original strain were selected by the increase or decrease of peak type and the change of relative area.

[0033] HPLC conditions: LC-20AT liquid chromatograph from Shimadzu, photodiode array ultraviolet-visible light detector SPD-M40, automatic sampler SIL-20A, chromatographic column Phenomenex Gemini NX-C18 (4.6 mm x 250 mm, 5 μm), injection volume 10 μL, flow rate 1.0 mL / min, column temperature 40°C, detection wavelength 210 nm, mobile phase acetonitrile: water (containing 0.1% formic acid) = 70:30 (v / v), isocratic elution.

[0034] (6) Select the high-SA-producing mutant strain corresponding to the plate colony in step (2) for subculturing. After six generations, select the sixth generation strain for culture and fermentation, and then perform HPLC analysis to screen for stable high-SA-producing mutant strains.

[0035] As a result, a high-SA-producing Fusarium mutant strain was screened and classified as Fusarium sp., strain number R1-UV-4-3a. It was deposited on July 15, 2024 at the China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO:M 20241573.

[0036] Example 2: Stability analysis of SA production by Fusarium R1-UV-4-3a

[0037] (1) Fusarium R1-UV-4-3a was inoculated into PDA medium and activated for 3 days at 30°C, and then passaged to the sixth generation.

[0038] (2) The strains of the first and sixth generation in step (1) were inoculated into PDB medium and cultured at 35°C and 180 rpm for 72 h to obtain seed liquid.

[0039] (3) The seed culture from step (2) was inoculated into the fermentation medium at a volume concentration of 5%, and fermented statically at 25°C for 13 days to obtain the fermentation broth. SA yield was detected using the method described in Example 1, with the original strain as a control. Each strain was replicated three times, and the results are shown in Table 1. The HPLC analysis of the first-generation fermentation broth of the original strain is shown in Table 1. Figure 3 The first-generation fermentation broth of Fusarium R1-UV-4-3a was analyzed by HPLC. Figure 4 .

[0040] Table 1: SA yield of strain SA

[0041]

[0042] The data in Table 1 show that the yield of compound SA in the fermentation product of the original strain was 126 ± 1.06 mg / L;

[0043] The yield of compound SA in the fermentation product of strain R1-UV-4-3a was 182±1.98 mg / L, which was about 48% higher than that of the original strain, and strain R1-UV-4-3a was genetically stable.

Claims

1. A Fusarium species that produces high levels of cyclophenolphthalein compounds ( Fusarium sp.) R1-UV-4-3a, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20241573, deposited on July 15, 2024, address: Wuhan University, Wuhan, China, 430072, China.

2. The application of Fusarium R1-UV-4-3a as described in claim 1 in the fermentation production of cyclic phenolphthalein compounds, wherein the cyclic phenolphthalein compounds are:

3. The application as described in claim 2, characterized in that, The application The process includes the following steps: (1) Inoculating Fusarium R1-UV-4-3a into PDA medium, activating and culturing at 30-37℃ for 3-5 days, then inoculating into PDB liquid medium, culturing at 30-37℃ and 150-200rpm for 60-80h to obtain seed culture; (2) Inoculating the seed culture into fermentation medium at a volume concentration of 1-10%, statically fermenting at 20-30℃ for 10-15 days to obtain fermentation broth containing cyclophenolphthalein compounds, separating and purifying to obtain cyclophenolphthalein compounds; The fermentation medium composition is: sucrose 20-30 g / L, tryptone 10-20 g / L, yeast extract 20-40 mg / L, sea salt 10-30 g / L, pH 6.5, solvent is water, the volume of fermentation medium is 50% of the container capacity, sterilizing at 121℃ for 20 min.

4. The application as described in claim 3, characterized in that, The PDA culture medium consists of 200.0 g / L potato, 20.0 g / L glucose, and 15.0~20.0 g / L agar, with a natural pH and water as the solvent; the PDB liquid culture medium consists of 200.0 g / L potato and 20.0 g / L glucose, with a natural pH and water as the solvent.

5. The application as described in claim 3, characterized in that, In step (2), the seed liquid volume inoculation amount is 5%.

6. The application as described in claim 3, characterized in that, The fermentation medium consisted of: 22.5 g / L sucrose, 16.5 g / L tryptone, 24 mg / L yeast extract, 20.0 g / L sea salt, pH 6.5, and water as the solvent.

7. The application as described in claim 3, characterized in that, Step (2) The fermentation culture temperature is 25 ℃, and the culture is left to stand for 13 days.

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