Streptomyces avermitilis AVE-16 with high yield of avermectin B2 and preparation method thereof

Abstract

The present application relates to a kind of high-yield abamectin B2 Streptomyces avermitilis AVE-16 and its preparation method, the Streptomyces avermitilis AVE-16 is preserved in China Microbial Culture Collection Center, and the preservation number is CGMCC No.38200.The present application uses Streptomyces avermitilis XB-12 as starting strain, uses ethyl methanesulfonate and atmospheric pressure plasma combined mutagenesis technology to obtain a Streptomyces avermitilis AVE-16, and the yield of abamectin B2a is increased by 145% compared with starting strain, and the total yield of abamectin (B1a+B2a) is increased by 44% compared with starting strain.

Description

Technical Field

[0001] This invention belongs to the field of avermectin B2 mutagenesis breeding technology, specifically relating to an avermectin B2-producing avermectin B2 strain AVE-16 and its preparation method. Background Technology

[0002] Avermectin is a sixteen-membered macrocyclic lactone secondary metabolite produced by Streptomyces avermitilis. It is a novel, pollution-free biological pesticide and veterinary drug with the characteristics of high efficiency, low toxicity, and environmental friendliness. Currently, avermectin produced by *Streptomyces avermectin* fermentation includes eight structurally similar components: AVMA1a, AVMA1b, AVMA2a, AVMA2b, AVMB1a, AVMB1b, AVMB2a, and AVMB2b. Although there are many types, among the avermectin produced by *Streptomyces avermectin* selected from nature, B1a has the highest yield, reaching 50%-70%, followed by B2a at 20%-40%, while other avermectins such as B1b and B2b account for approximately 5%-15% in total. Furthermore, among these eight components, component B has better biological activity than component A. Although it is a broad-spectrum insecticide, the activity of different components varies significantly against different pests. For example, B1 is more effective than B2 against lepidopteran, coleopteran, and dipteran pests, but its efficacy against nematodes is poor. B2, on the other hand, has specific efficacy against root-knot nematodes and root-rot nematodes, and its toxicity is lower than that of component B1. In addition, in recent years, with the development of its derivatives, derivative products specifically using avermectin (usually referring to avermectin B2a) as raw material have been continuously researched. Therefore, this has greatly increased the demand for avermectin B2. The by-product B2 produced by fermenting avermectin B1 alone is far from meeting the current demand for avermectin B2.

[0003] Microbial mutagenesis breeding is a microbial breeding technique that uses gene mutation as a means. It mainly includes physical mutagenesis, chemical mutagenesis, and biological mutagenesis. It is simple to operate, has a high mutation rate, and a wide mutation spectrum. It can not only increase yield and improve quality, but also expand product varieties and simplify process conditions. Therefore, using mutagenesis breeding technology to mutate Streptomyces avermectin is expected to change the product type composition of the original strain and obtain Streptomyces avermectin B2-producing strains to meet the problem of insufficient avermectin B2 production. Summary of the Invention

[0004] The purpose of this invention is to overcome the defects in the prior art and provide an abamectin B2-producing Streptomyces AVE-16 and its preparation method, which greatly improves the yield of abamectin B2 and the total yield of abamectin.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A high-yielding avermectin B2-producing Streptomyces AVE-16 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38200.

[0006] As a further technical solution, it was obtained by combined mutagenesis treatment with ethyl methanesulfonate and atmospheric pressure plasma.

[0007] A method for preparing the aforementioned Streptomyces avermectin AVE-16 includes the following steps: Step 1, Activation and seed culture of the starting strain: After activating the Streptomyces avermitilis starting strain, it was inoculated into seed culture medium for fermentation to obtain seed liquid; Step 2, Ethyl methanesulfonate mutagenesis treatment: After washing the seed liquid with sterile physiological saline and resuspending it, bacterial suspension A was prepared. Then, ethyl methanesulfonate was added to bacterial suspension A and shaken. After the treatment, 2-mercaptoethanol was added to terminate the reaction. The bacterial cells were collected by solid-liquid separation and washed with sterile physiological saline to obtain ethyl methanesulfonate-mutated bacterial cells. Step 3, Atmospheric pressure plasma mutagenesis treatment: The ethyl methanesulfonate mutagenized bacteria were resuspended in sterile physiological saline to obtain bacterial suspension B. Then, bacterial suspension B was evenly spread on a sterile slide and subjected to atmospheric pressure plasma mutagenesis treatment. After the mutagenesis treatment was completed, the bacteria on the slide were transferred to sterile physiological saline to obtain the mutant library bacterial suspension.

[0008] Step 4: Screening and purification of mutant strains: After isolating single colonies from the mutant strain suspension, the obtained single strains were inoculated into shake-flask fermentation medium for fermentation culture. After the fermentation culture was completed, the yield of avermectin B2 in the fermentation broth was measured. Based on the yield of avermectin B2, strains were screened, and finally avermectin streptomyces AVE-16 was obtained.

[0009] As a further technical solution, when screening strains, it is also necessary to determine the yield of avermectin B1 in the fermentation broth, and to comprehensively consider the yield of avermectin B2 and the total yield of avermectin for strain screening.

[0010] As a further technical solution, in the ethyl methanesulfonate mutagenesis system, the final volume concentration of ethyl methanesulfonate is 0.1-0.5%.

[0011] As a further technical solution, the conditions for atmospheric pressure plasma mutagenesis treatment are as follows: the distance between the plasma source and the sterile slide is 2-5 mm, the discharge power is 30-60 W, the discharge gas is argon, the gas flow rate is 5-15 L / min, and the mutagenesis treatment lasts for 30-120 s.

[0012] As a further technical solution, the concentration of bacterial suspension A is 10.6 -10 7 CFU / mL.

[0013] As a further technical solution, the concentration of bacterial suspension B is 10. 5 -10 6 CFU / mL.

[0014] The use of Streptomyces AVE-16 as described in claim 1 in the production of avermectin B2.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses Streptomyces avermectin XB-12 as the starting strain and obtains a Streptomyces avermectin AVE-16 using a combined mutagenesis technique of ethyl methanesulfonate and atmospheric pressure plasma. Compared with the starting strain, the yield of avermectin B2a is increased by 145%, and the total yield of avermectin (B1a+B2a) is increased by 44%. Attached Figure Description

[0016] Figure 1 High-performance liquid chromatography (HPLC) chromatogram of a sample obtained after processing the fermentation broth of Streptomyces avermitilis AVE-16. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0018] 1. Unless otherwise specified, all raw materials used in this invention are commercially available.

[0019] Example 1: Mutagenesis breeding of Streptomyces avermitilis 1. Activation and culture of the starting strain: The starting strain of Streptomyces avermitilis XB-12 (from Hebei Xingbai) was inoculated into slant medium and cultured at 28℃ for 7 days to obtain the activated slant strain; single colonies of the slant strain were picked and inoculated into seed medium and cultured at 28℃ and 180r / min for 60h to obtain seed liquid; The slant culture medium is formulated as follows: glucose 15 g / L, peptone 5 g / L, yeast extract 10 g / L, MgSO4·7H2O 0.5-1 g / L, KH2PO4 1 g / L, agar 20 g / L, pH 7.2; the seed culture medium is formulated as follows: glucose 15 g / L, soybean meal 20 g / L, yeast extract 10 g / L, MgSO4·7H2O 1 g / L, KH2PO4 1 g / L, pH 7.2.

[0020] 2. Ethyl methanesulfonate (EMS) mutagenesis treatment: Collect bacterial cells by centrifugation of the seed culture, wash 2-3 times with sterile physiological saline, and resuspend in sterile physiological saline to prepare a 10% concentration. 6 -10 7 CFU / mL bacterial suspension A; EMS solution was added to bacterial suspension A to make the final EMS concentration 0.1% (v / v), and the mixture was shaken at 28℃ and 150r / min for 20min. After the treatment, 2-mercaptoethanol was added to terminate the reaction, the bacterial cells were collected by centrifugation, and washed 3 times with sterile physiological saline to obtain EMS-mutated bacterial cells. The EMS solution was prepared using sterile physiological saline at a concentration of 10% (v / v); the final concentration of the 2-mercaptoethanol was 1% (v / v), and the reaction time was 10 min.

[0021] 3. Atmospheric pressure plasma (APP) mutagenesis treatment: The EMS-mutated bacterial bodies were suspended in sterile physiological saline to prepare a concentration of 10. 5 -10 6 CFU / mL bacterial suspension B; 100 μL of bacterial suspension B was evenly spread on a sterile slide and placed on the sample stage of an atmospheric pressure plasma mutagenesis device. The distance between the plasma source and the slide was adjusted to 5 mm. The mutagenesis was performed at a discharge power of 60 W and an argon gas flow rate of 10 L / min for 90 minutes. After the mutagenesis was completed, the bacteria on the slide were transferred to sterile physiological saline to obtain the mutant library bacterial suspension.

[0022] 4. Screening and purification of mutant strains: The mutant library bacterial suspension was serially diluted with sterile physiological saline. The bacterial suspension of appropriate dilution was spread on the isolation medium with the same formula as the slant medium and cultured at 28℃ for 7 days. Single colonies were picked and inoculated into shake flask fermentation medium with the following formula: glucose 25g / L, soybean meal 30g / L, yeast extract 10g / L, MgSO4·7H2O 1g / L, KH2PO4 2g / L, CaCO3 5g / L, pH 7.2. The mixture was fermented at 28℃ and 200 r / min for 8 days with shaking. The yields of avermectin B1a and B2a in the fermentation broth were determined by external standard method using high performance liquid chromatography (HPLC). Strains with high yields of both avermectin B2a and total avermectin (B1a+B2a) were screened to obtain the mutant strain Streptomyces avermectin AVE-16. The changes in the yields of avermectin B2a and B1a in the mutant strain Streptomyces avermectin AVE-16 compared to the starting strain are shown in Table 1. The conditions for determining the yield of abamectin were as follows: a C18 column (250 mm × 4.6 mm, 5 μm), a mobile phase of methanol-water (85:15, v / v), a flow rate of 1.0 mL / min, a detection wavelength of 245 nm, and a column temperature of 30 °C. The high-performance liquid chromatogram of the sample obtained after treatment with the fermentation broth of the mutant strain *Streptomyces avermitilis* AVE-16 is shown below. Figure 1 Among them, 5.459 min was avermectin B2b, 6.463 min was avermectin B2a, 10.189 min was avermectin B1b, and 12.955 min was avermectin B1a.

[0023] Table 1 As can be seen from the data in Table 1, compared with the starting strain XB-12, the mutant strain AVE-16 obtained by mutagenesis in this invention has a 145% increase in the yield of avermectin B2a and a 44% increase in the total yield of avermectin (B1a+B2a).

[0024] The mutant strain AVE-16 of Streptomyces avermectin obtained in this invention greatly increases the yield of avermectin B2, meeting the increasing demand for avermectin B2.

[0025] Example 2: Morphological and Physiological Biochemical Studies Morphological and physiological-biochemical studies were conducted on the *Streptomyces avermitilis* AVE-16 obtained in Example 1, and the results are as follows: (1) Colony color: grayish-white.

[0026] (2) Aerobic growth: growth.

[0027] (3) Cell size / mm: 2~8mm.

[0028] (4) Suitable growth temperature: 26.0-29℃.

[0029] (5) Suitable growth pH: 6.2-7.4.

[0030] Bacterial appearance: The colonies are round, wrinkled in the later stages of growth, dull, and with neat edges.

[0031] Gram staining: positive. Example 3: Preservation

[0032] The Streptomyces AVE-16 obtained in Example 1, which still belongs to Streptomyces avermitilis, was deposited at the China General Microbiological Culture Collection Center; accession number CGMCC No. 38200, deposit date March 30, 2026; deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0033] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. An abamectin B2-producing Streptomyces AVE-16, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 38200.

2. The *Streptomyces avermitilis* AVE-16 as described in claim 1, characterized in that, It was obtained by combined mutagenesis treatment with ethyl methanesulfonate and atmospheric pressure plasma.

3. A method for preparing *Streptomyces avermitilis* AVE-16 as described in claim 1, characterized in that, Includes the following steps: Step 1, Activation and seed culture of the starting strain: After activating the Streptomyces avermitilis starting strain, it was inoculated into seed culture medium for fermentation to obtain seed liquid; Step 2, Ethyl methanesulfonate mutagenesis treatment: After washing the seed liquid with sterile physiological saline and resuspending it, bacterial suspension A was prepared. Then, ethyl methanesulfonate was added to bacterial suspension A and shaken. After the treatment, 2-mercaptoethanol was added to terminate the reaction. The bacterial cells were collected by solid-liquid separation and washed with sterile physiological saline to obtain ethyl methanesulfonate-mutated bacterial cells. Step 3, Atmospheric pressure plasma mutagenesis treatment: The ethyl methanesulfonate mutagen bacteria were resuspended in sterile physiological saline to obtain bacterial suspension B. Then, bacterial suspension B was evenly spread on a sterile slide and subjected to atmospheric pressure plasma mutagenesis treatment. After the mutagenesis treatment was completed, the bacteria on the slide were transferred to sterile physiological saline to obtain the mutant library bacterial suspension. Step 4: Screening and purification of mutant strains: After isolating single colonies from the mutant strain suspension, the obtained single strains were inoculated into shake-flask fermentation medium for fermentation culture. After the fermentation culture was completed, the yield of avermectin B2 in the fermentation broth was measured. Based on the yield of avermectin B2, strains were screened, and finally avermectin streptomyces AVE-16 was obtained.

4. The method for preparing Streptomyces avermitilis AVE-16 according to claim 3, characterized in that, In the ethyl methanesulfonate mutagenesis system, the final volume concentration of ethyl methanesulfonate is 0.1-0.5%.

5. The method for preparing Streptomyces avermitilis AVE-16 according to claim 3, characterized in that, The conditions for atmospheric pressure plasma mutagenesis treatment are as follows: the distance between the plasma source and the sterile slide is 2-5 mm, the discharge power is 30-60 W, the discharge gas is argon, the gas flow rate is 5-15 L / min, and the mutagenesis treatment lasts for 30-120 s.

6. The method for preparing Streptomyces avermitilis AVE-16 according to claim 3, characterized in that, The concentration of bacterial suspension A is 10. 6 -10 7 CFU / mL.

7. The method for preparing Streptomyces avermitilis AVE-16 according to claim 3, characterized in that, The concentration of bacterial suspension B is 10. 5 -10 6 CFU / mL.

8. The use of Streptomyces AVE-16 as described in claim 1 in the production of avermectin B2.

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