Use of an acadm gene to increase production of a polyketide in paenibacillus polymyxa

Abstract

The application relates to application of an ACADM gene in improving the production of spinosyn in Saccharopolyspora spinosa, and belongs to the field of biological fermentation. The method mainly comprises overexpressing the ACADM gene in the Saccharopolyspora spinosa. Specifically, by introducing the ACADM gene into the Saccharopolyspora spinosa and enabling the ACADM gene to realize efficient expression in the host bacteria, the biosynthesis pathway of the spinosyn can be effectively promoted, so that the final yield of the spinosyn is significantly improved. Experiments show that the method is feasible in operation and remarkable in effect, and provides an effective genetic modification strategy for industrial production of the spinosyn.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and microbial fermentation technology, specifically relating to the application of the ACADM gene in increasing spinosad production in Saccharopolyspora spinosa, and more particularly to a method and its application for improving Saccharopolyspora spinosa using genetic engineering techniques to increase spinosad production. Background Technology

[0002] Spinosad is a secondary metabolite produced by aerobic fermentation of *Saccharopolysporaspinosa*, a member of the actinomycete genus. Its core skeleton is a twelve-membered macrocyclic lactone structure composed of 21 carbon atoms, with spinosad A and D as its main components. Due to its broad insecticidal spectrum, environmental friendliness, and low toxicity to mammals, spinosad has been widely used in the field of biopesticides.

[0003] The biosynthesis of spinosad is a complex metabolic process, primarily catalyzed by polyketide synthase (PKS) to synthesize a polyketide backbone. This process requires the consumption of large amounts of metabolic precursors, such as malonyl-CoA and methylmalonyl-CoA. However, the secondary metabolic regulatory mechanisms of the original strain of *Saccharomyces cerevisiae* are extremely complex, and the supply of endogenous metabolic precursors is often insufficient to meet the demands of high yields. Currently, although traditional physicochemical mutagenesis breeding and some genetic engineering techniques (such as overexpression of regulatory genes in the PKS gene cluster) can increase yields to some extent, it is still difficult to fully meet the growing global market demand, and the difficulty in increasing yield limits its industrialization level.

[0004] However, how to precisely regulate this metabolic node in specific production strains (such as Polysporus sacchariformis NRRL18395) and break through the yield bottleneck through efficient expression systems (such as introducing strong promoters) remains a key technical challenge that urgently needs to be solved in the field of bioengineering. Summary of the Invention

[0005] The purpose of this invention is to address the problems existing in the prior art by providing an application of the ACADM gene in increasing spinosad production in *Saccharopolyspora spinosa*. Specifically, it provides a method for increasing spinosad production in *Saccharopolyspora spinosa*, an engineered strain, and its application. The inventors unexpectedly discovered during their research that enhancing the expression of specific functional genes in *Saccharopolyspora spinosa* can significantly improve its spinosad synthesis capacity.

[0006] The objective of this invention can be achieved through the following methods:

[0007] In a first aspect, the present invention provides an application of the ACADM gene in increasing the yield of spinosad in *Saccharomyces cerevisiae*, wherein the application is to overexpress the ACADM gene in *Saccharomyces cerevisiae*. The amino acid sequence encoded by the nucleotide sequence of the ACADM gene contains an amino acid sequence that is at least 90% identical to the sequence shown in SEQ ID NO.2.

[0008] The amino acid sequence encoded by the ACADM gene contains an amino acid sequence that has at least 95%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO.2.

[0009] The amino acid sequence encoded by the ACADM gene is shown in SEQ ID NO.2.

[0010] As one embodiment of the present invention, the nucleotide sequence of the ACADM gene contains a nucleotide sequence that has at least 90% identity with the sequence shown in SEQ ID NO.1.

[0011] The nucleotide sequence of the ACADM gene contains a nucleotide sequence that has at least 95%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO.1.

[0012] The nucleotide sequence of the ACADM gene is shown in SEQ ID NO.1. The amino acid sequence encoded by the ACADM gene nucleotide sequence shown in SEQ ID NO.1 is the same as that shown in SEQ ID NO.2.

[0013] In one embodiment of the present invention, the polysaccharidobacterium spp. is polysaccharidobacterium NRRL18395 or WHU1107.

[0014] The *Saccharomyces cerevisiae* NRRL18395 used in this invention is a commonly used commercially available *Saccharomyces cerevisiae*; the *Saccharomyces cerevisiae* strain WHU1107 used has the preservation number CCTCC NO:M2021307.

[0015] As one embodiment of the present invention, the spinosad includes spinosad A and spinosad D.

[0016] Secondly, the present invention provides a method for increasing spinosad production in *Saccharomyces cerevisiae*, wherein the method involves enhancing the expression of the ACADM gene (overexpression of the ACADM gene) in *Saccharomyces cerevisiae*.

[0017] As one embodiment of the present invention, the overexpression of the ACADM gene is integrated into the genome of *Saccharomyces cerevisiae* via homologous recombination; the homologous arm used in the homologous recombination includes one of homologous arm 1 and homologous arm 2, preferably homologous arm 1.

[0018] Homologous arm 1 includes left homologous arm 1 and right homologous arm 1; the sequence of left homologous arm 1 is shown in SEQ ID NO.3, and the sequence of right homologous arm 1 is shown in SEQ ID NO.4.

[0019] Homologous arm 2 includes left homologous arm 2 and right homologous arm 2; the sequence of left homologous arm 2 is shown in SEQ ID NO.5, and the sequence of right homologous arm 2 is shown in SEQ ID NO.6; In one embodiment of the present invention, when the ACADM gene is overexpressed, a strong promoter is connected upstream of the ACADM gene; the strong promoter includes one of the kasOp* promoter and the ermEp* promoter, preferably the kasOp* promoter.

[0020] In some embodiments, the ACADM gene driven by the kasOp* promoter and the ermEp* promoter is integrated into the genome of the *Saccharomyces cerevisiae* via homologous recombination.

[0021] Thirdly, the present invention provides an engineered bacterium that produces high levels of spinosad; the engineered bacterium is obtained by overexpressing the ACADM gene in a strain of Polysaccharidium erythrosporum.

[0022] The engineered bacteria were obtained by introducing an expression vector containing the ACADM gene into *Saccharomyces cerevisiae*, or by genetically modifying *Saccharomyces cerevisiae* to enhance the expression activity of the ACADM gene.

[0023] The spinosad production capacity of the engineered bacteria obtained by this invention is significantly improved compared with its original strain, with an increase in yield of at least 30%.

[0024] Fourthly, an expression cassette for overexpressing the ACADM gene is provided, which is one of expression cassette 1, expression cassette 2, and expression cassette 3.

[0025] The expression cassette 1 contains a homologous arm 1, a kasOp* promoter, and an ACADM gene expressed by the promoter.

[0026] The expression cassette 2 contains a homologous arm 1, an ermEp* promoter, and an ACADM gene expressed by the promoter.

[0027] The expression cassette 3 contains a homologous arm 2, a kasOp* promoter, and an ACADM gene expressed by the promoter.

[0028] Fifthly, a vector or recombinant plasmid is provided.

[0029] The vector or recombinant plasmid contains the expression cassette and is stably inherited in *Saccharomyces cerevisiae*. The vector contains the expression cassette and homologous sequences required for genome integration, enabling the expression cassette to be site-specifically integrated into the *Saccharomyces cerevisiae* genome via homologous recombination.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The ACADM overexpression mutant strain constructed through genetic modification showed a significant increase in spinosad potency compared to the original strain under the same fermentation conditions, demonstrating extremely high industrial application value. Attached Figure Description

[0031] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram of the construction process of the overexpression plasmid pF20 in Example 1.

[0032] Figure 2 This is a schematic diagram illustrating the principle of homology exchange assay for the modified strain in Example 1.

[0033] Figure 3 This is a PCR verification diagram of the kasOp*-ACADM double-crossover strain overexpressing at position 1 of homologous arm 1 of *Saccharomyces cerevisiae* NRRL18395, as shown in Example 1.

[0034] Figure 4 The results show the spinosad yield of Polysaccharidone NRRL18395 and the strain F20-S1 overexpressing kasOp*-ACADM at position 1 of homologous arm 1 in Example 1.

[0035] Figure 5 The results show the spinosad yield of Polysaccharidone WHU1107 and the strain F20-S2 overexpressing kasOp*-ACADM at position 1 of homologous arm 2 in Example 2.

[0036] Figure 6 This is a schematic diagram of the construction process of the overexpression plasmid pF47 in Example 3.

[0037] Figure 7 This is a schematic diagram illustrating the principle of homology exchange assay for the modified strain in Example 3.

[0038] Figure 8 This is a PCR verification diagram of the strain that overexpressed ermEp*-ACADM double-crossover in homologous arm 1 of *Saccharomyces cerevisiae* NRRL18395, as described in Example 3.

[0039] Figure 9The results show the spinosad yield of Polysaccharidone NRRL18395 and the strain F47-S1 overexpressing ermEp*-ACADM at position 1 of homologous arm 3 in Example 3.

[0040] Figure 10 The results show the spinosad yield of Polysaccharidone WHU1107 and the strain F47-S2 overexpressing ermEp*-ACADM at position 1 of homologous arm 4 in Example 4.

[0041] Figure 11 This is a schematic diagram of the construction process of the overexpression plasmid pF48 in Example 5.

[0042] Figure 12 This is a schematic diagram illustrating the principle of homology exchange assay for the modified strain in Example 5.

[0043] Figure 13 This is a PCR verification diagram of the kasOp*-ACADM double-crossover strain overexpressing in the homologous arm 2 position of *Saccharomyces cerevisiae* NRRL18395, as shown in Example 5.

[0044] Figure 14 The results show the spinosad yield of Polysaccharidone NRRL18395 and the overexpression of kasOp*-ACADM strain F48-S1 at position 2 of homologous arm in Example 5.

[0045] Figure 15 The results show the spinosad yield of Polysaccharidone WHU1107 and the strain F48-S2 overexpressing kasOp*-ACADM at position 2 of the homologous arm in Example 6. Detailed Implementation

[0046] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0047] definition: As used herein, the terms “an” and “a kind”, as well as “the” and similar pronouns, indicate singular and plural, unless otherwise specified herein or the context clearly contradicts them. The terms “about,” “substantially,” and “similar to” mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which may depend in part on the method of measurement or determination of the value, or on the limitations of the measurement system.

[0048] The term "overexpression" generally refers to any amount greater than or equal to the expression level shown by a reference standard. In this invention, the term "overexpression" means that a gene product or polypeptide is expressed at a level higher than the expression level of the same gene product or protein in a comparable host cell before genetic alteration or under defined conditions without genetic alteration. In this context, the term "overexpression" includes any detectable expression. Gene overexpression can be achieved by using a promoter that induces high levels of expression of said gene. For example, the promoter of an endogenous gene can be replaced with a stronger promoter, i.e., a promoter that induces a higher expression level. In some embodiments of the invention, it is preferable to integrate an expression cassette containing said gene under the control of a strong promoter (such as the kasOp* promoter) into the genome.

[0049] A first aspect of this application provides a method for increasing spinosad production. The method includes enhancing the expression of the ACADM gene in a *Saccharomyces cerevisiae* starting strain. In some embodiments, the ACADM gene comprises a nucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO. 1; preferably, the ACADM gene has the nucleotide sequence shown in SEQ ID NO. 1.

[0050] ACADM gene nucleotide sequence (SEQ ID NO.1):

[0051] The ACADM gene nucleotide sequence shown in SEQ ID NO.1 encodes an amino acid sequence shown in SEQ ID NO.2.

[0052] The amino acid sequence of the ACADM gene contains an amino acid sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO.2; preferably, the amino acid sequence of the ACADM gene has the amino acid sequence shown in SEQ ID NO.2.

[0053] ACADM gene amino acid sequence (SEQ ID NO.2): MDFGYDERTEELRKQLLDFMDSHVYPAEPVFLEQLAEADDPWSAHPPVIEELKAEARNRGLWNLFLPGEGEHGAGLTNLQYAPLAEIMGRVGRLAPEATNCAAPDTGNMEVLALFGTEQQKKQWLQPLLDGEIRSAFCMTEPAVASSDATNIGTRIERDGDNYVINGRKWFSSGAMSPRCKILIVMGKTDPNAERHRQQSQIL VPKDTPGVQIKRSMNVFGYTDADHGGHAEIVFENARVPVENIIAGEGEGFSIAQARLGPGRIHHCMRAIGMAERALELMCRRANERIAFGKPLADQGVVQHWIAESRVKIEQARLLVLKTAWLMDTAGNQGAHTEIQAIKVGVPQMATWVIDRAIQAHGGGGVSQEFPLAHLYAHARTLHIVDGPDEVHRRSLARRELKKYK*.

[0054] In some embodiments, the enhanced expression is achieved by operatively linking a strong promoter upstream of the gene, preferably a kasOp* or ermEp* promoter.

[0055] In some embodiments, the sequence of the left homologous arm 1 is (SEQ ID NO.3), and the sequence of the right homologous arm 1 is (SEQ ID NO.4). Left homologous arm 1 (SEQ ID NO.3):

[0056] Right homologous arm 1 (SEQ ID NO.4):

[0057] The sequence of the left homologous arm 2 is (SEQ ID NO.5) and the sequence of the right homologous arm 2 is (SEQ ID NO.6). Left homologous arm 2 (SEQ ID NO.5):

[0058] Right homologous arm 2 (SEQ ID NO.6):

[0059] In some embodiments, the starting strain is *Polyspora sacchariformis* NRRL18395 or WHU1107. The inventors discovered that by introducing the promoters kasOp* and ermEp* upstream of this gene and overexpressing them in *Polyspora sacchariformis*, specifically at the homologous arm 1 or homologous arm 2 position in the genome, the yield of spinosad can be significantly increased. Experiments confirmed that overexpressing this gene using the kasOp* promoter at position 1 of the homologous arm of *Polysporium saccharidum* NRRL18395 and WHU1107 increased spinosad production by approximately 105.13% and 66.18%, respectively; overexpressing this gene using the ermEp* promoter at position 1 of the homologous arm of *Polysporium saccharidum* NRRL18395 and WHU1107 increased spinosad production by approximately 76.65% and 43.72%, respectively; and overexpressing this gene using the kasOp* promoter at position 2 of the homologous arm of *Polysporium saccharidum* NRRL18395 and WHU1107 increased spinosad production by approximately 57.28% and 23.15%, respectively.

[0060] A second aspect of this application provides an engineered bacterium that produces high levels of spinosad.

[0061] The engineered bacteria were obtained by introducing an expression vector containing the ACADM gene into the starting strain of *Saccharomyces cerevisiae*, and by enhancing the expression activity of the ACADM gene in the starting strain through genetic modification.

[0062] In some embodiments, the ACADM gene is expressed by a kasOp* promoter. The kasOp* promoter-driven ACADM gene is integrated into the genome of *Saccharomyces cerevisiae* via homologous recombination.

[0063] In some embodiments, the ACADM gene is expressed by the ermEp* promoter. The ACADM gene driven by the ermEp* promoter is integrated into the genome of the *Saccharomyces cerevisiae* via homologous recombination.

[0064] In some embodiments, the ACADM gene driven by the kasOp* promoter is integrated into the genomic homology arm 1 of the *Saccharomyces cerevisiae* via homologous recombination.

[0065] In some embodiments, the ACADM gene driven by the kasOp* promoter is integrated into the homologous arm 2 of the genome of the *Saccharomyces cerevisiae* via homologous recombination.

[0066] In some embodiments, the ACADM gene has at least two copies in the genome of the engineered bacteria. Increasing the copy number of the ACADM gene in the strain is beneficial to further improve the yield of spinosad.

[0067] In some embodiments, the *Polysporium sacchariformis* is *Polysporium sacchariformis* NRRL18395 or WHU1107. The *Polysporium sacchariformis* strain WHU1107 used in the following examples has the accession number CCTCC NO: M2021307.

[0068] The third aspect of this paper provides the application of the ACADM gene in increasing the production of spinosad by Polysporus spp.

[0069] Example 1: Overexpression of kasOp*-ACADM at position 1 of homologous arm increases spinosad production in Polysaccharidobacterium NRRL18395 1.1 Construction of recombinant plasmid pF20 Using the Gibson assembly principle, 20 bp homologous sequences were attached to both ends of the left homologous arm 1 (Larm1), kasOp* promoter, ACADM gene (SEQ ID NO.1), right homologous arm 1 (Rarm1), and pOJ260 vector (containing the apramycin resistance gene Apr) using primers.

[0070] Using the genome of *Saccharomyces cerevisiae* NRRL18395 obtained by phenol-chloroform extraction as a template, The left homologous arm 1 was amplified using the upstream primer F20-Larm-F, 5'-cgacggccagtgccaagcttgaccccctcctgcagacggc-3' (SEQ ID NO. 7) and the downstream primer F20-Larm-R, 5'-tcacccgccccagctgtgcc' (SEQ ID NO. 8).

[0071] The ACADM gene was amplified using the upstream primer F20-ACADM-F, 5'-ggcacagctggggcgggtgattacttgtacttcttcagttc-3' (SEQ ID NO.9) and the downstream primer F20-ACADM-R, 5'-gtggatttcggctacgacgaac-3' (SEQ ID NO.10).

[0072] The right homologous arm 1 was amplified using the upstream primer F20-Rarm-F, 5'-gaccacccacaccgatagac-3' (SEQ ID NO. 11) and the downstream primer F20-Rarm-R, 5'-ctatgacatgattacgaattccactggtacaggtgtgtacg-3' (SEQ ID NO. 12).

[0073] Using the kasOp* promoter as a template, the kasOp* promoter was amplified using the upstream primer F20-kas-F, 5'-tcgtcgtagccgaaatccacATGGCGTATCCCCTTTCAGA-3' (SEQ ID NO. 13) and the downstream primer F20-Kas-R, 5'-gtctatcggtgtgggtggtcGGAACGATCGTTGGCTGTGTTC-3' (SEQ ID NO. 14).

[0074] The pOJ260 vector was linearized using the EcoRI / HindIII double restriction enzyme sites. The recombinant plasmid pF20 was obtained via multi-fragment Gibson assembly. The constructed plasmid was validated using first-generation sequencing.

[0075] A schematic diagram of pF20 plasmid construction is shown below. Figure 1 .

[0076] 1.2 Construction of a strain overexpressing kasOp*-ACADM at position 1 of homologous arm A schematic diagram illustrating the homology exchange assay principle of the modified strain overexpressing kasOp*-ACADM at position 1 of homology arm is shown below. Figure 2 .

[0077] 1.2.1 Conjugation transfer of transformed Escherichia coli and Polysporus spp. Preparation of E. coli donor bacteria: The plasmid pF20 was transformed into ET12567 (pUZ8002) to obtain pF20 in ET12567 (pUZ8002) containing the plasmid: E. coli T12567 (pUZ8002) competent cells were taken out from -80℃ and thawed on ice; 1 μL of pF20 plasmid was added and gently mixed; the cells were placed on ice for 30 min, heat-shocked in a 42℃ water bath for 90 s, and then in an ice bath for 3 min; 900 μL of LB medium was added and mixed; the cells were then recovered in a shaker at 37℃*220 rpm for 45 min. After resuscitation, the bacterial cells were collected by centrifugation at 6000 rpm for 3 min. The supernatant was discarded, leaving approximately 100 μL of liquid to resuspend the cells. This resuspended cells were then evenly spread onto LB agar plates containing chloramphenicol (34 µg / mL), kanamycin (50 µg / mL), and apramycin (50 µg / mL). The plates were incubated at 37°C for 16 h. The resulting single colonies were designated pF20 inET12567 (pUZ8002). A single colony was picked and inoculated into 10 mL of LB agar (containing the three antibiotics mentioned above), and incubated overnight at 37°C with shaking at 220 rpm.

[0078] Preparation of recipient bacteria for Polysporus spp.: Activate 100 μL of glycerol inoculum of Polysporus saccharidus NRRL18395, stored at -80℃, by spreading it onto a solid agar plate (2 g / L glucose, 10 g / L soybean peptone, 2 g / L yeast extract, 2 g / L 3-morpholine propanesulfonic acid (MOPS), 3 g / L calcium carbonate, 0.5 g / L magnesium sulfate, 0.5 g / L dipotassium hydrogen phosphate, 0.046 g / L ferrous sulfate heptahydrate, 15 g / L agar powder, pH adjusted to 7.2, and sterilized at 115℃ for 30 min). Incubate the plates at 28℃ for 7 days. The spores on the plate were then collected with a cotton swab into 5 mL of TES buffer (0.05 M TES, pH 8.0), treated in a 50°C water bath for 10 minutes, and an equal volume of pre-germination medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L sodium chloride) was added. The plate was then incubated at 37°C with shaking for 4 hours. The spores were collected by centrifugation and resuspended in an appropriate amount of TES for later use.

[0079] Bonding transfer: Take 1.5 mL of overnight cultured E. coli (OD600 ≈ 0.4-0.6) and centrifuge at 4000 rpm for 5 minutes. Discard the supernatant and wash the cells twice with an equal volume of LB medium. Finally, resuspend the cells in 1 mL of pre-germination medium.

[0080] Take 500 µL of pre-germinating spore suspension and 500 µL of washed E. coli suspension. Gently mix the two together. Take 200 µL of the mixture and drop it onto the center of a dry, antibiotic-free solid agar plate (pre-dried in a laminar flow hood for 30 minutes to remove surface moisture). Gently spread the mixture evenly with a sterile spreader until the liquid is completely absorbed. Incubate the plate upright in a 28°C incubator for 20 hours.

[0081] Based on a solid plate volume of 25 mL, add 50 mg / L of trimethoprim and 15 mg / L of Apr antibiotics to 2 mL of sterile water, mix well, and cover the plate. Incubate the plate at 28°C for 10 days until single colonies grow.

[0082] 1.2.2 Screening of single-exchange strains Single colonies grown on the ABB13 plates were selected and cultured on ABB13 plates containing 15 mg / L apramycin and 50 mg / L trimethoprim for 6 days. Using the genome of the strain obtained by phenol-chloroform extraction as a template, the single-exchange strain was verified using the upstream primer F20-ACADM-F, 5'-ggcacagctggggcgggtgattacttgtacttcttcagttc-3' (SEQ ID NO. 9) and the downstream primer F20-QC-R1, 5'-gaggcgtccgacggctac-3' (SEQ ID NO. 15). The result showed a 1535bp verification group, confirming it as the correct single-exchange modified strain.

[0083] 1.2.3 Construction of a double-crossover strain overexpressing kasOp*-ACADM at position 1 of homologous arm Single colonies of the correct single-exchange strain were isolated on antibiotic-free solid agar plates. After 7 days of incubation, individual colonies were selected, and each colony was simultaneously spread onto both solid agar plates containing 15 mg / L apramycin (2 g / L glucose, 10 g / L soybean peptone, 2 g / L yeast extract, 2 g / L 3-morpholinopropanesulfonic acid (MOPS), 3 g / L calcium carbonate, 0.5 g / L magnesium sulfate, 0.5 g / L dipotassium hydrogen phosphate, 0.046 g / L ferrous sulfate heptahydrate, 15 g / L agar powder, pH adjusted to 7.2, and sterilized at 115℃ for 30 min) and antibiotic-free solid agar plates. The spread plates were observed after 7 days of incubation. Double-crossover strains grown only on antibiotic-free solid agar plates were expanded. A portion of these strains was selected, and DNA was extracted and used as a template. The candidate double-crossover strains were validated using upstream primer F20-QC-F3, 5'-GGACCGTTCCTTCCGTCTAC-3' (SEQ ID NO. 16) and downstream primer F20-QC-R1, 5'-gaggcgtccgacggctac-3' (SEQ ID NO. 15) as validation primers. The validation group showing 2292 bp in the agarose gel electrophoresis results was the correctly overexpressing kasOp*-ACADM double-crossover modified strain at position 1 of the homologous arm. This strain was named F20-S1. Figure 3 ).

[0084] 1.3 Fermentation verification of recombinant *Saccharomyces cerevisiae* strain F20-S1 100 μL of NRRL18395 and F20-S1 strains were plated onto fermentation plates for activation. The fermentation plate formulation was: 5 g / L glucose, 3 g / L yeast extract, 5 g / L soybean peptone, 20 g / L soybean meal, 20 g / L agar, pH adjusted to 7.2 with NaOH, and sterilized at 115℃ for 30 min. The plated plates were then incubated at 28℃ for 7 days. A 1 cm sample was then drawn using a pipette tip. 2Microbial blocks of various sizes were placed in seed culture medium. The seed culture medium formula was: 15 g / L soluble starch, 10 g / L corn steep liquor powder, 12 g / L cottonseed peptone, 1.5 g / L ammonium sulfate, 1.5 g / L dipotassium hydrogen phosphate, 3 g / L calcium carbonate, 0.5 g / L magnesium sulfate heptahydrate, and the pH was adjusted to 7.2. 30 mL portions of the medium were dispensed into 250 mL shake flasks and sterilized at 121 °C for 40 min. The seeds were cultured at 220 rpm, 28 °C, and 50% humidity for 72 h, and then transferred to fermentation medium at a rate of 5%. The fermentation medium was formulated as follows: 25 g / L glucose, 25 g / L corn starch, 30 g / L cottonseed meal, 2 g / L yeast extract, 10 g / L corn steep liquor, 10 g / L soybean oil, 1.5 g / L dipotassium hydrogen phosphate, 0.5 g / L ferrous sulfate, and 6 g / L calcium carbonate. The pH was adjusted to 7.2. 25 mL of the medium was dispensed into 250 mL shake flasks and sterilized at 121 °C for 40 min. The medium was then cultured for 12 days at 220 rpm, 28 °C, and 50% humidity. The spinosad yield was determined using HPLC according to the detection method described in the reference (Gao-Yi, Kunhua, et al. Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Ployketide Synthase Gene Cluster Reconstitution in Streptomyces[J]. AcsSynth Biol, 2017.).

[0085] The results showed that the average spinosad yield (in the examples, the sum of spinosad A and spinosad D in each example) of the starting strain *Polyspora sacchariformis* NRRL 18395 was 102.41 mg / L, while the spinosad yield of the obtained strain F20-S1, which overexpressed kasOp*-ACADM at position 1 of the homologous arm, was 210.07 mg / L, representing a 105.13% increase compared to the starting strain *Polyspora sacchariformis* NRRL18395. Figure 4 ) Example 2: Overexpression of kasOp*-ACADM at position 1 of homologous arm increases spinosad production in Polysporus spp. WHU1107 The same method as in Example 1 was used to construct a recombinant strain of Polysaccharidobacterium sarcodactylis WHU1107 that overexpressed kasOp*-ACADM at position 1 of the homologous arm. The difference was that Polysaccharidobacterium sarcodactylis NRRL18395 in Example 1 was replaced with the high-yielding strain Polysaccharidobacterium sarcodactylis WHU1107. The verified modified strain was named F20-S2.

[0086] The fermentation yield of the overexpressing strain F20-S2 was detected using the same fermentation method and detection conditions as in Example 1. The results showed that the average spinosad yield of the starting strain *Polyspora sacchariformis* WHU1107 was 4346.57 mg / L, while the spinosad yield of the F20-S2 strain, which overexpressed kasOp*-ACADM at position 1 of the homologous arm, was 7222.94 mg / L, representing a 66.18% increase compared to the starting strain *Polyspora sacchariformis* WHU1107. Figure 5 ) Example 3: Overexpression of ermEp*-ACADM at position 1 of homologous arm increases spinosad production in Polysaccharidobacterium NRRL18395 3.1 Construction of recombinant plasmid pF47 The plasmid construction method in this embodiment is the same as that in Embodiment 1, except that the promoter is replaced by ernEp* instead of kasOp*.

[0087] Using the ermEp* promoter as a template, the ermEp* promoter was amplified using the upstream primer F47-erm-F, 5'-gttcgtcgtagccgaaatccacTGGATCCTACCAACCGGCAC' (SEQ ID NO. 17) and the downstream primer F47-erm-R, 5'-gtctatcggtgtgggtggtcGCGAGTGTCCGTTCGAGTGG' (SEQ ID NO. 18). The recombinant plasmid pF47 was obtained via Gibson assembly. A schematic diagram of pF47 plasmid construction is shown below. Figure 6 .

[0088] 3.2 Construction of a strain overexpressing ermEp*-ACADM double-crossover at position 1 of homologous arm The strain construction and testing methods in this embodiment are the same as those in Example 1, except that the promoter is replaced by ernEp* instead of kasOp*.

[0089] A schematic diagram of the construction of the ermEp*-ACADM double-crossover strain overexpressing at position 1 of homologous arm is shown below. Figure 7 Multiple candidate double-crossover strains were validated using upstream primer F20-QC-F3, 5'-GGACCGTTCCTTCCGTCTAC-3' (SEQ ID NO. 16) and downstream primer F20-QC-R1, 5'-gaggcgtccgacggctac-3' (SEQ ID NO. 15) as validation primers. The validation group showing a 2355bp agarose gel electrophoresis result was the strain correctly overexpressing ermEp*-ACADM double-crossover at position 1 of the homologous arm. This strain was named F47-S1. Figure 8 ).

[0090] The fermentation yield of the overexpressing strain F47-S1 was detected using the same fermentation method and detection conditions as in Example 1. The results showed that the average spinosad yield of the starting strain *Polyspora sacchariformis* NRRL 18395 was 102.41 mg / L, while the spinosad yield of the F47-S1 strain overexpressing ermEp*-ACADM at position 1 of the homologous arm was 180.91 mg / L, representing a 76.65% increase compared to the starting strain *Polyspora sacchariformis* NRRL18395. Figure 9 ) Example 4: Overexpression of ermEp*-ACADM at position 1 of homologous arm increases spinosad production in Polysporus spp. WHU1107 The same method as in Example 3 was used to construct a recombinant strain of Polysaccharidobacterium sarcodactylon WHU1107 that overexpressed ermEp*-ACADM at position 1 of the homologous arm. The difference was that the Polysaccharidobacterium sarcodactylon NRRL18395 in Example 3 was replaced with the high-yielding strain Polysaccharidobacterium sarcodactylon WHU1107. The resulting modified strain was named F47-S2.

[0091] The fermentation yield of the overexpressing strain F47-S2 was detected using the same fermentation method and detection conditions as in Example 1. The results showed that the average spinosad yield of the starting strain *Polyspora sacchariformis* WHU1107 was 4346.57 mg / L, while the spinosad yield of the obtained strain F47-S2, which overexpressed ermEp*-ACADM at position 1 of the homologous arm, was 6246.70 mg / L, representing a 43.72% increase compared to the starting strain *Polyspora sacchariformis* WHU1107. Figure 10 ) Example 5: Overexpression of kasOp*-ACADM at position 2 of homologous arm increases spinosad production in Polysporus saccharidus NRRL18395 5.1 Construction of recombinant plasmid pF48 The plasmid construction method in this embodiment is the same as that in Example 1, except that: the left homologous arm 1 and the right homologous arm 1 are replaced with the left homologous arm 2 and the right homologous arm 2: the left homologous arm 2 is amplified by the upstream primer F48-Larm-F, 5'-cgacggccagtgccaagcttgtgctggagggcacgctggag-3' (SEQ ID NO. 19) and the downstream primer F48-Larm-R, 5'-tcacgccgccggcctcgaac' (SEQ ID NO. 20).

[0092] The right homologous arm 2 was amplified using upstream primer F48-Rarm-F, 5'-ttagcgcagctcgtctgcgg-3' (SEQ ID NO. 21) and downstream primer F48-Rarm-R, 5'-gctatgacatgattacgaattcgtgcagacccggattccgga-3' (SEQ ID NO. 22). The recombinant plasmid pF48 was obtained via fragment Gibson assembly. A schematic diagram of pF48 plasmid construction is shown below. Figure 11 .

[0093] 5.2 Construction of strains overexpressing kasOp*-ACADM at position 2 of homologous arm The strain construction and testing methods in this embodiment are the same as those in Example 1, except that: left homologous arm 1 and right homologous arm 1 are replaced with left homologous arm 2 and right homologous arm 2.

[0094] A schematic diagram of the construction of the kasOp*-ACADM double-crossover strain overexpressing at position 2 of homologous arm is shown below. Figure 12 Multiple candidate double-crossover strains were validated using upstream primer F48-QC-F, 5'-tcgactacgacttcacagaac-3' (SEQ ID NO. 23) and downstream primer F48-QC-R, 5'-tcggacaagacctgcacgatc-3' (SEQ ID NO. 24) as validation primers. The validation group showing a 1970bp agarose gel electrophoresis result was the strain correctly overexpressing kasOp*-ACADM double-crossover at position 2 of the homologous arm; this strain was named F48-S1. Figure 13 ).

[0095] The fermentation yield of the overexpressing strain F48-S1 was detected using the same fermentation method and detection conditions as in Example 1. The results showed that the average spinosad yield of the starting strain *Polyspora sacchariformis* NRRL 18395 was 102.41 mg / L, while the spinosad yield of the F48-S1 strain overexpressing kasOp*-ACADM at position 2 of the homologous arm was 161.08 mg / L, representing a 57.28% increase compared to the starting strain *Polyspora sacchariformis* NRRL18395. Figure 14 ) Example 6: Overexpression of kasOp*-ACADM at position 2 of homologous arm increases spinosad production in Polysaccharidobacterium spinosum WHU1107 The same method as in Example 5 was used to construct a recombinant strain of Polysaccharidobacterium sarcodactylon WHU1107 that overexpressed kasOp*-ACADM at position 2 of the homologous arm. The difference was that Polysaccharidobacterium sarcodactylon NRRL18395 in Example 5 was replaced with the high-yielding strain Polysaccharidobacterium sarcodactylon WHU1107. The resulting modified strain was named F48-S2.

[0096] The fermentation yield of the overexpressing strain F48-S2 was detected using the same fermentation method and detection conditions as in Example 1. The results showed that the average spinosad yield of the starting strain *Polyspora sacchariformis* WHU1107 was 4346.57 mg / L, while the spinosad yield of the F48-S2 strain, which overexpressed kasOp*-ACADM at position 2 of the homologous arm, was 5352.98 mg / L, representing a 23.15% increase compared to the starting strain *Polyspora sacchariformis* WHU1107. Figure 15 ) The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An application of the ACADM gene in increasing spinosad production in *Saccharomyces cerevisiae*, wherein the application involves overexpressing the ACADM gene in *Saccharomyces cerevisiae*; The amino acid sequence encoded by the ACADM gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the ACADM gene is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The *Saccharomyces cerevisiae* is either *Saccharomyces cerevisiae* NRRL18395 or WHU1107. And / or, the spinosad includes spinosad A and spinosad D; And / or, the overexpression of the ACADM gene is achieved through homologous recombination, integrating it into the genome of *Saccharomyces cerevisiae*. The homologous arms used in the homologous recombination include one of homologous arm 1 and homologous arm 2. Homologous arm 1 includes a left homologous arm 1 and a right homologous arm 1. The sequence of the left homologous arm 1 is shown in SEQ ID NO. 3, and the sequence of the right homologous arm 1 is shown in SEQ ID NO.

4. Homologous arm 2 includes a left homologous arm 2 and a right homologous arm 2. The sequence of the left homologous arm 2 is shown in SEQ ID NO. 5, and the sequence of the right homologous arm 2 is shown in SEQ ID NO.

6. And / or, when the ACADM gene is overexpressed, a strong promoter is attached upstream of the ACADM gene; the strong promoter includes one of the kasOp* promoter and the ermEp* promoter.

4. An engineered bacterium that produces high levels of spinosad, characterized in that, The engineered bacteria were obtained by overexpressing the ACADM gene in a strain of Polysporum spinosum. The amino acid sequence encoded by the ACADM gene is shown in SEQ ID NO.

2.

5. An expression cassette for overexpressing the ACADM gene, characterized in that, The expression box is one of expression box 1, expression box 2, and expression box 3; The expression cassette 1 contains a homologous arm 1, a kasOp* promoter, and an ACADM gene expressed by the promoter. The expression cassette 2 contains a homology arm 1, an ermEp* promoter, and an ACADM gene expressed by the promoter. The expression cassette 3 contains a homologous arm 2, a kasOp* promoter, and an ACADM gene expressed by the promoter. The amino acid sequence encoded by the ACADM gene is shown in SEQ ID NO.2; Homologous arm 1 includes a left homologous arm 1 and a right homologous arm 1; the sequence of the left homologous arm 1 is shown in SEQ ID NO.3, and the sequence of the right homologous arm 1 is shown in SEQ ID NO.4; homologous arm 2 includes a left homologous arm 2 and a right homologous arm 2; the sequence of the left homologous arm 2 is shown in SEQ ID NO.5, and the sequence of the right homologous arm 2 is shown in SEQ ID NO.

6.

6. A vector or recombinant plasmid, characterized in that, The vector or recombinant plasmid contains the expression cassette as described in claim 5.

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