Preparation method for saccharopolyspora spinosa spn016 and spinosad and screening method for saccharopolyspora spinosa

Abstract

A preparation method for Saccharopolyspora spinosa Spn016 and spinosad, and a screening method for Saccharopolyspora spinosa. The described Saccharopolyspora spinosa Spn016 has a classification name of Saccharopolyspora spinosa and was deposited in the China General Microbiological Culture Collection Center in Beijing, China under CGMCC accession No. 30471 on April 28, 2024. The present invention can solve the problem of Saccharopolyspora spinosa growing slowly in the prior art and pertains to the field of Saccharopolyspora spinosa.

Description

Preparation methods of Polysporus spinosae Spn016 and spinosad, and screening methods of Polysporus spinosae.

[0001] This application is based on and claims priority to Chinese application CN application number 202411003252.X, filed on July 25, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of Polysacchariformis, specifically to a method for preparing Polysacchariformis Spn016 and spinosad, and a method for screening Polysacchariformis. Background Technology

[0003] Saccharopolyspora spinosa is a soil actinomycete that produces spinosad, a novel green biopesticide. Spinosad is a macrolide secondary metabolite formed through aerobic fermentation. Spinosad belongs to the macrolide antibiotic class and mainly comprises spinosad A and spinosad D. As a biopesticide, it has many advantages, such as a broad insecticidal spectrum, high insecticidal activity, short half-life, and easy biodegradability. Therefore, it has become one of the most promising biopesticides internationally and has twice won the U.S. Presidential Green Chemicals Challenge Award.

[0004] The biosynthetic pathway of spinosad has been elucidated, but its regulatory mechanism is not yet fully understood. The construction of related engineered bacteria is complex, and the current production level cannot meet industrial needs. Furthermore, *Saccharomyces cerevisiae* grows slowly and has a long growth cycle, requiring 7-10 days for plate growth and more than 10 days for fermentation. These conditions not only limit the improvement of industrial production but also increase the cycle of molecular modification of chassis strains.

[0005] Therefore, screening out rapidly growing chassis strains with spinosad capabilities from the soil is crucial for industrial applications and strain modification. At the same time, rapid screening methods are also necessary and have significant advantages in terms of cost control.

[0006] Summary of the Invention

[0007] The main objective of this invention is to provide a method for preparing Spn016 of Polysaccharidone and spinosad, as well as a method for screening Polysaccharidone, to solve the problem of slow growth of Polysaccharidone in the prior art.

[0008] To achieve the above objectives, according to a first aspect of the present invention, a *Saccharopolyspora spinosa* Spn016 is provided, classified as *Saccharopolyspora spinosa*, which was deposited on April 28, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 30471.

[0009] To achieve the above objective, according to a second aspect of the present invention, a method for preparing spinosad is provided, the method comprising: culturing the above-mentioned Polysporus spinosus Spn016, and isolating spinosad from the culture system; wherein the spinosad comprises spinosad A and spinosad D.

[0010] Furthermore, *Saccharomyces cerevisiae* Spn016 contains the DNA sequence shown in SEQ ID NO: 1.

[0011] Further, the cultivation method of *Spn016* includes: streaking *Spn016* on a solid culture plate to obtain a sporulating plate; inoculating the spores of *Spn016* on the sporulating plate into a seed culture medium to obtain a seed liquid; and inoculating the seed liquid into a fermentation medium to obtain a culture system. Preferably, the cultivation method includes: streaking *Spn016* on a solid culture plate and culturing at 25–35°C for 3–5 days. After 1 day, obtain a plate containing spores; take spores of *Spn016* from the plate containing spores and inoculate them into a seed culture medium, and culture at 25–35°C and 100–300 rpm for 12–60 h to obtain a seed solution; add the seed solution to the fermentation medium at a volume percentage of 1–20% (i.e., the volume ratio of seed solution to fermentation medium is 1–20:100), and culture at 25–35°C and 100–300 rpm for 3–7 days to obtain a culture system.

[0012] Furthermore, the fermentation medium, by mass-volume ratio, includes 20–100 g / L glucose, 5–50 g / L cottonseed meal, 2–20 g / L corn steep liquor, 2–20 g / L soluble starch, and 1–10 g / L CaCO3, with a pH of 7.0–7.4.

[0013] Further, the solid culture plates include modified TSB solid medium or Bennett solid medium; preferably, by weight-volume ratio, the modified TSB solid medium includes 5-20 g / L corn steep liquor powder, 17 g / L tryptone, 3 g / L soybean peptone, 2.5 g / L D-glucose, 5.0 g / L NaCl, 2.5 g / L K₂HPO₄, and 25 g / L agar powder, with a pH of 7.2 ± 0.5. Preferably, by weight-volume ratio, the Bennett solid medium includes 1 g / L yeast extract, 1 g / L beef extract, 2 g / L enzymatically hydrolyzed casein A, 10 g / L glucose, and 15 g / L agar, with a pH of 7.2-7.4.

[0014] Furthermore, the seed culture medium includes TSB liquid medium, which, by mass-volume ratio, comprises: 17 g / L tryptone, 3 g / L soybean peptone, 2.5 g / L D-glucose, 5.0 g / L NaCl, 2.5 g / L K2HPO4, and pH 7.2 ± 0.5.

[0015] Furthermore, after obtaining the culture system, the bacterial cells and / or bacterial solution in the culture system are broken and / or extracted to separate spinosad.

[0016] To achieve the above objective, according to a third aspect of the present invention, a method for screening *Saccharomyces cerevisiae* is provided, the method comprising: performing PCR amplification on the genome of a candidate strain using primers targeting the spnG gene to obtain a first amplification product; performing PCR amplification on the genome of the candidate strain using primers targeting the spnP gene to obtain a second amplification product; if the first amplification product is 1058 bp and the second amplification product is 1060 bp, the candidate strain is *Saccharomyces cerevisiae*.

[0017] Furthermore, the primers for the spnG gene are the DNA sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3; and the primers for the spnP gene are the DNA sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0018] Using the technical solution of the present invention, a strain of Polysporus spp. Spn016 was obtained through screening. This strain has a fast growth rate, can rapidly proliferate and produce spores, and can synthesize spinosad A and spinosad D. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0020] Figure 1 shows the phylogenetic tree of the 16S rDNA gene of *Spn016*, a polyspora spp. according to Example 2 of the present invention.

[0021] Figure 2 shows the growth status of *Spn016* and 4.1365 of *Saccharomyces cerevisiae* according to Example 3 of the present invention at 4 and 6 days.

[0022] Figure 3 shows microscopic morphological images of *Saccharomyces cerevisiae* Spn016 and 4.1365 according to Example 3 of the present invention.

[0023] Figure 4 shows the wet weight results of *Saccharomyces cerevisiae* Spn016 and 4.1365 according to Example 3 of the present invention.

[0024] Figure 5 shows the spinosad production results of Spn016 and 4.1365 of Polysporum spinosae according to Example 4 of the present invention.

[0025] [Revised according to Rule 26, September 2024] Biomaterial Information: A species of Saccharopolyspora spinosa, Spn016, was deposited on April 28, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China. The depository address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. The accession number is CGMCC No. 30471. Detailed Implementation

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0027] As mentioned in the background section, the growth of *Polysporium spp.* in the prior art is slow, which is insufficient to meet the needs of genetic modification and fermentation production of the secondary metabolite spinosad. Therefore, in this application, the inventors screened and obtained a strain of *Polysporium spp.* Spn016 that can proliferate rapidly and shorten the required culture time. Based on this, a series of protection schemes for this application have been proposed.

[0028] In a first typical embodiment of this application, a *Saccharopolyspora spinosa* Spn016 is provided, classified as *Saccharopolyspora spinosa*, which was deposited on April 28, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 30471.

[0029] In the field of biotechnology, *Saccharomyces cerevisiae* is an important microbial resource, attracting much attention due to its unique metabolic capabilities and the production of bioactive substances. However, the relatively slow growth rate of *Saccharomyces cerevisiae* limits its application in industrial production and scientific research. Especially during gene manipulation processes such as gene cloning, expression vector construction, and gene editing, the slow growth of *Saccharomyces cerevisiae* directly leads to extended experimental cycles. This not only increases research costs but also affects research efficiency. Therefore, improving the growth rate of *Saccharomyces cerevisiae* and shortening the gene manipulation cycle has become an important research topic. In this application, the inventors discovered that the aforementioned *Saccharomyces cerevisiae* Spn016 exhibits rapid growth, strong sporulation capacity, and the ability to produce spinosad A and D, making it a potential industrial application value as a chassis strain.

[0030] In a preferred embodiment, *Saccharomyces cerevisiae* Spn016 contains the DNA sequence shown in SEQ ID NO: 1.

[0031] SEQ ID NO: 1:

[0032] In a second typical embodiment of this application, a method for preparing spinosad is provided, the method comprising: culturing the above-mentioned Polysporus spinosus Spn016, and isolating spinosad from the culture system; wherein, spinosad includes spinosad A and spinosad D.

[0033] In the above preparation method, compared with other polysacchariformis species, the *Spn016* strain can proliferate and produce spinosad more quickly. Therefore, culturing *Spn016* and isolating spinosad from the culture system results in higher production efficiency and greater industrial application value. After cultivation, those skilled in the art can flexibly select existing separation methods, including but not limited to cell disruption, extraction, and column chromatography, to isolate spinosad from the culture system (including but not limited to bacterial cells or bacterial suspension).

[0034] In a preferred embodiment, the method for culturing *Spn016*, a polysporum spp., includes: streaking *Spn016* onto a solid culture plate to obtain a sporulating plate; inoculating the spores of *Spn016* from the sporulating plate into a seed culture medium to obtain a seed solution; and inoculating the seed solution into a fermentation medium to obtain a culture system. Preferably, the above-mentioned culturing method includes: streaking *Spn016* onto a solid culture plate and culturing at 25–35°C (including but not limited to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35°C) for 3–5 days to obtain a sporulating plate; taking spores of *Spn016* from the sporulating plate and inoculating them into a seed culture medium at 25–35°C (including but not limited to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35°C). Incubate at 100-300 rpm (including but not limited to 100, 125, 150, 175, 200, 225, 250, 275 or 300 rpm) for 12-60 h (including but not limited to 12, 18, 24, 30, 36, 42, 48, 54 or 60 h) to obtain seed culture; divide the seed culture into 1-20% (v / v) (including but not limited to 1, 2, 3, 5, 7, 10, 15 or 20%). Add an inoculum of (v / v) to the fermentation medium and incubate at 25–35°C (including but not limited to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C) and 100–300 rpm (including but not limited to 100, 125, 150, 175, 200, 225, 250, 275 or 300 rpm) for 3–7 days (including but not limited to 3, 4, 5, 6 or 7 days) to obtain the culture system.

[0035] In a preferred embodiment, the fermentation medium comprises, by weight-volume ratio, 20-100 g / L glucose (including but not limited to 20, 30, 40, 50, 60, 70, 80, 90, or 100 g / L), 5-50 g / L cottonseed meal (including but not limited to 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L), 2-20 g / L corn steep liquor (including but not limited to 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 g / L), 2-20 g / L soluble starch (including but not limited to 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 g / L), and 1-10 g / L CaCO3 (including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 g / L), pH 7.0 to 7.4 (including but not limited to 7.0, 7.1, 7.2, 7.3 or 7.4).

[0036] In a preferred embodiment, the solid culture plate comprises modified TSB solid medium or Bennett solid medium; preferably, by weight-volume ratio, the modified TSB solid medium comprises 5-20 g / L corn steep liquor powder (including but not limited to 5, 6, 8, 10, 12, 14, 16, 18 or 20 g / L), 17 g / L tryptone, 3 g / L soybean peptone, 2.5 g / L D-glucose, 5.0 g / L NaCl, 2.5 g / L K2HPO4 and 25 g / L agar powder, pH 7.2±0.5; preferably, by weight-volume ratio, the Bennett solid medium comprises 1 g / L yeast extract, 1 g / L beef extract, 2 g / L enzymatically hydrolyzed casein type A (NZ Amine Type A), 10 g / L glucose, 15 g / L agar, pH 7.2-7.4 (including but not limited to 7.2, 7.3 or 7.4).

[0037] In a preferred embodiment, the seed culture medium comprises TSB liquid medium, which, by mass-volume ratio, comprises: 17 g / L tryptone, 3 g / L soybean peptone, 2.5 g / L D-glucose, 5.0 g / L NaCl, 2.5 g / L K2HPO4, and pH 7.2 ± 0.5.

[0038] In a preferred embodiment, after obtaining the culture system, the bacterial cells and / or bacterial solution in the culture system are broken and / or extracted to separate spinosad.

[0039] In the culture system of *Spn016*, both the bacterial cells and the bacterial solution contain spinosad. Therefore, those skilled in the art can flexibly select extraction methods from the prior art, such as extracting the bacterial solution, extracting the bacterial cells, or first breaking the bacterial cells and then extracting them, to obtain spinosad from the bacterial cells and / or bacterial solution through separation methods commonly used in the prior art, such as chromatography and adsorption.

[0040] In a third typical embodiment of this application, a screening method for *Saccharomyces cerevisiae* is provided. The screening method includes: performing PCR amplification on the genome of the candidate strain using primers targeting the spnG gene to obtain a first amplification product; performing PCR amplification on the genome of the candidate strain using primers targeting the spnP gene to obtain a second amplification product; if the first amplification product is 1058 bp and the second amplification product is 1060 bp, the candidate strain is *Saccharomyces cerevisiae*.

[0041] Like other macrolide-synthesizing bacteria, *Saccharomyces cerevisiae* has its biosynthetic genes clustered together, approximately 70–80 kb in length. At the center of this gene sequence are 3–5 highly conserved genes encoding type I polyketide synthase (PKS), a key enzyme in the synthesis of the spinosad lactone ring. In this application, the inventors discovered that when two of these genes, spnG and spnP, are disrupted, the strain cannot transform exogenous aglycones and will not produce spinosad. Therefore, these two genes were selected as specific identification genes, enabling rapid screening of spinosad-producing *Saccharomyces cerevisiae*. Compared to the traditional colony PCR + sequencing method, this saves the sequencing step, shortens the identification time, reduces costs, and improves efficiency.

[0042] In a preferred embodiment, the primers for the spnG gene are the DNA sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3; and the primers for the spnP gene are the DNA sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5.

[0043] SEQ ID NO: 2: ATGCGCGTACTCGTCGTTC.

[0044] SEQ ID NO: 3: AGCACCGTTGCGATTGAGTC.

[0045] SEQ ID NO: 4: CGGCGAGTTCGCACTTCTTC.

[0046] SEQ ID NO: 5: TCCGCCCTACGAGATTCGTC.

[0047] In a preferred embodiment, the *Spinosa polyspora* obtained by the screening method is a *Spinosa polyspora* capable of producing spinosad; spinosad includes spinosad A and spinosad D.

[0048] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0049] Example 1: Obtaining *Spn016*, a type of polyspora sacchariformis.

[0050] 1. Sampling and Separation:

[0051] The dilution method was used to isolate Polysporus spp. from soil. 250 soil samples (7g each) from different regions were weighed and placed in 250mL Erlenmeyer flasks containing 90mL of sterile water. The samples were mixed on a shaker at 28℃ and 200rpm for 30min. The mixtures were then diluted 100, 1000, and 10000 times with sterile water, respectively, and spread onto modified TSB agar plates containing 50μg / mL nystatin, 25μg / mL nalidixic acid, and 10μg / mL potassium dichromate. The plates were incubated at 30℃ for 3–10 days.

[0052] The modified TSB solid culture medium consists of the following components: corn steep liquor powder 10 g / L, tryptone 17 g / L, soybean peptone 3 g / L, D-glucose 2.5 g / L, NaCl 5.0 g / L, K2HPO4 2.5 g / L, agar powder 25 g / L, pH 7.2 ± 0.5. The dispensed solid culture medium is sterilized at 121°C for 20 min. After the temperature drops to about 50°C, antibiotics are added, mixed well, and dispensed into petri dishes.

[0053] 2. Strain purification:

[0054] Using the white colonies that 4.1365 presents on agar plates as a reference, samples with white spores were selected from the final grown colonies for streak separation in three zones to obtain purified single clones. These clones were then activated in TSB liquid medium for preservation and stored at -80°C.

[0055] Example 2: Rapid identification of Polysporum spinosum Spn016:

[0056] The selected white colonies were identified by PCR. The initial spnG and spnP specific gene PCR confirmed them to be *Saccharomyces cerevisiae*. Primer sequences are shown in Table 1 below.

[0057] Table 1 Primer sequences

[0058] The 16S rRNA gene sequence was obtained by a second PCR on colonies that showed bands for both spnG and spnP genes. The universal primer sequences are shown in Table 2 below.

[0059] Table 2 Primer sequences

[0060] After obtaining the 16S rRNA gene sequence of the strain through gene amplification, the nucleotide sequence was obtained by Anshengda Company. The strain information was then obtained through BLAST analysis on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The results showed that the strain Spn016, isolated from soil in Kaifeng City, Henan Province, had the highest homology (99%) with *Saccharopolyspora spinosa* JCM 9375. rDNA sequences with high similarity to this sequence were selected, and a phylogenetic tree was constructed, as shown in Figure 1. Based on these results, Spn016 belongs to *Saccharopolyspora spinosa*.

[0061] The 16S rRNA gene sequence of Spn016 is SEQ ID NO: 1.

[0062] Example 3: Determination of the growth curve of Polysporum spinosum Spn016

[0063] Glycerol tubes of Saccharopolyspora spinosa Spn016 and the purchased standard strain Saccharopolyspora spinosa 4.1365 (purchased from CGMCC) were taken out of the -80℃ freezer and streaked in modified TSB solid medium for enrichment. They were cultured at 30℃ for 4 days and 6 days, respectively. Figure 2 shows that the growth and sporulation capacity of Spn016 at 4 days were significantly better than that of 4.1365 at 6 days.

[0064] One loopful of spores each from Spn016 and 4.1365 plates was scraped and transferred to 250 mL Erlenmeyer flasks containing 25 mL of TSB liquid medium, and cultured at 30 °C and 200 rpm on a shaker. Mycelial growth was observed under a microscope at 5.5 h, 22.5 h, 28 h, 46 h, and 53 h, and wet weight samples were taken to plot growth curves.

[0065] The results are shown in Figures 3 and 4. Spn016 exhibited longer and more extended hyphae at 28 hours, while 4.1365 hyphae were shorter and fewer. Throughout the fermentation process, the growth rate of Spn016 was significantly higher than that of 4.1365, indicating that Spn016 was more adaptable to fermentation culture than the standard strain 4.1365. Figure 3 shows a microscopic comparison (×100x) of the morphology of strain Spn016 after 4 days of plate culture and that of the standard strain 4.1365 after 6 days of plate culture, followed by inoculation into a shake flask 28 hours later.

[0066] Example 4: Fermentation culture and yield determination of Polysporum spinosum Spn016

[0067] Seed culture medium: Scrape one loopful of spores from Spn016 and 4.1365 plates and transfer them to 250 mL Erlenmeyer flasks containing 25 mL of TSB liquid medium. Incubate at 30 °C and 200 rpm for 24 h.

[0068] Fermentation medium: The seed culture was transferred at an inoculum rate of 10% to a 500 mL Erlenmeyer flask containing 50 mL of fermentation medium (50 g / L glucose, 32 g / L cottonseed meal, 8 g / L corn steep liquor, 6 g / L soluble starch and 4 g / L CaCO3, pH 7.2) and fermented at 30 °C and 220 rpm for 5 days.

[0069] Detection of spinosad: 2 mL of sample was added to 4 mL of methanol, shaken thoroughly, centrifuged, and the supernatant was collected for HPLC analysis. Results showed that after 5 days of fermentation, the main products of Spn016 were spinosad A and spinosad D, with a total yield of approximately 22.96 mg / L. Component A accounted for approximately 93.2%, and component D accounted for approximately 6.8%, while the total yield of spinosad A and D in the control 4.1365 was only 11.32 mg / L. The spinosad yield results of strain Spn016 and *Polyspora spp.* 4.1365 are shown in Figure 5.

[0070] From the above description, it can be seen that the embodiments of the present invention achieve the following technical effects: Utilizing the *Spn016* strain obtained through screening, which has a rapid growth rate, can quickly proliferate and produce spores, and can synthesize spinosad A and D, it can be used in the production of spinosad, thereby producing large quantities of spinosad in a short time. Furthermore, due to the rapid growth rate of *Spn016*, the culture time required for proliferation and sporulation is short, making it more suitable as a chassis cell than other *Spn016* strains for use in genetic manipulation experiments, thus shortening the experimental time and improving efficiency. In particular, the success rate of gene manipulation using *Spn016* strains in the prior art is not high, often requiring repeated experiments to successfully obtain the target strain in gene editing and other operations. Therefore, the rapid proliferation characteristic of *Spn016* strain is more important and significant in the genetic manipulation of this strain, and *Spn016* strain has good application prospects in genetic manipulation.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A Saccharopolyspora spinosa, classified as Saccharopolyspora spinosa, deposited in the China General Microbiological Culture Collection Center on April 28, 2024, located in Beijing, China, and assigned the accession number CGMCC No. 30471.

2. Sugonella spnO 16 according to claim 1, characterized in that The Saccharopolyspora spinosa contains a DNA sequence shown in SEQ ID NO:

1.

3. A method of preparing a spinosyn, characterized by, The preparation method comprises: culturing the Saccharopolyspora spinosa of claim 1 or 2 to obtain the polyketide from a culture system; wherein the polyketide comprises polyketide A and polyketide D.

4. The production method according to claim 3, characterized by, The culture method of the Saccharopolyspora spinosa comprises: streaking the Saccharopolyspora spinosa on a solid culture plate to obtain a sporulation plate; inoculating spores of the Saccharopolyspora spinosa on the sporulation plate into a seed culture medium to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium to obtain the culture system.

5. The production method according to claim 4, characterized by, The culture method comprises: streaking the Saccharopolyspora spinosa on the solid culture plate, culturing at 25-35°C for 3-5 days to obtain a sporulation plate; inoculating spores of the Saccharopolyspora spinosa on the sporulation plate into the seed culture medium, culturing at 25-35°C and 100-300 rpm for 12-60 h to obtain the seed liquid; adding the seed liquid into the fermentation culture medium at a volume percentage of 1-20% inoculation amount, culturing at 25-35°C and 100-300 rpm for 3-7 days to obtain the culture system.

6. The production method according to claim 4 or 5, characterized by, The fermentation culture medium comprises, by mass volume ratio, glucose 20-100 g / L, cottonseed cake powder 5-50 g / L, corn syrup powder 2-20 g / L, soluble starch 2-20 g / L, and CaCO31-10 g / L, and has a pH of 7.0-7.

4.

7. The production method according to claim 4 or 5, characterized by, The solid culture plate comprises a modified TSB solid culture medium or a Bennett solid culture medium; The modified TSB solid culture medium comprises, by mass volume ratio, corn syrup powder 5-20 g / L, tryptone 17 g / L, soybean peptone 3 g / L, D-glucose 2.5 g / L, NaCl 5.0 g / L, K2HPO42.5 g / L, and agar powder 25 g / L, and has a pH of 7.2±0.5; The Bennett solid culture medium comprises, by mass volume ratio, yeast extract 1 g / L, beef extract 1 g / L, enzymatic casein A 2 g / L, glucose 10 g / L, and agar powder 15 g / L, and has a pH of 7.2-7.

4.

8. The production method according to claim 4 or 5, characterized by, The seed culture medium comprises a TSB liquid culture medium, The TSB liquid culture medium comprises, by mass volume ratio, tryptone 17 g / L, soybean peptone 3 g / L, D-glucose 2.5 g / L, NaCl 5.0 g / L, K2HPO42.5 g / L, and has a pH of 7.2±0.

5.

9. The production method according to claim 4 or 5, characterized by, After the culture system is obtained, the mycelia and / or the broth in the culture system are disrupted and / or extracted, and the spinosyn is separated and obtained.

10. A method for screening of Actinosynnema pretiosum, characterized by, The screening method comprises the following steps: PCR amplification is performed on the genome of the candidate strain by using primers for the spnG gene to obtain a first amplification product; PCR amplification is performed on the genome of the candidate strain by using primers for the spnP gene to obtain a second amplification product; If the first amplification product is 1058 bp and the second amplification product is 1060 bp, the candidate strain is the Saccharopolyspora spinosa.

11. The screening method according to claim 10, characterized in that, The primers for the spnG gene are DNA sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 3; The primers for the spnP gene are DNA sequences as shown in SEQ ID NO: 4 and SEQ ID NO: 5.

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