A method of increasing production of paenibacillus polymyxa polymyxin

Abstract

The application discloses a method for improving the production of spinosad of Saccharopolyspora spinosa, and belongs to the field of genetic engineering. The method is to overexpress spnF genes and / or spnP genes in the Saccharopolyspora spinosa, and the Saccharopolyspora spinosa genetic engineering bacteria with high yield of spinosad can be obtained by overexpressing the spnF genes and / or spnP genes in the Saccharopolyspora spinosa. It is found that overexpression of the spnF genes and / or spnP genes can significantly improve the production of spinosad of the Saccharopolyspora spinosa.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of genetic engineering, and particularly relates to a method for improving the yield of spinosyn produced by Saccharopolyspora spinosa. BACKGROUND

[0002] Spinosyns are secondary metabolites obtained after aerobic fermentation by Saccharopolyspora spinosa, and are a new type of green broad-spectrum biopesticide, belonging to macrolide antibiotics. Spinosyns exhibit the safety of biological pesticides and the rapidity of chemical pesticides due to their unique insecticidal mechanism, and are harmless to the environment, crops and mammals due to their easy degradability, and have no carcinogenicity, teratogenicity, mutagenicity or neurotoxicity, and thus have won the US President's Green Chemistry Challenge Award three times. At present, spinosyns are still produced by Saccharopolyspora spinosa through aerobic fermentation. Although multiple patents report that the yield of spinosyns can be improved by improving the culture medium or the control method of the fermenter, the fermentation yield of the strain itself is poor, and thus it is difficult to realize large-scale production through fermentation optimization, and therefore the industrialization of this product has not been realized in China. SUMMARY

[0003] The application aims to solve the problem of low yield of spinosyn produced by Saccharopolyspora spinosa through fermentation, and provides a method for improving the yield of spinosyn produced by Saccharopolyspora spinosa.

[0004] The object of the application is achieved by the following technical scheme:

[0005] A method for improving the yield of spinosyn produced by Saccharopolyspora spinosa, which is overexpressing spnF gene and / or spnP gene in Saccharopolyspora spinosa.

[0006] The spnF gene catalyzes the intermolecular cross-linking reaction of spinosyn macrolide.

[0007]

[0008] The spnP gene catalyzes the loading of forosamine in spinosyn.

[0009]

[0010] A genetically engineered Saccharopolyspora spinosa with high yield of spinosyn, which is Saccharopolyspora spinosa overexpressing spnF gene and / or spnP gene.

[0011] In some embodiments, the method for improving the production of spinosad in Saccharopolyspora spinosa is overexpression of spnF gene and / or spnP gene in Saccharopolyspora spinosa WHU1107 strain.

[0012] The Saccharopolyspora spinosa WHU1107 strain mentioned above was deposited in the China Center for Type Culture Collection (CCTCC) on March 31, 2021, and was named Saccharopolyspora spinosa WHU1107, with the accession number CCTCC NO: M 2021307. The Saccharopolyspora spinosa WHU1107 strain produces spinosad at a yield much higher than that of the existing reported strains. Overexpression of spnF gene and / or spnP gene in the WHU1107 strain can further improve the yield of spinosad.

[0013] The genetically engineered Saccharopolyspora spinosa strain with high yield of spinosad is used in the production of spinosad.

[0014] A method for producing spinosad includes the following steps: inoculating the genetically engineered Saccharopolyspora spinosa strain with high yield of spinosad into a fermentation medium to obtain a fermentation product containing spinosad through fermentation.

[0015] In some embodiments, the formula of the fermentation medium is: 8 g of glucose, 2 g of cottonseed meal, 1 g of protein powder, 0.5 g of yeast powder, 0.4 g of trisodium citrate, 0.2 g of dipotassium hydrogen phosphate, 0.3 g of calcium carbonate, 0.2 g of ammonium sulfate, and 5 g of rapeseed oil are added to 100 mL of water, with pH 7.0.

[0016] In some embodiments, the fermentation conditions are: 250 rpm, 28℃, and 60% humidity.

[0017] Advantages and beneficial effects of the present application: The present application finds that overexpression of spnF gene and / or spnP gene can significantly improve the yield of spinosad in Saccharopolyspora spinosa. The present application provides a new modification method and new material for the industrial production of spinosad. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a result graph of LC-MS detection of the components of the fermentation broth of Saccharopolyspora spinosa WHU1107 strain.

[0019] Figure 2 is a result graph of HPLC determination of the content of spinosad in the fermentation broth of Saccharopolyspora spinosa WHU1107 strain.

[0020] Figure 3 is the construction map of pIB139-spnF plasmid. A: pIB139-spnF plasmid schematic diagram; B: pIB139-spnF plasmid enzyme cutting verification analysis map.

[0021] Figure 4 is the construction map of pSET152-spnP plasmid. A: pSET152-spnP plasmid schematic diagram; B: pSET152-spnP plasmid enzyme cutting verification analysis map.

[0022] Figure 5 is the pIB139-spnF-spnP plasmid schematic diagram.

[0023] Figure 6 is the result map of the content of spinosyn in different Saccharopolyspora spinosa fermentation broth. DETAILED DESCRIPTION

[0024] The following examples are used to further illustrate the present application, but should not be construed as limiting the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art.

[0025] The Saccharopolyspora spinosa WHU1107 strain used in the following examples was deposited with the China Center for Type Culture Collection (CCTCC) on March 31, 2021, and was named Saccharopolyspora spinosa WHU1107, with the accession number CCTCC NO: M 2021307. After fermentation culture in a culture bottle containing 25 mL of fermentation medium, the shake flask yield of spinosyn from the strain was as high as 4 g / L or more.

[0026] Example 1 Screening of Saccharopolyspora spinosa WHU1107 strain

[0027] 1. Isolation of spinosyn strain

[0028] Soil samples were collected from multiple regions in July 2017, and actinomycetes were isolated therefrom. The isolation method was as follows: the soil was divided into 0.1 g / mL with sterile water, and then 10 glass beads were added after the soil was suspended with sterile water, and then it was diluted by 10 times step by step, and 10 pieces of separation medium were coated at each concentration. The basic formula of the separation medium was as follows: 5 g of glucose, 3 g of yeast extract, 10 g of enzymatic casein (N-Z amine type A) were added into 1 L of distilled water, and then the mixture was dissolved thoroughly, and the pH was adjusted to 7.0 with NaOH; 200 mL was divided, and 4 g of agar was added to each bottle; sterilization was performed at 115 DEG C for 30 min. After sterilization, it was cooled to about 65 DEG C, and naphthyldin was added to a final concentration of 25 μg / mL to inhibit the growth of gram-negative bacteria, and nystatin was added to a final concentration of 50 μg / mL to inhibit the growth of fungi. Among the colonies that could grow finally, the colonies with the morphology of streptomycin samples producing spores were selected for preservation, and a total of 78 strains were collected.

[0029] Each 5 strains were mixed for fermentation, and the fermentation method was as follows:

[0030] (1) Related medium

[0031] Seed medium: 1 g of glucose, 1 g of yeast extract, 0.2 g of N-Zamine type A, 2.5 g of cottonseed meal, 2 g of corn starch, 0.2 g of magnesium sulfate heptahydrate and 0.1 g of ammonium sulfate were added into 100 mL of distilled water, and then the mixture was dissolved thoroughly, and the pH was adjusted to 7.0 with NaOH; 25 mL of seed medium was divided in each bottle for seed culture; sterilization was performed at 121 DEG C for 30 min.

[0032] Fermentation medium: 8 g of glucose, 2 g of cottonseed meal, 1 g of protein powder (Hai Dong brand), 0.5 g of yeast powder, 0.4 g of trisodium citrate, 0.2 g of dipotassium hydrogen phosphate, 0.3 g of calcium carbonate, 0.2 g of ammonium sulfate and 5 g of rapeseed oil were added into 100 mL of distilled water, and then the mixture was dissolved thoroughly, and the pH was adjusted to 7.0 with NaOH; sterilization was performed at 121 DEG C for 30 min; 25 mL of fermentation medium was divided in each bottle for fermentation culture; note: calcium carbonate was divided into each bottle.

[0033] (2) Fermentation method

[0034] Each isolated strain was picked and inoculated on a new plate for growth. When the growth was suitable, a 1 cm*1 cm piece of the colony was inoculated in the seed culture medium. Five strains were inoculated in one culture bottle (a total of 16 bottles). The culture was incubated at 250 rpm, 28°C, and 60% humidity for 96 hours. The first-stage seed was observed by microscopy to determine whether it was contaminated and the growth state. If the seed was not contaminated and the growth state was good, the second-stage seed was inoculated at a ratio of 1%. The culture conditions of the second-stage seed were the same as those of the first-stage seed. The second-stage seed was observed by microscopy at 60 hours, and the seed was observed to determine whether it was contaminated. If the seed was normal, the fermentation was inoculated at a ratio of 5% in a culture bottle containing fermentation medium. Each bottle of bacteria was inoculated in triplicate. The fermentation culture conditions were as follows: 250 rpm, 28°C, and 60% humidity for 12 days.

[0035] The detection method in the reference (Gao-Yi, Tan, Kunhua, et al. Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces [J]. Acs Synth Biol, 2017.) was used to analyze whether the fermentation broth contained spinosad. It was found that spinosad was produced in the first and seventh bottles. The 10 strains in the first and seventh bottles were fermented one by one according to the above method. Finally, it was found that 3 strains could produce spinosad, with yields of 51.2 mg / L, 253 mg / L, and 336 mg / L, respectively, which were named WHU1100, WHU1101, and WHU1102.

[0036] 2. Strain mutagenesis

[0037] The slant of the WHU1102 strain was prepared into a spore suspension with normal saline. After filtration with absorbent cotton, the spore concentration was adjusted to 10 -6 ~10 -7 mL of single spore suspension was placed in a sterile 9 cm diameter plate, and irradiated at a distance of 20 cm from a 15 W ultraviolet lamp for 30 s. Then, it was diluted by 10 times in stages and plated.

[0038] Every 20 strains were divided into a group, and fermented and screened according to the above method. A total of about 3000 strains were screened. The highest-yielding group of 20 strains was selected as the preliminary screening strain. The 20 strains were mixed and subjected to a second round of mutagenesis according to the above method. Similarly, about 3000 strains were screened, and the highest-yielding group of 20 strains was selected as the screening strain (named WHU1103-WHU1122, respectively).

[0039] The 20 strains were fermented one by one according to the above method, and the highest yield strain was WHU1107. The detection method in the reference (Gao-Yi, Tan, Kunhua, et al. Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces [J]. Acs Synth Biol, 2017.) was used to detect the components of the fermentation product by LC-MS. The results ( Figure 1 ) showed that spinosyn A and spinosyn D could be detected in the fermentation broth; then HPLC ( Figure 2 ) analysis showed that the total yield of spinosyn A and spinosyn D was about 4.1 g / L, of which the A component accounted for about 95%, and the D component accounted for about 5%.

[0040] 3. Identification of strain WHU1107

[0041] Strain WHU1107 was identified. The 16s rRNA sequence of strain WHU1107 is shown as SEQ ID NO. 1, which has the highest sequence similarity with GENBANK ACCESSION NR_024839.1, with a similarity of 99%. According to the above results, strain WHU1107 belongs to Saccharopolyspora spinosa.

[0042] 4. Preservation of strain WHU1107

[0043] Strain WHU1107 belongs to Saccharopolyspora spinosa, and was preserved in China Center for Type Culture Collection (CCTCC) on March 31, 2021, with the preservation number of CCTCC NO: M 2021307.

[0044] 5. Comparison of the yield of strain WHU1107 and other Saccharopolyspora spinosa strains

[0045] The yield of the multi-killing high-yield strain obtained by mutagenesis and other methods reported in previous documents or patents is about 1-2 g / L, which is far lower than the yield of 4 g / L of the WHU1107 strain of the present application. The wild-type strain of S. cichorii NRRL18395 commercially available on the market was detected for the production of multi-killing bacteria by the fermentation method described above, and the yield was 78.7 mg / L, of which the A component accounted for about 84%, and the D component accounted for about 16%, which was also far lower than the yield of 4 g / L of the WHU1107 strain. These results show that the yield of WHU1107 as high as 4 g / L is the performance of its own high-yield multi-killing bacteria, and it can be developed as an industrial strain for high-yield multi-killing bacteria.

[0046] Example 2 Construction of genetically engineered S. cichorii strain overexpressing spnF gene and / or spnP gene

[0047] (1) Construction of plasmids pIB139-spnF, pSET152-spnP and pIB139-spnF-spnP

[0048] In this embodiment, attB-attP sites are used for integration, and the promoter ermEp is selected to overexpress spnF, and the original promoter PspnP is selected to overexpress spnP.

[0049] 1) Construction of plasmid pIB139-spnF

[0050] The extracted genomic DNA of S. cichorii WHU1107 was used as a PCR template, and the cloned fragment spnF primer (upstream primer: 5'-gtgccggttggtaggatccacatatggtgttgccaggtggcgcaccaac-3', downstream primer: 5'-tatgacatgattacgaattcgatatctcagccgaccggcttccgcgccgtc-3') was used to amplify the target band spnF of 880 bp in size, and the target band was subjected to Gibson assembly with the pIB139 vector digested with NdeI / EcoRV to obtain a vector overexpressing spnF with the strong promoter ermEp. The assembly product was transformed into E. coli DH10B competent cells, and after random picking of E. coli monoclonal culture and extraction of plasmid, the plasmid was identified by double digestion with NcoI and EcoRI (B), and two target fragments of 4462 bp and 2256 bp in size were obtained, and the plasmid correctly identified by digestion was selected. Subsequently, a first-generation sequencing method was used for verification, and the plasmid verified successfully was pIB139-spnF (A). Figure 3 Figure 3 A).

[0051] 2) Construction of plasmid pSET152-spnP

[0052] ​The genomic DNA of extracted Saccharopolyspora spinosa WHU1107 was used as a PCR template, and the cloning fragment Pspnp-spnP primer (upstream primer: 5'-gactctagagcaggcgacgatcagtcttcgcgc-3', downstream primer: 5'-cgcgcggccgctcacggatggccatcagactgcccag-3') was used to amplify the target fragment Pspnp-spnP with a size of 1719 bp. The target band was recovered together with the pSET152 vector digested with XbaI and NotI, and then T4 ligase was used for enzyme ligation to obtain the vector overexpressing spnP with the original promoter PspnP of spnP. The vector was digested with EcoRV for identification Figure 4 B) Two target fragments with sizes of 6012 bp and 1396 bp were obtained, and the plasmid pSET152-spnP was successfully constructed. Subsequently, a first-generation sequencing method was used for verification, and the verified plasmid was pIB139-spnP Figure 4 A).

[0053] 3) Construction of plasmid pIB139-spnF-spnP

[0054] The genomic DNA of extracted Saccharopolyspora spinosa WHU1107 was used as a PCR template, and the cloning fragment spnF primer (upstream primer: 5'-gtgccggttggtaggatccacatatggtgttgccaggtggcgcaccaac-3', downstream primer: 5'-cgtcgcctgctcagccgaccggcttccgcgccgtc-3') was used to amplify the target band spnF with a size of 864 bp. The cloning fragment Pspnp-spnP primer (upstream primer: 5'-ggtcggctgagcaggcgacgatcagtcttc-3', downstream primer: 5'-aacagctatgacatgattacgaattctcacggatggccatcagactg-3') was used to amplify the target band spnP with a size of 1735 bp. The target bands spnF and Pspnp-spnP were Gibson assembled with the pIB139 vector digested with NdeI / EcoRI, and the Gibson assembly product was transformed into E. coli DH10B competent cells. After random picking of E. coli monoclonal culture and extraction of plasmids, one of the plasmids with correct enzyme digestion was sequenced, and the plasmid pIB139-spnF-spnP was successfully constructed Figure 5 ).

[0055] (2) Screening of spnF and spnP overexpression strains

[0056] The constructed plasmid and pSET152 were transformed into competent E. cooli ET12567 / pUZ8002, respectively. Single clones were selected and expanded in LB medium as donor bacteria for conjugative transfer. Inter-generic conjugative transfer between the two parents was performed with Saccharopolyspora spinosa WHU1107. The selected conjugates were cultured in ABB13 (5 g / L soluble starch, 5 g / L soy peptone, 2.1 g / L MOPS, 3 g / L CaCO3, 0.01 g / L thiamine hydrochloride, 0.046 g / L After expansion culture on (FeSO4·7H2O, 20 g / L agar) plates, some colonies of each conjugate were picked and activated in TSB-M liquid medium (30 g / L tryptic soy broth, 50 g / L mannitol) for 3 days. Genomic DNA was extracted as a template and the upstream primer: 5'-cagcggtggagtgcaatgtcgt-3' and the downstream primer: 5'-cagaggcgggatgcgaagaatg-3' were used to amplify the apramycin resistance gene of 750 bp. The strains were the correct strains overexpressing spnF or spnP, or spnF and spnP. They were named WHU1107 / pIB139-spnF, WHU1107 / pSET152-spnP, and WHU1107 / pIB139-spnF-spnP, respectively. The WHU1107 / pSET152 strain was used as the control group.

[0057] (3) Fermentation of strains overexpressing spnF and spnP

[0058] The strains overexpressing spnF and spnP were fermented and tested according to the method in Example 1. Figure 6 ) showed that in a culture flask filled with 25 mL of fermentation medium, overexpression of the spnF gene using the strong ermEp promoter resulted in a spinosad yield of 4410 mg / L, a 7.4% increase compared to the control strain WHU1107 / pSET1524, which produced 105 mg / L of spinosad. Overexpression of the spnP gene using the native spnP promoter resulted in a spinosad yield of 4499 mg / L, a 9.6% increase. Simultaneous overexpression of both the spnF and spnP genes resulted in a spinosad yield of 4725 mg / L, a 15.1% increase.

[0059] SpnF and spnP both belong to the modification genes after the synthesis of the polyketide skeleton in the spinosyn biosynthesis. In the spinosyn biosynthesis genes, in addition to spnF and spnP, there are also seven genes belonging to the modification genes after the synthesis of the polyketide skeleton, including spnJ, spnM, spnG, spnL, spnI, spnK and spnH. In the literature (Gao-Yi, Tan, Kunhua, et al. Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces [J]. Acs Synth Biol, 2017.), it is proved that overexpression of spnI in the heterologous expression of spinosyn in Streptomyces albus can improve the yield of spinosyn. Therefore, we tried to construct plasmids for overexpressing spnI, spnH and spnJ using ermEp as a vector in WHU1107 according to the above method, and transferred them into WHU1107 according to the above conjugation method. The detection showed that the yield of spinosyn in the strain overexpressing spnI was 2317 mg / L, the yield of spinosyn in the strain overexpressing spnJ was 2259 mg / L, and the yield of spinosyn in the strain overexpressing spnH was 2301 mg / L. The yields of the three strains were lower than that of the original strain. These results show that on the basis of the prior art, it cannot be reasonably predicted that overexpression of spinosyn biosynthesis-related genes is related to improving the yield of spinosyn in Saccharopolyspora spinosa.

[0060] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and shall be included in the protection scope of the present application. SEQUENCE LIST INFORMATION: DTD Version: V1_3 File Name: spinosyns.xml Software Name: WIPO Sequence Software Version: 2.1.1 Creation Date: 2023-01-28 Basic Information: Current Application / Applicant File Name: Wuhan University Applicant Name or Designation: Wuhan University Applicant Name or Designation / Language: zh Applicant Name or Designation / Latin Name: Wuhan University Inventor Name: Liu Tianqiang Inventor Name / Language: zh Inventor Name / Latin Name: liutiangang Invention Title: A method for improving the production of spinosyn by saccharopolyspora spinosa (zh) Total Sequence Amount: 11 Sequence: Sequence Number (ID): 1 Length: 1518 Molecule Type: DNA Feature Position / Qualifier: - source, 1..1518 > mol_type, genomic DNA > organism, Saccharopolyspora spinosa Residues: aaaggaggtg atccagccgc accttccggt acggctacct tgttacgacttcgtcccaat 60 cgccagtccc accttcgacc actcccccca caagggttgg gccatgggcttcgggtgtta 120 ccgactttca tgacgtgacg ggcggtgtgt aaggcccg ggaacgtattcaccgcagca 180 atgctgatct gcgattacta gcgactccga cttcacgagg tcgagttgcagaccccgatc 240 cgaactgaga ccggctttaa gggattcgct ccacctcacg atatcgccaccctctgtacc 300 agccattgta gcatgtgtga agccctgggc ataaggggca tgatgacttgacgtcatccc 360 caccttcctc cgagttgacc ccggcagtcc cccacgagtc cccggcataacccgctggca 420 acatagggca agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg 480 acgacagcca tgcaccacct gtacaccaac cacaagggaa accccatctctggagctgtc 540 tagtgcatgt caaacccagg taaggttctt cgcgttgcat cgaattaatccacatgctcc 600 gccgcttgtg cgggcccccg tcaattcctt tgagttttag ccttgcggccgtactcccca 660 ggcggggcgc ttaatgcgtt agctacggca cggaaacagt ggaacccatccccacaccta 720 gcgcccaacg tttacggcgt ggactaccag ggtatctaat cctgttcgctccccacgctt 780 tcgctcctca gcgtcagtat cggcccagag acccgccttc gccaccggtgttcctcctga 840 tatctgcgca tttcaccgct acaccaggaa ttccagtctc ccctaccgaactcaagtctg 900 cccgtatcga ccgcaagccc acagttaagc tgcaggtttt cacggccgacgcgacaaacc 960 gcctacgagc tctttacgcc caataaatcc ggacaacgct cgcacctacgtagtaccgcg 1020 gctgctggca cgtagttagc cggtgcttct tctacaccta ccgtcacccgaaggcttcgt 1080 cgatgtcgaa agaggtttac aacccgaagg ccgtcatccc ccacgcggcgttgctgcgtc 1140 aggctttcgc ccattgcgca agattcccca ctgctgcctc ccgtaggagt ctgggccgtg 1200 tctcagtccc agtgtggccg gtcaccctct caggccggct acccgtcgtc gccttggtag 1260 gccatcaccc caccaacaag ctgataggcc gcggactcat cctgcaccgccagaactttc 1320 cacacaccac catgcgataa tgtgtcatat ccggtattag accccgtttccaaggcttat 1380 cccagagtgc agggcagatt acccacgtgt tactcacccg ttcgccactcatccacaccc 1440 gaagatgctt cagcgttcga cttgcatgtg ttaagcacgc cgccagcgttcgtcctgagc 1500 caggatcaaa ctctccaa 1518 Sequence ID (ID): 2 Length: 49 Molecule Type: DNA Feature Location / Qualifier: - source, 1..49 > mol_type, other DNA > organism, synthetic construct Residues: gtgccggttg gtaggatcca catatggtgt tgccaggtgg cgcaccaac 49 Sequence ID (ID): 3 Length: 51 Molecule Type: DNA Feature Location / Qualifier: - source, 1..51 > mol_type, other DNA > organism, synthetic construct Residue: tatgacatga ttacgaattc gatatctcag ccgaccggct tccgcgccgt c 51 Sequence ID (ID): 4 Length: 33 Molecule type: DNA Feature Location / Qualifier: - source, 1..33 > mol_type, other DNA > organism, synthetic construct Residue: gactctagag caggcgacga tcagtcttcg cgc 33 Sequence ID (ID): 5 Length: 37 Molecule type: DNA Feature Location / Qualifier: - source, 1..37 > mol_type, other DNA > organism, synthetic construct Residue: cgcgcggccg ctcacggatg gccatcagac tgcccag 37 Sequence ID (ID): 6 Length: 49 Molecule type: DNA Feature Location / Qualifier: - source, 1..49 > mol_type, other DNA > organism, synthetic construct Residue: gtgccggttg gtaggatcca catatggtgt tgccaggtgg cgcaccaac 49 Sequence ID (ID): 7 Length: 35 Molecule type: DNA Feature Location / Qualifier: - source, 1..35 > mol_type, other DNA > organism, synthetic construct Residues: cgtcgcctgc tcagccgacc ggcttccgcg ccgtc 35 Sequence ID (ID): 8 Length: 30 Molecule type: DNA Feature Location / Qualifier: - source, 1..30 > mol_type, other DNA > organism, synthetic construct Residues: ggtcggctga gcaggcgacg atcagtcttc 30 Sequence ID (ID): 9 Length: 47 Molecule type: DNA Feature Location / Qualifier: - source, 1..47 > mol_type, other DNA > organism, synthetic construct Residues: aacagctatg acatgattac gaattctcac ggatggccat cagactg 47 Sequence ID (ID): 10 Length: 22 Molecule type: DNA Feature Location / Qualifier: - source, 1..22 > mol_type, other DNA > organism, synthetic construct Residues: cagcggtgga gtgcaatgtc gt 22 Sequence ID (ID): 11 Length: 22 Molecule type: DNA Feature Position / Qualifier: - source, 1..22 > mol_type, other DNA > organism, synthetic construct Residues: cagaggcggg atgcgaagaa tg 22 END

Claims

1. A method for increasing the yield of spinosad from Saccharopolyspora spinosa, characterized by: To overexpress in Saccharopolyspora spinosa spf Genes and / or spnP Gene; The spiny Saccharopolyspora is a spiny Saccharopolyspora with a deposit number of CCTCC NO: M 2021307 ( Saccharopolyspora spinosa )WHU1107.

2. A genetically engineered strain of Saccharopolyspora spinosa that produces high levels of spinosad, characterized by: For overexpression spf Genes and / or spnP Gene of Saccharopolyspora spinosa; The high-yield spinosad genetic engineering bacteria is overexpressed in the spinosad Saccharopolyspora WHU1107 with a deposit number of CCTCC NO: M2021307. spf Genes and / or spnP Gene acquisition.

3. Use of the genetically engineered Saccharopolyspora spinosa according to claim 2 in the production of spinosad.

4. A method for producing spinosad, characterized in that: The following steps are involved: The genetically engineered Saccharopolyspora spinosa described in claim 2 is inoculated into a fermentation medium, and a fermentation product containing spinosad is obtained by fermentation.

5. The method for producing spinosad according to claim 4, wherein: The fermentation medium is formulated as follows: 8 g of glucose, 2 g of cottonseed meal, 1 g of protein powder, 0.5 g of yeast powder, 0.4 g of trisodium citrate, 0.2 g of dipotassium hydrogen phosphate, 0.3 g of calcium carbonate, 0.2 g of ammonium sulfate, and 5 g of rapeseed oil are added to every 100 mL of water, with a pH of 7.

0.

6. The method for producing spinosad according to claim 4, wherein: The fermentation conditions are: 250 rpm, 28°C, and 60% humidity.

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