Saccharopolyspora high-yield engineering strain for producing polyoxin j / l and construction method and application thereof

Abstract

The application discloses an engineering strain of Saccharopolyspora spinosa for high yield of spinosyn J / L, a construction method and application thereof. The engineering strain is obtained by precisely knocking out a conservative functional fragment in a rhamnose 3'-O-methyltransferase activity center of a spnK gene from a Saccharopolyspora spinosa HU14590 as a starting strain. The strain has been preserved in the China Center for Type Culture Collection (CCTCC) on May 13, 2026, and the preservation number is CCTCC M 2026943. The application significantly improves the yield of spinosyn J / L by using a spnK partial gene knockout silencing strategy, and the yield of spinosyn J / L is 20-30 times that of the wild type Saccharopolyspora spinosa. The engineering strain has strong genetic stability and still maintains the high yield characteristics after 10 generations. The engineering strain can be used for industrialized fermentation production of spinosyn J / L, and provides a precursor for synthesis of ethyl spinosyn, and has important application value.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and microbial fermentation, specifically to a genetically engineered strain of *Saccharomyces cerevisiae* that produces high levels of spinosad J / L, as well as the construction method and application of this engineered strain. Background Technology

[0002] Spinosad is a macrolide biopesticide produced by fermentation of *Saccharopolyspora spinosa*. It possesses advantages such as high efficiency, low toxicity, easy degradation, and safety for non-target organisms, making it widely used in agricultural green pest control and pet parasite control. Spinosad is a mixture, with spinosad A and D as the main components and spinosad J and L as minor components. Spinosad J / L is a key precursor for the synthesis of ethyl spinosad (a second-generation spinosad insecticide). Ethyl spinosad has a broader insecticidal spectrum and higher biological activity, resulting in strong market demand. The structures of spinosad compounds are as follows:

[0003]

[0004]

[0005] Currently, the core problem restricting the industrial production of spinosad J / L is the extremely low J / L yield of wild-type or conventional mutant *Saccharomyces cerevisiae*, which cannot meet the industrial demand for ethyl spinosad synthesis. In existing technologies, the main method to increase spinosad J / L yield is to silence the spnK gene by knocking out a portion of it, thus blocking the synthesis pathway of spinosad A / D and directing metabolic flow towards J / L accumulation.

[0006] However, existing technologies have significant drawbacks: while the strategy of silencing the spnK gene by knocking out a portion of it can improve J / L yield to some extent, the improvement is limited (only 8-9 times that of the wild type), and still falls short of industrialization requirements. Therefore, optimizing the spnK gene knockout scheme to further improve spinosad J / L yield has become a pressing technical challenge in this field. Summary of the Invention

[0007] The purpose of this invention is to provide an engineered strain of *Saccharomyces cerevisiae* that produces high levels of spinosad (J / L), its construction method, and its application.

[0008] To achieve the above and other related objectives, the technical solution provided by this invention is: a high-yield polysaccharide-producing engineered strain of *Polysporium spinosum*, wherein the engineered strain is *Polysporium spinosum* HU01785, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026943 and deposit date of May 13, 2026.

[0009] To achieve the above and other related objectives, the technical solution provided by this invention is: a method for constructing a high-yield polysaccharide-producing engineered strain of *Polysporium spp.*, wherein the engineered strain is constructed using *Polysporium spp.* HU14590 as the starting strain, by knocking out part of the spnK gene to silence the spnK gene, and is named *Polysporium spp.* HU01785. It has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026943 and deposit date of May 13, 2026.

[0010] The preferred technical solution includes the following steps:

[0011] Step 1: Using the spnK gene as the target gene, a conserved 41 amino acid sequence was selected within the open reading frame. The upper and lower homologous arms were amplified using primer annealing. These were then sequentially ligated into the *E. coli*-*Streptomyces* shuttle vector 3-57 to construct the recombinant knockout plasmid 3-57-ΔspnK, which inhibits spnK gene expression in *Polyspora sacchariformis* HU14590. The conserved nucleotide sequence is as follows:

[0012] The nucleotide sequence encoding the conserved 41 amino acid sequence is as follows:

[0013] TACGGACCGT TCGACATCAT CATCGACGAT GGCAGCCACA TCAACGGACA CGTGCGCACATCCCTGGAAA CGCTGTTCCC CCGGTTGCGC AGCGGTGGCG TATACGTGAT CGAGGATCTG TGG.

[0014] Step 2: Vector conjugation and transfer: The plasmid 3-57-ΔspnK constructed in Step 1 was transformed into Escherichia coli S17 to obtain recombinant Escherichia coli; the recombinant Escherichia coli was conjugated and transferred with Polysporus spp. HU14590, plated on LB medium containing apramycin, and cultured for 12-14 days to obtain conjugates;

[0015] Step 3: Screening and verification of engineered bacteria: PCR verification was performed on the conjugates to confirm partial gene knockout of spnK. The strain with the highest spinosad J / L yield was screened by shake-flask fermentation. This strain was further passaged to increase spinosad yield, which is the target engineered bacteria, Polyspora spinosad HU01785.

[0016] The preferred technical solution is that, in step 2, the specific conditions for conjugation transfer are as follows: the recombinant Escherichia coli and the mycelial suspension of Polysporus spp. HU14590 are mixed at a volume ratio of 1:2-5, spread on LB medium, and cultured at 25-32℃ for 10-30 hours. Then, the antibiotic Apramycin is added, and the culture is continued for 7-14 days.

[0017] To achieve the above and other related objectives, the technical solution provided by the present invention is: the application of the engineered strain of Polysaccharidium erythropoietin with high spinosad J / L production in the production of spinosad J / L.

[0018] The preferred technical solution includes the following steps: inoculating Polysporus spp. HU01785 onto a slant culture medium and culturing at 25-32℃ for 10-14 days; transferring to a seed culture medium and culturing at 25-32℃ and 200-300rpm for 60-80 hours; and inoculating into a fermentation culture medium at an inoculation rate of 8-12% and culturing at 25-32℃ and 200-300rpm for 10-30 days.

[0019] The preferred technical solution is that the fermentation medium per liter consists of: 60-100g glucose, 30-35g cottonseed meal powder, 5-7g soybean meal powder, 5-8g corn steep liquor, 8-12g corn starch, 1.5-2.5g yeast extract, 3-6g CaCO3, 38-45g methyl oleate, and pH 6.8-7.2.

[0020] Due to the application of the above technical solution, the advantages of this invention compared with the prior art are:

[0021] 1. Using Polysporus spp. HU14590 as the starting strain, the spnK gene was silenced by knocking out part of the spnK gene, which blocked the synthesis of spinosad A / D and made the metabolic flux preferentially flow to J / L. At the same time, the length of the knockout fragment and the design of homologous arms were optimized to further improve the J / L yield.

[0022] 2. Significantly increased yield: This invention optimizes the spnK partial gene knockout scheme, achieving a spinosad J / L yield of 3000~4000 mg / L, which is 20~30 times that of wild-type Polysporus spinosae, and more than 20% higher than existing single knockout strategies.

[0023] 3. Strong genetic stability: After 10 consecutive generations, the J / L yield of the engineered bacteria does not fluctuate by more than 5%, meeting the stability requirements for industrial production.

[0024] 4. Significant application value: It provides sufficient precursors for the synthesis of ethyl spinosad, reduces the production cost of ethyl spinosad, and promotes the upgrading of the green biological pesticide industry. Attached Figure Description

[0025] Figure 1 Construction map of vector 3-57-PermE.

[0026] Figure 2 : Schematic diagram of spnK knockout vector construction.

[0027] Figure 3 : spnK knockout vector construction related band map, M: DNA marker (Takara DL5000).

[0028] Figure 4 Schematic diagram of the construction of the knockout strain Polyspora hygroscopica HU01785.

[0029] Figure 5 PCR verification map of related sequences of the knockout strain Polyspora hygrostis HU01785.

[0030] Figure 6 The genome sequencing of the knockout strain *Polyspora sacchariformis* HU01785 and the original strain HU14590 were compared and verified.

[0031] Figure 7 HPLC chromatogram of spinosad A / D produced by fermentation of the originating bacterium *Polyspora spp.* HU14590.

[0032] Figure 8 HPLC analysis chromatogram of spinosad J / L produced by fermentation of engineered strain Polysaccharidobacterium spinosum HU01785. Detailed Implementation

[0033] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0034] Please see Figure 1-8 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes and to aid those skilled in the art in understanding and reading the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0035] Preservation of the strain: Saccharopolyspora spinosa HU01785 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026943 and deposit date of May 13, 2026; deposit address: Wuhan University, Wuhan, China.

[0036] Unless otherwise specified, all reagents or materials described in the following examples are commercially available.

[0037] Example 1: Construction of a high-yield spinosad J / L strain

[0038] 1. Obtaining the starting strain

[0039] The starting strain was Polysacchariformis HU14590.

[0040] 2. PCR amplification to obtain the upper and lower homologous arms of spnK

[0041] Primer design: Gene ID: IAG28_RS01785 coding region DNA sequence, two pairs of primers were designed for amplification of it respectively.

[0042] The primer sequences are as follows:

[0043] Primer sequences used to amplify a 2.020kb spnK homologous arm fragment:

[0044] Primer 1 (fragment 1. forward primer, as shown in the sequence listing SED ID NO.1):

[0045] 5´- ttggatccgatatcagatctgccctcgggcaggtgtaccg-3´;

[0046] Primer 2 (fragment 1. reverse primer, as shown in the sequence listing SED ID NO.2): 5´-aatccgggagcataggtcgtcttgtcgtcaacggcggccagc-3´;

[0047] Primer sequences used to amplify a 2.051kb spnK homologous arm fragment:

[0048] Primer 3 (fragment 2. forward primer, as shown in sequence listing SED ID NO.3):

[0049] 5´-tggccgccgttgacgacaagacgacctatgctcccggattc-3´;

[0050] Primer 4 (fragment 2. Reverse primer, as shown in sequence listing SED ID NO.4):

[0051] 5´-gaccatggggtacccatatgaagtgtcgaggaagtcgctc-3´;

[0052] 2. Amplify the upper and lower homologous arms of the spnK gene separately.

[0053] Amplification of homologous arms on spnK: Using *Polysporium spp.* genomic DNA as a template, primers Primer 1 and Primer 2 were used. PCR amplification was performed using HS DNA polymerase (TaKaRa). The amplification conditions were: 94℃ for 5 minutes; then 98℃ for 10 seconds, annealing at 63℃ for 20 seconds, 72℃ for 2 minutes, for a total of 29 cycles; finally, 72℃ for 5 minutes. After the reaction, the PCR products were detected by 1% agarose gel electrophoresis. The expected specific bands appeared at the 2kb marker size, as shown below. Figure 3 The target band A was gel-excised and recovered for subsequent vector construction. The sequences of the homologous arm gene fragments on spnK obtained are as follows.

[0054] GCCCTCGGGC AGGTGTACCG AACCCTGTTC AAGAACTGGG GCGTGACGAT CCCGACCGGCGCATGTCCCG GGGTGGGCGC AGGCGGGCAC ATCCTCGGCG GGGGATACGG CCCGCTGTCG CGCCGATTCGGTTCGGTCGT CGACTACCTT CAAGGCGTCG AGGTCGTCGT GGTCGACCAG GCCGGTGAAG TGCACATCGTCGAGGCCGAC CGGAACTCCA CGGGCGCCGG TCACGACTTG TGGTGGGCGC ACACCGGTGG CGGTGGCGGCAACTTCGGGA TCGTCACCAG GTTTTGGCTC CGAACGCCGG ACGTGGTCAG CACCGACGCC GCAGAGCTCCTGCCACGGCC GCCCGCGACA GTGCTGCTCC GATCGTTCCA CTGGCCGTGG CACGAACTGA CAGAGCAGTCATTCGCCGTG CTCCTACAGA ACTTCGGCAA TTGGTACGAG CAGCACAGCG CGCCTGAATC CACGCAACTCGGGTTGTTCA GCACGCTCGT CTGCGCACAC CGGCAAGCTG GCTACGTCAC GCTGAACGTT CACCTGGACGGCACGGATCC GAACGCGGAA CGCACCCTGG CCGAACACCT GTCGGCGATC AACGCCCAGG TCGGCGTGACTCCAGCCGAA GGGCTGCGGG AAACCCTGCC GTGGTTGCGA TCGACCCAGG TGGCCGGGGC GATCGCCGAAGGCGGCGAAC CGGGCATGCA ACGGACCAAG GTCAAAGCCG CCTACTTGCG CACCGGGCTG TCCGAAGCTCAACTAGCCAC GGTTTACCGG CGGCTGACCG TCTACGGATA CGACAACCCT GCGGCGGCGC TGTTGCTGCTCGGTTACGGC GGTATGGCGA ATGCCGTGGC TCCGTCGGCC ACCGCACTCG CTCAGCGCGA CTCGGTTCTCAAAGCGCTGT TCGTCACGAACTGGTCGGAG CCCGCCGAGG ACGAGCGGCA TCTGACCTGG ATTCGCGGTTTCTACCGCGA GATGTACGCC GAAACCGGCG GAGTTCCGGT GCCAGGTACC CGTGTCGACG GCTCCTACATCAACTACCCG GACACCGACT TGGCCGATCC ATTGTGGAAC ACCTCCGGTG TTGCCTGGCA CGACCTGTACTACAAAGACA ACTACCCGCG GCTGCAGCGG GCCAAAGCGC GGTGGGATCC GCAGAACATC TTCCAGCACGGCCTGTCGAT CAAACCGCCG GCACGGCTTT CACCCGGTCA GCCATGAGGA GCTCATCACG ATGTCCACAACGCACGAGAT CGAAACCGTG GAACGCATCA TCCTCGCCGC CGGATCCAGT GCGGCGAGCC TGGCCGACCTGACCACCGAA CTCGGACTCG CCAGGATCGC ACCCGTGCTG ATCGACGAGA TCCTCTTCCG CGCGGAACCGGCCCCCGACA TCGAACGGAC CGAGGTCGCG GTCCAGATCA CCCACCGAGG CGAGACCGTT GACTTCGTCCTGACGCTACA GTCCGGTGAG CTGATCAAGG CCGAGCAACG ACCGGTCGGA GACGTCCCGC TGCGGATCGGTTACGAGCTC ACCGATCTCA TCGCCGAGTT GTTCGGCCCA GGAGCTCCCA GGGCCGTCGG CGCCCGGAGCACCAACTTCC TCCGAACCAC CACATCCGGT TCGATACCCG GTCCGTCGGA ACTGTCCGAT GGCTTCCAGGCCATCTCCGC AGTGGTCGCC GGCTGCGGGC ACCGACGTCC CGACCTCAAC TTGCTCGCCT CCCACTACCGCACGGACAAG TGGGGCGGCC TGCACTGGTT CACCCCGCTA TACGAGCGAC ACCTCGGCGA GTTCCGTGATCGCCCGGTGC GCATCCTGGA GATCGGTGTCGGTGGCTACA ACTTCGACGG TGGCGGCGGC GAATCCCTGAAGATGTGGAA GCGCTACTTC CACCGCGGCC TCGTGTTCGG GATGGACGTT TTCGACAAGT CCTTCCTCGACCAGCAGAGG CTCTGCACCG TCCGCGCCGA CCAGAGCAAG CCCGAGGAGC TGGCCGCCGT TGACGACAAG.

[0055] Amplification of homologous arms under spnK: Using *Saccharomyces cerevisiae* genomic DNA as a template, primers Primer 3 and Primer 4 were used. PCR amplification was performed using HS DNA polymerase (TaKaRa). The amplification conditions were: 94℃ for 5 minutes; then 98℃ for 10 seconds, annealing at 60℃ for 20 seconds, 72℃ for 2 minutes, for a total of 29 cycles; finally, 72℃ for 5 minutes. After the reaction, the PCR products were detected by 1% agarose gel electrophoresis, and the expected specific bands appeared at the marker size of 2kb, as shown below. Figure 3 The target band A was gel-excised and recovered for subsequent vector construction. The sequences of the homologous arm gene fragments obtained under spnK are as follows.

[0056] ACGACCTATG CTCCCGGATT CGGCGGGCAG GCGCAGTGCC CGGCCGCACC CGGCACCACGGTCAGCCTGC TCAAGAACCT GTTGGAAGGC GTTCAGCACG AGGAGCAGCC GCATGCGGGC TCGTACGAGCCGAGCTACCT GGAACGCAAT TTGGTCGGCC TCCACACCTA CCACAACATC GCGTTCCTGG AGAAAGGCGTCAACGCCGAA GGCGGCGTTC CTGCTTGGGT GCCAAGGAGT CTGGACGACA TATTGCACCT GGCCGACGTGAACAGCGCGG AGGACGAGTG AACAGCAGAG GGGCGAACAC ACAGGCATTT CCGACCGCGG ATCAGGTGGAGTCCATCTTC GATGCGTTGG CGCACGGGCG TCCCCTGCAC CACGGTTACT GGGCGGGCGG GTATCGGGAGGATGCCGGTG CCACACCGTG GTCGGATGCT GCCGACCAAC TGACCGACCT GTTCATCGAC AAGGCCGCGCTCCGTCCCGG AGCGCACCTG TTCGACCTGG GCTGCGGCAA TGGGCAGCCC GTAGTCCGTG CGGCATGCGCCAGCGGCGTT CGAGTCACCG GAATCACCGT GAACGCCCAG CATCTCGCCG CCGCCACCAG GCTCGCCAACGAGACCGGAC TGGCCGGCAG TCTTGAGTTC GATCTAGTCG ACGGCGCCCA GCTGCCCTAC CCGGACGGTTTCTTTCAGGC CGCATGGGCG ATGCAGTCCG TCGTGCAGAT CGTGGACCAG GCCGCCGCGA TCCGCGAGGTCCACCGAATC CTGGAACCCG GCGGCCGGTT CGTCCTCGGA GACATCATCA CTCGGGTTCG ACTCCCGGAAGAGTACGCGG CGGTTTGGAC GGGCACGACC GCCCATACCT TGAACAGCTT CACGGCGCTG GTCAGCGAAGCCGGGTTCGA GATTCTCGAAGTCACCGACC TCACGGCACA GACCAGGTGC ATGGTCTCCT GGTACGTCGACGAGTTGCTC CGGAAACTCG ATGAGCTCGC CGGCGTCGAG CCTGCGGCTG TCGGCACCTA CCAGCAACGCTACTTGGGAG ACATCGCGGC GAAGCACGGA CCGGGACCAG CACAGCTGAT CGCCGCGGTT GCGGAATACCGGAAACATCC GGATTACGCC AGAAACGAGG AAAGCATGGG TTTCATGCTC CTGCAGGCTC GAAAGAAGCAGTCCTGATGG CCTCCGAGCA CGCCAGCCTG GTCGGCGACG ATCTGCGGGC ACCCGCGGAT GATCCCTTCTACCGACCGCC GACGCCGCTA CCGCCGGGTG TCCCGGGCAC GCTCCTCAGG GCCCGGCCCG TCTCGGCACTGCGCGGCACG GGCGAACCCG TCGCAGCCAA GGCCTGGCAA ATCCTCTACC GGTCCAACTC CGCCCTTGGCATGCCGAACG CCGTCTCCGG CACCGTTCTG GTGCCGAACA TCCCGTGGCC GCGCGAAGAT CGCCCCATCATCACTTTCGC AGTGGGCACC CACGGCCTCG GTAGCCAAGT TGCCCCGTCG TACCTGCTTC GAACCGGAACCGAGCCGGAG ACCGAGCTGA TCGCCGTGGC CCTCGACCGC GGGTGGGCCG TGGTCATCAC CGACTACGAAGGCCTCGGTA CTCCTGGAAC CCACACCTAC ACCGTCGGCA GGGCGCAGGG ACACGCCATG CTCGATGCCGCCCGCGCTGC GCAACGGCTA CCGGGCTCCG GCCTGACGAC CGACTGCCCG GTCGGCATCT GGGGCTATGCGCAGGGTGGG CAAGCGTCGG CCTTCGCCGG CGAACTGCAC CCCACCTACG CACCTGAACT GCGAATCCGCGCTGCGGCCG CAGGTGCGGT GCCGATCGATCTGCTGGACA TCATCCACCG AAATGACGGG GTGTTCACCGGGCCGGTGCT GGCCGGCCTG GTCGGGCATG CCGCTGCCTA CCCCGATCTG CCATTCGACG AGCTTCTCACCGAAGCGGGT CGTACCGCCG TTGATCAAGT GCGCGAGCTC GGTGCACCGG AGCTCGTCAC CCGCTTCCTCGGCCGCGAGC TGAGCGACTT CCTCGACACT T.

[0057] 3. Construction of the 3-57-PermE-ΔspnK plasmid

[0058] spnK gene silencing vector 3-57-PermE, such as Figure 2 As shown, the specific method is as follows: the vector poJ260 was digested with NdeI enzyme, the digestion product was recovered by gel extraction, and the recovered product was ligated using Gibson Assembly technology. The ligation product was transformed into E. coli Top10 competent cells and cultured overnight at 37°C on LB agar plates containing 50 μg / mL apramycin (5 g yeast extract, 10 g tryptone, 10 g NaCl, 20 g agar, water added to 1 L, pH adjusted to 7.2).

[0059] The primer sequences are as follows:

[0060] Select a single colony for primer sequence amplification of a 745bp DNA fragment:

[0061] Primer 5 (41aa-YZ-F, forward primer, as shown in SED ID NO.5 of the sequence listing): 5´-accaacttcctccgaaccac-3´

[0062] Primer 6 (41aa-YZ-R, reverse primer, as shown in SED ID NO.6 of the sequence listing): 5´-gtcggaaatgcctgtgtgtt-3´

[0063] The correctness of plasmid construction was verified by amplification with specific primers for Primer 5 and Primer 6. Figure 3 (B): The transformant PCR product was consistent with the expected size (745 bp). No frameshift or point mutations were detected by sequencing analysis, and the overknockout vector was formally named 3-57-PermE-ΔspnK.

[0064] 4. Obtaining high-yield spinosad J / L strains

[0065] The plasmid 3-57-PermE-ΔspnK was transformed into E. coli S17, plated on LB agar containing 50 μg / mL Amprolium, and the transformants were picked to extract the plasmid. The plasmid was then digested and verified, thus completing the construction of the donor strain E. coli S17 3-57-PermE-ΔspnK.

[0066] Hyphae conjugation transfer method:

[0067] E. coli S17 donor strain 3-57-PermE-ΔspnK culture, harvesting, and washing: 1) Pick three healthy E. coli S17 single clones from the transformed LB agar plate and inoculate them into LB medium containing the corresponding antibiotic. Incubate overnight (approximately 12 h) at 37℃ and 220 rpm; 2) Perform PCR verification on the three clones to determine if the inoculated E. coli clones are correct; 3) Add 10% of the bacterial culture to LB liquid medium containing antibiotics. Incubate at 37℃ and 220 rpm for approximately 3 h, and measure whether the OD600 is 0.4–0.6; 4) Aliquot the bacterial culture into two 1.5 mL EP tubes, briefly centrifuge at 12000 rpm, and remove the supernatant in a clean bench; 5) Add 1 mL of sterile, antibiotic-free LB liquid medium to a single tube, mix thoroughly by pipetting, and wash the bacterial cells; 6) Repeat the washing process three times, resuspend the bacterial cells in 600 μL of liquid LB medium, and store at room temperature for later use. Cultured in LB liquid medium containing the above-mentioned apramycin until OD. 600 =0.4-0.6.

[0068] Treatment method for recipient bacteria HU14590: 1) Transfer the seed culture frozen at -20℃ to 20mL TSB medium (TSB 30g / L, sterilized at 121℃ for 20min) at a 1% inoculation rate and incubate at 28℃ for 48h; 2) Transfer the above bacterial culture to 18mL TSB medium at a 10% inoculation rate and incubate at 28℃ for 24h; 3) Transfer the above bacterial culture to 16mL TSB medium at a 25% inoculation rate and incubate at 28℃ for 6h to obtain the recipient bacteria; 4) Collect the mycelium by centrifugation at 8000rpm for 5min, discard the supernatant, and resuspend in 1mL TSB medium. 12000 rpm, briefly centrifuge, discard supernatant; 5) Take 300 μL of the treated E. coli and resuspend the Saccharopolysporum precipitate, spread evenly on R6 conjugation transfer plate, and incubate at 28℃ for 16 h; 6) Add 25 μL each of NA and Apra to 300 mL of sterile water, mix well, pour into R6 medium (Sucrose 200 g, Dextrilin 10 g, Casamino Acids 1 g, K2SO4 0.1 g, FeSO4•7H2O 0.1 g, MgSO4•7H2O 0.05 g, MnCl2•4H2O 0.001 g, ZnSO4•7H2O 0.001 g, Agar 20 g, add ddH2O to make up to 950 mL, sterilize at 115℃ for 25 min. Add 32.5 mL of 2M L-glutamate, 10 mL of 5M CaCl2•2H2O and 1M On the surface of MOPS (5 mL), after the adsorption layer is completely dry, it is incubated at 28℃ for 7-14 days. The resulting colonies are conjugates containing poJ260-PermE-14590.

[0069] The conjugate fragments were inoculated onto plates containing Apra (50 μg / mL) and cultured at 28°C for 10 days. Based on the characteristics of the 3-57-PermE suicide vector (it cannot replicate autonomously, and its stable existence depends on genome integration), the continuous growth of positive clones confirmed that the recombinant plasmid pOJ260-PermE-14590 had been stably integrated into the host genome via homologous recombination. Figure 4 As shown.

[0070] PCR verification of conjugating strains:

[0071] Conjugated zygotes were selected and passaged in fresh solid agar plates containing 50 μg / mL apramycin (5g soluble starch, 5g glucose, 5g mannitol, 5g yeast extract, 10g malt extract, 7.5g enzymatically hydrolyzed casein, 2g MgSO4·7H2O, 20g agar, water to 1L, pH adjusted to 7.2, sterilized at 115℃ for 30 min). Spores were obtained after about 10 days of culture. The grown spores were then passaged in antibiotic-free solid agar for three consecutive passages. Single colonies with good growth were inoculated into TSB medium and cultured at 28℃ for 2-3 days. Whole genome DNA was extracted and PCR verification was performed to confirm that the zygotes did not lose plasmids during antibiotic-free passage. The specific steps are as follows: Using the extracted bacterial DNA as a template, PCR verification of the spnK knockout site within the strain was performed using primer pairs Primer 5 / Primer 6 and Primer 7 / Primer 8.

[0072] The primer sequences are as follows:

[0073] Primer 5 (41aa-YZ-F, forward primer, as shown in SED ID NO.5 of the sequence listing): 5´-accaacttcctccgaaccac-3´

[0074] Primer 6 (41aa-YZ-R, reverse primer, as shown in SED ID NO.6 of the sequence listing): 5´-gtcggaaatgcctgtgtgtt-3´

[0075] Primer 7 (41aa-#spnK#-F1, forward primer, as shown in SED ID NO.7 of the sequence listing): 5´-ccaacttcctccgaaccacc-3´

[0076] Primer 8 (41aa-#spnK#-R1, reverse primer, as shown in SED ID NO.8 of the sequence listing): 5´-cgtgtccgttgatgtggctg-3´

[0077] PCR amplification of HU14590, verified by agarose gel electrophoresis, showed that the genomic PCR amplification product of the strain's conjugate exhibited a specific band at approximately 700 bp, while the negative control (the genome of the high-yielding mutant strain of *Saccharomyces cerevisiae*) showed a specific band at approximately 800 bp. Figure 5 As shown in -A. Using the HU01785 genome as a template, PCR amplification of the Primer 7 / Primer 8 fragment at the junction of the upstream and downstream homologous arms yielded no relevant DNA bands, as shown in Figure 1. Figure 5As shown in -B. Sequencing analysis did not detect frameshifts or point mutations, indicating the successful construction of the high-producing spinosad J / L strain HU01785. Figure 6 As shown.

[0078] Example 2: Stability Verification of Engineered Bacteria

[0079] The selected engineered bacteria were passaged 10 times on antibiotic-free solid plates, with shake-flask fermentation performed after each passage, and the J / L yield was measured. The results showed that after 10 passages, the J / L yield of the engineered bacteria was 3621.5 mg / L, with a fluctuation of only 4.3% compared to the initial yield, indicating that the engineered bacteria have good genetic stability.

[0080] Example 3: Shake-flask fermentation of ΔspnK modified strain and original strain

[0081] 1. Shake-flask fermentation of the starting strain and the ΔspnK modified strain

[0082] Spores of the starting strain and engineered strain of *Saccharomyces cerevisiae* were diluted and spread onto solid agar plates. After incubation at 28°C for 6 days, single colonies were picked and cultured on solid agar plates for 12 days.

[0083] Seed culture of *Polysporium spp.*: Healthy single-clone colonies were inoculated into 250 mL Erlenmeyer flasks containing 20 mL of seed culture medium and cultured at 28℃ and 220 rpm for 72 h to obtain *Polysporium spp.* seed culture medium (1 L): glucose 20 g, dextrin 20 g, yeast extract 15 g, cottonseed meal 25 g, ammonium sulfate 1 g, MgSO4·7H2O 2 g, pH=7.0. Sterilized at 121℃ for 20 min.

[0084] Fermentation culture of *Polysporium argentea*: Seed culture was inoculated at a rate of 10% into a 250 mL shake flask containing 25 mL of fermentation medium and cultured at 28 °C and 220 rpm for 14 days. Fermentation medium (1 L): 80 g glucose, 32.5 g cottonseed meal, 6.5 g soybean meal, 7 g corn steep liquor, 10 g corn starch, 2 g yeast extract, 5 g CaCO3, 42 mL methyl oleate, adjusted to pH 7.0, and sterilized at 121 °C for 20 min.

[0085] Sampling was conducted at different time points (0, 3d, 7d, 10d, 12d, and 14d) during the fermentation of the starting strain HU14590 and the engineered strain HU01785. Three shake-flask replicates were designed for each strain at each sampling point. The HPLC fermentation parameters of spinosad were determined after 14 days of fermentation using the following methods:

[0086] HPLC detection of spinosad: Mix 1 mL of fermentation broth with 4 mL of methanol, dilute 5 times, and shake at 2500 rpm for 30 min. Centrifuge at 12000 rpm for 5 min. Collect the supernatant with a syringe and filter through a 0.22 μm organic phase filter membrane. Use a Welch Ultimate® XB-C18 column (5 μm, 4.6 × 250 mm); column temperature 25℃; mobile phase: methanol-acetonitrile-water (45:45:10) containing 10% 6 mM ammonium acetate; flow rate 1.0 mL / min; injection volume 20 μL; wavelength 250 nm.

[0087] Example 3: A high-yield polysaccharide-producing engineered strain of *Saccharomyces cerevisiae* (J / L), its construction method, and its application.

[0088] A high-yield polysaccharide-producing engineered strain of *Polysporium spinosum*, namely *Polysporium spinosum* HU01785, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026943 and deposit date of May 13, 2026.

[0089] To achieve the above and other related objectives, the technical solution provided by this invention is: a method for constructing a high-yield polysaccharide-producing engineered strain of *Polysporium spp.*, wherein the engineered strain is constructed using *Polysporium spp.* HU14590 as the starting strain, by knocking out part of the spnK gene to silence the spnK gene, and is named *Polysporium spp.* HU01785. It has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026943 and deposit date of May 13, 2026.

[0090] A preferred embodiment includes the following steps:

[0091] Step 1: Using the spnK gene as the target gene, a conserved 41 amino acid sequence was selected within the open reading frame. The upper and lower homologous arms were amplified using primer annealing. These were then sequentially ligated into the *E. coli*-*Streptomyces* shuttle vector 3-57 to construct the recombinant knockout plasmid 3-57-ΔspnK, which inhibits spnK gene expression in *Polyspora sacchariformis* HU14590. The conserved nucleotide sequence is as follows:

[0092] The nucleotide sequence encoding the conserved 41 amino acid sequence is as follows:

[0093] TACGGACCGT TCGACATCAT CATCGACGAT GGCAGCCACA TCAACGGACA CGTGCGCACATCCCTGGAAA CGCTGTTCCC CCGGTTGCGC AGCGGTGGCG TATACGTGAT CGAGGATCTG TGG.

[0094] Step 2: Vector conjugation and transfer: The plasmid 3-57-ΔspnK constructed in Step 1 was transformed into Escherichia coli S17 to obtain recombinant Escherichia coli; the recombinant Escherichia coli was conjugated and transferred with Polysporus spp. HU14590, plated on LB medium containing apramycin, and cultured for 12 days to obtain conjugates;

[0095] Step 3: Screening and verification of engineered bacteria: PCR verification was performed on the conjugates to confirm partial gene knockout of spnK. The strain with the highest spinosad J / L yield was screened by shake-flask fermentation. This strain was further passaged to increase spinosad yield, which is the target engineered bacteria, Polyspora spinosad HU01785.

[0096] In a preferred embodiment, the specific conditions for conjugation transfer in step 2 are as follows: the recombinant Escherichia coli and the mycelial suspension of Polysporus spp. HU14590 are mixed at a volume ratio of 1:2-5, spread on LB medium, and cultured at 25-32℃ for 10-30 hours. Then, the antibiotic Apramycin is added, and the culture is continued for 7-14 days.

[0097] This describes the application of the engineered strain of Polysporus spp. that produces high levels of spinosad (J / L) in the production of spinosad (J / L).

[0098] A preferred embodiment includes the following steps: inoculating Polysporus spp. HU01785 onto a slant culture medium and culturing at 25°C for 10 days; transferring to a seed culture medium and culturing at 25°C and 200 rpm for 60 hours; and inoculating into a fermentation culture medium at an 8% inoculation rate and culturing at 25-32°C and 200 rpm for 1 day.

[0099] A preferred embodiment is that each liter of the fermentation medium consists of: 60g glucose, 30g cottonseed meal powder, 5g soybean meal powder, 5g corn steep liquor, 8g corn starch, 1.5g yeast extract, 3g CaCO3, 38g methyl oleate, and pH 6.8.

[0100] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A high-yield polyspora spinosad strain producing spinosad J / L, characterized in that: The engineered strain of *Polysporium spp.* is *Polysporium spp.* HU01785, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M2026943 and deposit date of May 13, 2026.

2. A method for constructing an engineered strain of *Saccharomyces cerevisiae* that produces high levels of spinosad (J / L), characterized in that: The engineered strain of *Polysporium saccharidum* was constructed using *Polysporium saccharidum* HU14590 as the starting strain, by knocking out part of the spnK gene to silence the spnK gene. It was named *Polysporium saccharidum* HU01785 and has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2026943 and deposit date of May 13, 2026.

3. The method for constructing a high-yield polyspora spinosad engineered strain according to claim 2, characterized in that, Includes the following steps: Step 1: Using the spnK gene as the target gene, 41 conserved amino acid sequences were selected within the open reading frame. The upper and lower homologous arm fragments were amplified using primer annealing. The two fragments were then sequentially ligated into the Escherichia coli-Streptomyces shuttle vector 3-57 to construct the spnK gene expression recombinant knockout plasmid 3-57-ΔspnK, which inhibits the expression of the spnK gene in Polysporus saccharidus HU14590. Step 2: Vector conjugation and transfer: The plasmid 3-57-ΔspnK constructed in Step 1 was transformed into Escherichia coli S17 to obtain recombinant Escherichia coli; the recombinant Escherichia coli was conjugated and transferred with Polysporus spp. HU14590, plated on LB medium containing apramycin, and cultured for 12-14 days to obtain conjugates; Step 3: Screening and verification of engineered bacteria: PCR verification was performed on the conjugates to confirm partial gene knockout of spnK. The strain with the highest spinosad J / L yield was screened by shake-flask fermentation. This strain was further passaged to increase spinosad yield, which is the target engineered bacteria, Polyspora spinosad HU01785.

4. The method for constructing the engineered strain of *Saccharomyces cerevisiae* with high spinosad yield (J / L) according to claim 3, characterized in that, In step 2, the specific conditions for conjugation transfer are as follows: the recombinant Escherichia coli and the mycelial suspension of Polysporum spp. HU14590 are mixed at a volume ratio of 1:2-5, spread on LB medium, and cultured at 25-32℃ for 10-30 hours. Then, the antibiotic Apramycin is added, and the culture is continued for 7-14 days.

5. The application of the engineered strain of Polysporus spp. with high spinosad J / L production as described in claim 1 in the production of spinosad J / L.

6. The application of the engineered strain of *Saccharomyces cerevisiae* with high spinosad yield (J / L) as described in claim 5 in the production of spinosad (J / L), characterized in that, The steps include: inoculating Polysporus spp. HU01785 onto a slant culture medium and culturing at 25-32℃ for 10-14 days; transferring to a seed culture medium and culturing at 25-32℃ and 200-300rpm for 60-80 hours; and inoculating into a fermentation culture medium at an inoculation rate of 8-12% and culturing at 25-32℃ and 200-300rpm for 10-30 days.

7. The application of the engineered strain of *Saccharomyces cerevisiae* with high spinosad yield (J / L) as described in claim 5 in the production of spinosad (J / L), wherein each liter of the fermentation medium comprises: 60-100g glucose, 30-35g cottonseed meal, 5-7g soybean meal, 5-8g corn steep liquor, 8-12g corn starch, 1.5-2.5g yeast extract, 3-6g CaCO3, 38-45g methyl oleate, and pH 6.8-7.2.

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