Engineering modified saccharopolyspora spinosa and method for producing spinosad by using engineering modified saccharopolyspora spinosa

Abstract

The invention provides engineering modified saccharopolyspora spinosa and a method for producing spinosad by using the engineering modified saccharopolyspora spinosa. The saccharopolyspora spinosa overexpresses g6pdh-1. The content of NADPH (nicotinamide adenine dinucleotide phosphate) in the modified saccharopolyspora spinosa is increased, and the content of acyl coenzyme is increased, so that the synthesis of a lactone ring is improved, and finally, the increase of the yield of spinosad is realized. The transformed saccharopolyspora spinosa can greatly improve the yield of spinosad, and is of great significance to industrial production of spinosad.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an engineered polysporum oryzae and a method for producing spinosad. Background Technology

[0002] Spinosad is an intracellular secondary metabolite produced by the soil actinomycete *Saccharomyces cerevisiae* through aerobic fermentation. It belongs to the novel macrolide antibiotics. Due to its unique chemical structure and mechanism of action, spinosad can effectively control various lepidopteran pests such as the diamondback moth and beet armyworm, while remaining non-toxic to mammals and birds. However, the low yield of spinosad restricts its industrial production and widespread application. To date, researchers have explored various methods to increase spinosad yield, but genetic modification of *Saccharomyces cerevisiae* is difficult. Improvements to spinosad-producing strains have largely focused on strain mutagenesis. Despite these efforts, yields have remained below 1 g / L. Therefore, obtaining high-yielding spinosad strains has become a crucial research topic in recent years.

[0003] Streptomyces can produce abundant secondary metabolites. Each secondary metabolite is obtained through strain-specific synthesis and modification by specific enzymes, and its precursors and cofactors are derived from primary metabolism. Therefore, primary metabolism can have a significant impact on secondary metabolism. In Streptomyces, there are reports on increasing the yield of secondary metabolites by regulating cofactors produced in primary metabolism, such as ATP and NADPH: Li et al. (Li XB, Chen J, Andersen JM, et al. Cofactor engineering redirects secondary metabolism and enhances erythromycin production in Saccharopolyspora ery-thraea[J].ACS Synthetic Biology.2020,9(3):655–670) increased the yield of erythromycin by 28% by altering the ATP content of erythromycin-producing strains through genetic engineering. Zheng Zijing et al. (Zheng Zijing, Yu Lan, Tang Zhenyu, et al. Effects of blocking the zwf1 gene in industrial Streptomyces lividans on oxytetracycline biosynthesis [J]. Chinese Journal of Antibiotics. 2012, 37: 35-38.) reported that blocking the zwf1 gene in industrial Streptomyces lividans increased oxytetracycline production by 36.2%. Jin et al. (Jin XM, Chang YK, Lee JH, et al. Effects of increased NADPH concentration by metabolic engineering of thepentose phosphate pathway on antibiotic production and sporulation in Streptomyces lividans TK24 [J]. Journal of Microbiology and Biotechnology. 2017, 27: 1867-1876.) reported that heterologous expression of the zwf1, zwf2, and zwf3 genes of Streptomyces lividans in Streptomyces lividans increased the amount of undecylpyridin by 180%.

[0004] Therefore, starting from the precursors of spinosad synthesis, exploring a method and application to increase the yield of spinosad synthesis from *Spinosa multispora* is of great significance for the industrial production of spinosad. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an engineered *Saccharomyces cerevisiae* strain and a method for producing spinosad. The engineered *Saccharomyces cerevisiae* strain overexpresses g6pdh-1, increasing the NADPH level within the strain. NADPH, as a cofactor, facilitates the synthesis of acyl-CoA precursors. These precursors undergo condensation with polyketide synthase to form a lactone ring structure. This multi-component lactone ring structure, along with two hexaglycoses, constitutes spinosad. Therefore, overexpression of g6pdh-1 in *Saccharomyces cerevisiae* effectively increases the yield of spinosad, which has profound significance for the industrial production of spinosad.

[0006] In a first aspect, the present invention provides an engineered *Saccharomyces cerevisiae* strain that overexpresses g6pdh-1 and / or g6pdh-2, preferably, the strain overexpresses g6pdh-1.

[0007] The amino acid sequence of g6pdh-1 contains SEQ ID NO:1 or has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO:1.

[0008] The amino acid sequence of g6pdh-2 includes SEQ ID NO:2 or has 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with SEQ ID NO:2.

[0009] The g6pdh-1 and g6pdh-2 mentioned above encode glucose-6-phosphate dehydrogenase.

[0010] The g6pdh-1 and / or g6pdh-2 are expressed on plasmids or on chromosomes.

[0011] Preferably, the expression vector carrying g6pdh-1 and / or g6pdh-2 is introduced into Polysporus spp.

[0012] The overexpression includes increasing the promoter strength of g6pdh-1 and / or g6pdh-2, and / or introducing g6pdh-1 and / or g6pdh-2 into *Saccharomyces cerevisiae*.

[0013] A second aspect of the present invention provides a method for constructing *Saccharomyces cerevisiae* as described in the first aspect, the method comprising inserting the g6pdh-1 gene and / or the g6pdh-2 gene into an expression vector, and transferring the expression vector into *Saccharomyces cerevisiae*.

[0014] Preferably, the construction method includes inserting the g6pdh-1 gene into an expression vector and transforming the expression vector into Polysporus spp.

[0015] The expression vector is selected from pSET152, pNZ8148, pET28a, pNZ8048, and pNZ8150, with pSET152 being the preferred expression vector.

[0016] The g6pdh-1 or g6pdh-2 gene is regulated by a promoter, which includes an inducible promoter and / or a constitutive promoter. Preferably, the promoter is a constitutive promoter.

[0017] The inducible promoter is selected from P lux promoter, P lac One or more of the following: promoter, phaP promoter, or fadBA promoter;

[0018] The constitutive promoters mentioned are selected from wild-type P porin Or its mutants, ermE*, elongation factor-1α, cytomegalovirus promoter, ubiquitin C promoter or β-actin promoter, or one or more of these.

[0019] In one specific embodiment of the present invention, the promoter is ermE*.

[0020] A third aspect of the present invention provides a method for producing spinosad, the method comprising culturing *Polysporium spinosum* as described in the first aspect or *Polysporium spinosum* obtained by the construction method described in the second aspect.

[0021] The fermentation medium for culturing the *Saccharomyces cerevisiae* includes yeast extract, anhydrous glucose, peptone, cottonseed meal, dextrin, soybean oil, NaCl, MgSO4, KH2PO4, and CaCO3.

[0022] The concentration of the yeast extract is 1-30 g / L, preferably 2-20 g / L, such as 1 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, or 30 g / L.

[0023] The concentration of the anhydrous glucose is 30-90 g / L, preferably 40-80 g / L, such as 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, or 90 g / L.

[0024] The concentration of peptone is 1-30 g / L, preferably 2-20 g / L, such as 1 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, or 30 g / L.

[0025] The concentration of the cottonseed powder is 10-50 g / L, preferably 15-40 g / L, such as 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L.

[0026] The concentration of the dextrin is 10-50 g / L, preferably 15-40 g / L, for example 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L.

[0027] The concentration of the soybean oil is 0.01-10 g / L, preferably 0.1-5 g / L, such as 0.01 g / L, 0.1 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L.

[0028] In one specific embodiment of the present invention, the fermentation culture medium comprises 10 g / L yeast extract, 60 g / L anhydrous glucose, 10 g / L peptone, 20 g / L cottonseed meal, 20 g / L dextrin, and 1 g / L soybean oil.

[0029] The concentration of NaCl is 0.1-5 g / L, preferably 1 g / L; the concentration of MgSO4 is 0.01-1 g / L, preferably 0.5 g / L; the concentration of KH2PO4 is 0.01-1 g / L, preferably 0.5 g / L; and the concentration of CaCO3 is 1-5 g / L, preferably 3 g / L.

[0030] The pH of the fermentation medium is 6.8-7.8, preferably 7-7.6, such as 6.8, 7, 7.2, 7.4, 7.6, 7.8.

[0031] In one specific embodiment of the present invention, the pH of the fermentation medium is 7.2.

[0032] The spinosad mentioned includes spinosad A and spinosad D.

[0033] A fourth aspect of the present invention provides a method for producing spinosad, the method comprising regulating the pentose phosphate pathway of Polysaccharidobacterium spp.

[0034] The described pentose phosphate pathway for regulating *Saccharomyces cerevisiae* includes the introduction of g6pdh-1 and / or g6pdh-2, preferably, the pentose phosphate pathway for regulating *Saccharomyces cerevisiae* is the introduction of g6pdh-1.

[0035] A fifth aspect of the present invention provides a method for improving the supply of lactone ring precursors in *Saccharomyces cerevisiae*, the method comprising introducing g6pdh-1 and / or g6pdh-2 into *Saccharomyces cerevisiae*; preferably, the method comprises introducing g6pdh-1 into *Saccharomyces cerevisiae*.

[0036] In a sixth aspect, the present invention provides a method for increasing the content of acyl-CoA in *Saccharopolysacchariformis*, the method comprising introducing g6pdh-1 and / or g6pdh-2 into *Saccharopolysacchariformis*; preferably, the method comprises introducing g6pdh-1 into *Saccharopolysacchariformis*.

[0037] A seventh aspect of the present invention provides the application of g6pdh-1 in improving the production capacity of spinosad by Polysaccharidobacterium sarcodactylis, wherein Polysaccharidobacterium sarcodactylis highly expresses g6pdh-1.

[0038] The amino acid sequence of the g6pdh-1 contains SEQ ID NO: 1.

[0039] An eighth aspect of the present invention provides an expression vector comprising g6pdh-1 and / or g6pdh-2.

[0040] The expression vector further includes a promoter, which includes an inductive promoter and / or a constitutive promoter. Preferably, the promoter is a constitutive promoter.

[0041] The inducible promoter is selected from P lux promoter, P lac One or more of the following: promoter, phaP promoter, or fadBA promoter;

[0042] The constitutive promoters mentioned are selected from wild-type P porin Or its mutants, ermE*, elongation factor-1α, cytomegalovirus promoter, ubiquitin C promoter or β-actin promoter, or one or more of these.

[0043] The expression vector is selected from pSET152, pNZ8148, pET28a, pNZ8048, and pNZ8150, with pSET152 being the preferred expression vector.

[0044] Beneficial effects:

[0045] The lactone ring structure in spinosad is obtained by condensing short-chain fatty acids using polyketide synthase. The biosynthesis of fatty acids requires the cofactor NADPH as a hydrogen donor. This invention uses *Polyspora spp.* as the starting species and increases the amount of NADPH by enhancing the expression of the g6pdh-1 gene, thereby increasing the supply of the lactone ring precursor and ultimately improving the spinosad production capacity of *Polyspora spp.*

[0046] (1) This invention utilizes bioinformatics to discover two glucose-6-phosphate dehydrogenase genes, g6pdh-1 and g6pdh-2, in the genome of *Saccharopolyspora*. Using molecular biology methods, g6pdh-1 and g6pdh-2 genes were cloned into the shuttle vector pSET152-PermE* containing a strong promoter. Overexpression of these genes in *Saccharopolyspora* was then achieved using conjugative transfer. The results showed that enhanced expression of g6pdh-1 increased NADPH levels in *Saccharopolyspora*.

[0047] (2) The *Saccharomyces cerevisiae* strain constructed in this invention overexpresses g6pdh-1, which increases the NADPH level in the bacteria, increases the amount of acyl-CoA, and then increases the synthesis of lactone rings, ultimately increasing the yield of spinosad.

[0048] (3) In the shake flask fermentation of the present invention, the yield of spinosad reached 1.2 g / L, and in the fermenter experiment, the yield of spinosad was as high as 2.1 g / L, which is the highest yield reported in the present invention.

[0049] The above only summarizes some aspects of the present invention and is not, and should not be considered as limiting the present invention in any way.

[0050] All patents and publications mentioned in this application are incorporated herein by reference in their entirety. Those skilled in the art will recognize that certain modifications can be made to this invention without departing from its spirit or scope. The following embodiments further illustrate the invention in detail and should not be considered as limiting the scope of the invention or the specific methods described herein. Attached Figure Description

[0051] Figure 1 Alignment of protein sequences encoded by Streptomyces zwf1 and Polysporus saccharidus g6pdh-1 and g6pdh-2.

[0052] Figure 2 Agarose gel electrophoresis image of PCR verification of the g6pdh expression plasmid.

[0053] Figure 3 Screening plate diagram of engineered strains.

[0054] Figure 4 : NAPH content in Polysporum spp.

[0055] Figure 5 : Production of spinosad in shake flasks by original and engineered strains.

[0056] Figure 6 : Production figures of spinosad fermentation tank culture of original and engineered strains. Detailed Implementation

[0057] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] The present invention will be described in detail below by way of examples.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0060] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0061] The strains, culture media, and experimental methods involved in the examples are as follows:

[0062] 1. Strains

[0063] Saccharopolyspora spinosa YK219 is a spinosad-producing strain, deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO: 64919.

[0064] Escherichia coli BL21(DE3) was used for protein purification and expression.

[0065] Escherichia coli S17-1 was used for conjugation transfer of Polysporus spp.

[0066] 2. Culture medium

[0067] LB medium for Escherichia coli: NaCl 10 g / L, peptone 10 g / L, yeast extract 5 g / L;

[0068] MS medium for Polysporus spp.: agar powder 20 g / L, mannitol 20 g / L, soybean powder 20 g / L;

[0069] Spore-forming medium for Polysporus spp.: anhydrous glucose 5 g / L, yeast extract 3 g / L, whole milk powder 20 g / L, MgSO4·7H2O 2 g / L, agar powder 15 g / L, adjusted pH to 7.2;

[0070] Seed culture medium for Polysporus sacchariformis: yeast extract 3 g / L, maltose 4 g / L, anhydrous glucose 5 g / L, MgSO4 2 g / L, pH adjusted to 7.2;

[0071] Fermentation medium for *Saccharomyces cerevisiae*: yeast extract 10 g / L, anhydrous glucose 60 g / L, peptone 10 g / L, cottonseed meal 20 g / L, dextrin 20 g / L, soybean oil 1 g / L, NaCl 1 g / L, MgSO4 0.5 g / L, KH2PO4 0.5 g / L, CaCO3 3 g / L, adjusted to pH 7.2.

[0072] 3. Experimental Methods

[0073] 3.1 Conjugation Transfer Procedure between Escherichia coli and Polysporus spp.

[0074] Escherichia coli S17-1 was selected as the donor strain, and 25 μg / mL kanamycin and 25 μg / mL erythromycin were added during culture. Because the pCMint2-ermE* plasmid contains abelasma resistance, 50 μg / mL abelasma resistance was also added during culture to ensure that the plasmid was not lost.

[0075] The recipient strain, *Saccharomyces cerevisiae* YK219, was selected, and its spore suspension was used for conjugation transfer. The spore suspension preparation process is as follows: (1) 100 μL of spore suspension stored in the refrigerator was spread onto a slant sporulation medium and incubated at 30°C for 5 days. (2) Spores were scraped off using an inoculation frame and placed in 2×YT liquid medium. Sterile glass beads were added, and the mixture was vortexed for 5 min to fully disperse the spores. The spore suspension was obtained by filtration (the number of spores was 10⁸ after plate counting). (3) The spore suspension was dispensed into EP tubes at 500 μL / tube and incubated at 30°C and 250 rpm for 24 h for later use.

[0076] For conjugation transfer, donor and recipient bacteria were mixed at a 1:10 ratio, centrifuged at 8000 rpm for 5 min, and 200 μL was spread onto 2× carboxymethyl cellulose sodium (CMC) medium and incubated at 30 °C. At 16 h, antibiotic solution was applied for covering. The mixture was covered with 1 mL of the appropriate antibiotic solution (12.5 μg / mL abelasavir and 30 μg / mL nalidixic acid), dried, and incubated at 30 °C for approximately 10 days to observe the growth of the conjugates.

[0077] Conjugates were picked and placed on BHI solid plates containing 12.5 μg / mL abelasin and 30 μg / mL nalidixic acid. After incubation at 30°C for about one week, the conjugates were transferred again to BHI solid plates containing 25 μg / mL abelasin. This confirmed that the conjugate strain had been successfully transferred. The strain was then cultured on slant sporulation medium to prepare a spore suspension for fermentation testing.

[0078] 3.2 Construction of recombinant plasmids

[0079] Using the genome of *Polysporium spp.* as a template, the g6pdh1 and g6pdh2 fragments were amplified using primers containing corresponding restriction enzyme sites (g6pdh1-F / g6pdh1-R and g6pdh2-F / g6pdh2-R). The PCR products were detected by 1% agarose gel electrophoresis. The target fragments were recovered, ligated into the pCR-Blunt vector, transformed into *E. coli* DH10B competent cells, and screened using blue-white screening. Restriction enzyme digestion with NdeI and EcoRI was then performed for identification, and positive clones were selected for sequencing.

[0080] Positive clones with correct sequencing were selected and amplified. Plasmids were extracted using a plasmid extraction kit. pCR-Blunt-g6pdh1 / 2 and the expression vector pSET152-PermE* were double-digested with NdeI and EcoRI, and the gene and vector fragments were recovered. The digested g6pdh-1 / 2 gene fragment was ligated to the vector pSET152-PermE* using T4 ligase. The ligation was performed into *E. coli* competent cells DH10B. Positive clones were selected, amplified, and plasmids were extracted. After confirming correct enzyme digestion, the recombinant plasmid pSET152-PermE*-g6pdh1 / 2 was obtained and transformed into *E. coli* S17-1.

[0081] 3.3 Construction and Validation of Engineered Strains

[0082] The donor strain S17-1 containing the target plasmid pSET152-PermE*-g6pdh1 / 2 was mixed with the pre-germination spore solution of the recipient strain *Polyspora spp.* and spread on MS medium plates containing apramycin and streptomycin at final concentrations of 50 and 100 μg / mL, respectively. The mixture was incubated at 30°C for 7-10 days to obtain white conjugates. The conjugates were verified by enzyme digestion and sequencing. Positive conjugates were selected to obtain the modified strains *Polyspora spp.* YK219-G2 / YK219-G3.

[0083] 3.4 Fermentation culture of engineered strains and verification of plasmid stability

[0084] Single colonies of the engineered strains were randomly selected and inoculated into 5 mL of seed culture medium, and cultured at 260 rpm and 30 °C for 3 days. Then, a 1% inoculum was transferred from the seed culture medium to 25 mL of fermentation medium, and cultured at 260 rpm and 30 °C for 7 days. The spinosad titer of the fermentation broth was determined, with three replicates for each strain. The modified strains YK219-G2 and K219-G3 were inoculated into fermentation medium and fermented in shake flasks at 30 °C and 200 rpm for 48 h. They were then subcultured at a 15% inoculum in fresh medium for 10 generations. Afterward, the bacterial cells were collected, genomic DNA was extracted, and plasmid stability of the modified strains was verified by apramycin resistance testing.

[0085] 3.5 Determination of NADPH

[0086] Collect 3 mL of sample solution, centrifuge at 5000 x g for 10 min at 4 °C, and remove the supernatant. Add 300 μL of phosphate buffer (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM and KH2PO4 2 mM, pH 7.4), sonicate on ice for 3 min, then centrifuge at 15000 x g for 30 min at 4 °C, and remove the supernatant. Take 250 μL of precipitate, add 100 μL of Tris HCl (0.1 M, pH 8.0), 50 μL of MgCl2 (0.01 M), 50 μL of glucose-6-phosphate (0.0006 M), and 50 μL of NADP (0.0002 M), mix well, and react at 28 °C for 2 min for HPLC detection. Use gradient elution with a mobile phase of 0.1 M KH2PO4 (pH 6.0) and 0.1 M KH2PO4 (pH 6.0) containing 10% (V / V) CH3OH. The detection wavelength is 254 nm, and the flow rate is 1.3 mL / min.

[0087] 3.6 HPLC determination of spinosad yield

[0088] Sample preparation: Most of the spinosad synthesized during the fermentation of *Saccharomyces cerevisiae* is present in the fermentation broth, with a small amount remaining in the mycelium. Therefore, the method of extracting the entire fermentation broth was adopted. Take 5 mL of fermentation broth, add 5 mL of methanol, mix well, sonicate for 30 min, centrifuge at 10000 r / min for 10 min, and take the supernatant. Filter the supernatant through a 0.22 μm microporous membrane and perform HPLC analysis.

[0089] HPLC conditions: reversed-phase C18 column, mobile phase: methanol:acetonitrile:water = 40:55:5 (0.05% ammonium hexanoate), flow rate 0.5 mL / min, column temperature 30℃, injection volume 10 μL, UV scanning wavelength 246 nm.

[0090] Example 1: Identification of the glucose-6-phosphate dehydrogenase gene in *Saccharomyces cerevisiae*

[0091] The zwf1 gene (accession number: NP_626202.1) encoding glucose-6-phosphate dehydrogenase in *Streptomyces aquamarine* was compared with the genome sequence of *Polyspora sacchariformis*. Sequence alignment revealed 72% and 42% similarity to the protein sequences encoded by the g6pdh-1 gene (accession number: WP_101376973.1) and g6pdh-2 gene (accession number: WP_010314638.1), respectively. Figure 1 Therefore, it is speculated that these two genes are responsible for encoding glucose-6-phosphate dehydrogenase in *Saccharomyces cerevisiae*.

[0092] The amino acid sequence of g6pdh-1 is SEQ ID NO: 1:

[0093] MTRSEQWHNPLRDPRDKRLPRIAGPCGLTIFGVTGDLSRKKLMPAIYDLANRGLLPPGFALTGFARRDWADQDFGQVVYDAVREHARTPFHQNVWDRLAEGIRFVPGSFDDDDAFDRLAETVKQLDA ERGTGGNHAFYLSVPPSAFPTVLKQLSRSGLTDQSGDSWRRVVIEKPFGHDLESAQQLNGIVNEVFPEESVFRIDHYLGKETVQNILALRFANQLFEPIWNAHYVDHVQITMAEDIGLGGRAGYYDGI GAARDVIQNHLLQLLAFTAMEEPVSSFSPQDLRAEKIKVLSATKPVGPFDQTTARGQYTGGWQGGSLVPGLHEEGGFASDSITETYAAITLEVESRRWAGVPFYLRSGKRLGRRVTEIAVVFKRAPHLP FDSTTMTEELGQNALVIRVQPDEGITMRFGAKVPGTSMEVRDVTMDFGYGHAFTESSPEAYERLILDVLLGEPSLFPVNEEVELSWQILDPVLDHWSANGKPEPYKAGTWGPPSADAMLARTGRVWRRP

[0094] The amino acid sequence of g6pdh-2 is SEQ ID NO: 2:

[0095] MTRPDDHVIVLFGATGDLAKRKLLPGLFHLACAGLLPDGYRIVGSGRAHSALNDDAFRKHAHDAVSTFGRCEPSGPVWESFADSLSFAASDPPDAGPLLAAVRAAEDSLGGQPRRLFHLAVPPAAFGSIIGMLGDTGLAEKSKVIVEKPFGTDLASARALNQTIHAVFDESRIFRIDHFLGKESVDNILALRFANGMFEPIWNRDHISHVQIDVPETLGLEGRAHFYEGTGAFRDMIVTHLIQVLGIVAMEPPVSLAAQPLRDEKAKVFAALRPIETGHVVRGQYDGYRDEPGVAPDSQTETFTALRIEVDNWRWAGVPFYLRSGKKLAQHHEVITLGLREPPLRMFPIDLPQQENYRSNKIVIDFADPGWIAACFLAKEPGPTMRLAPEAMTFRYADSFCQEHGLEGYERLILDAMLGDQSLFTRSDGIERIWEASTPLLENPPPVHLYAPGSWGPEPAVSELIAPHQWYLEDAGDVVRR

[0096] The amino acid sequence of zwf1 is SEQ ID NO: 3:

[0097] MSSSNPLRDPADRRLPRIAGPSGLVIFGVTGDLSRKKLMPAVYDLANRGLLPPGFSLVGFARRDWEHEDFAQVVHDAVKEHSRTPFREEVWQQLIQGMRFVQGTFDDDDAFERLRGTIEELDKAQG TGGNFAFYLSVPPKSFPVVIQQLKKHGLADQTNGSWRRAVIEKPFGHDLKSAEELNAIVHEVFGSDQVFRIDHYLGKETVQNILALRFANTMFEPIWNRSYVDHIQITMAEDIGIGGRAGYYDGIGA ARDVIQNHLLQLLALTAMEEPASFDADALAAEKTKVLGAVRLPKDLGRDTVRGQYAAGWQGGAKAVGYLEEDGIDPKSKTDTYAAIKVGIDNRRWAGVPFYLRTGKRLGRRVTEIAVVFQRAPHSPF DTTATEELGSNAIVIRVQPDEGVTVRFGSKVPGTSMEIRDVSMDFAYGESFTESSPEAYERLILDVLLGDANLFPRTEEVELSWKILDPIEEYWDEHGTPAQYPAGTWGPVEADDMLERDGRSWRRP

[0098] Example 2: Construction of Polysporus spp. overexpressing glucose-6-phosphate dehydrogenase

[0099] First, the g6pdh-1 and g6pdh-2 genes from *Polysporium spp.* were cloned, and a constitutive strong promoter, ermE*, was added upstream of these genes. Then, expression vectors pSET152-PermE*-g6pdh1 and pSET152-PermE*-g6pdh2 were constructed. PCR verification of these vectors detected g6pdh-1 and g6pdh-2 fragments of approximately 1500 bp. Figure 2 The two expression vectors were then transformed into *Saccharomyces cerevisiae* YK219 via conjugation transfer, and positive conjugates YK219-G2 and YK219-G3 were obtained after antibiotic screening. Figure 3 ).

[0100] Example 3: Fermentation culture of *Saccharomyces cerevisiae* modified by engineering

[0101] First, shake-flask fermentation was performed on *Polysporium spp.* YK219 and its engineered strain. Fermentation broth was collected after 7 days of fermentation and, after multiple treatments, the NADPH content in the organisms was measured. In the two engineered strains, the NADPH content in YK219-G3 spores remained essentially unchanged compared to YK219, while the NADPH content in YK219-G2 was more than double that of YK219. Figure 4 The g6pdh-1 gene encodes glucose-6-phosphate dehydrogenase, an important gene in the pentose phosphate pathway (PPP pathway). When the g6pdh-1 gene is overexpressed, the corresponding enzyme content increases, and the NADPH level of *Saccharomyces cerevisiae* increases accordingly. The reason why the NADPH level of *Saccharomyces cerevisiae* overexpressing the g6pdh-2 gene did not increase may be that the dehydrogenase encoded by the g6pdh-2 gene is not involved in the PPP pathway of *Saccharomyces cerevisiae*, which was unexpected by the inventors. Furthermore, when detecting NADPH levels in *Saccharomyces cerevisiae* at different time points, it was found that the NADPH level of YK219-G2 increased rapidly after three days of fermentation, while strains YK219 and YK219-G3 did not show this trend.

[0102] After fermentation, the fermentation broth was subjected to multiple treatments, and the spinosad yield in *Saccharomyces cerevisiae* was determined by HPLC. The results are as follows: Figure 5 As shown, strain YK219-G2 exhibited a significantly increased spinosad yield, 19.6% higher than YK219, reaching 1.2 g / L, while the yield of YK219-G3 remained essentially the same as that of YK219. This indicates that the higher intracellular NADPH availability of YK219-G2 effectively enhances spinosad production. Comparing the spinosad synthesis capabilities of YK219 and YK219-G2 in a 50L fermenter, the results showed that on day 7 of fermentation, YK219-G2 achieved a spinosad yield of 4.2 g / L, a 50% increase compared to YK219 (2.8 g / L). Figure 6 (This represents a significant improvement for industrial strains producing spinosad, which is beneficial for increasing industrial output.)

[0103] Experimental results show that increasing the NADPH content in *Saccharomyces cerevisiae* spores can increase spinosad production. Overexpression of the g6pdh-1 gene can accelerate the conversion of glucose-6-phosphate to gluconolactone, enhance the expression level of the PPP pathway, produce a large amount of NADPH, increase the supply of acyl-CoA precursors, promote the conversion of acyl-CoA to macrolides, and thus increase the production of spinosad from *Saccharomyces cerevisiae*.

Claims

1. An engineered Emericella species, characterized in that, The g6pdh-1 overexpressed in the Saccharopolyspora contains an amino acid sequence of SEQ ID NO:

1.

2. The method of constructing Actinosynnema pretiosum according to claim 1, characterized in that, The method comprises inserting the g6pdh-1 gene into an expression vector and transforming the expression vector into the Saccharopolyspora.

3. The construction method of claim 2, wherein, The expression vector is selected from pSET152, pNZ8148, pET28a, pNZ8048, pNZ8150.

4. The construction method of claim 2, wherein, The g6pdh-1 gene is regulated by a promoter, and the promoter comprises an inducible promoter and / or a constitutive promoter.

5. The construction method according to claim 4, characterized in that, The inducible promoter is selected from one or more of Plux promoter, Plac promoter, phaP promoter or fadBA promoter; and the constitutive promoter is selected from one or more of wild-type Pporin or a mutant thereof, ermE*, elongation factor-1 alpha, cytomegalovirus promoter, ubiquitin C promoter or beta-actin promoter.

6. A method of producing a spinosyn, characterized by, The method comprises culturing the engineered bacteria of claim 1 or the engineered bacteria obtained by the construction method of claim 2.

7. The method of claim 6, wherein, The spinosyn is spinosyn A and spinosyn D.

8. A method of increasing the supply of lactone ring precursors in E. siraeus, comprising, The method comprises introducing g6pdh-1 into the Saccharopolyspora.

9. A method for increasing the content of acyl-CoA in Zymobacterium, characterized by, The method comprises introducing g6pdh-1 into the Saccharopolyspora.

10. Use of g6pdh-1 to improve the capacity of L. stipitatus to produce spinosyn, characterized in that, The g6pdh-1 overexpressed in the Saccharopolyspora contains an amino acid sequence of SEQ ID NO: 1.

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