Construction method of saccharopolyspora tuberosa high-yield genetic engineering bacteria

Abstract

The invention discloses a construction method of saccharopolyspora tuberosa high-yield genetic engineering bacteria, and belongs to the technical field of genetic engineering, the construction method comprises the following steps: by taking a strain genome of a mutant strain S.pogona aG6 as a template, transferring five overexpression plasmids into the mutant strain S.pogona aG6 by adopting a conjugational transfer method, respectively naming overexpression mutant strains as spO2611, spO1943, spO6746, spO4102 and spO1322, and according to an experimental result, constructing the saccharopolyspora tuberosa high-yield genetic engineering bacteria. According to the present invention, the sp1322 and the sp6746 are subjected to co-expression to construct the engineering strain O1322-6746, such that the construction of the next strain can be guided by analyzing the potential modification target, and the ideal phenotype can be constructed in the target strain so as to provide the feasible scheme for the rational design and the improvement of the butenyl spinosad yield;

Description

Technical Field

[0001] This application belongs to the technical field of genetic engineering. Specifically, it relates to a method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora pogona. Background Art

[0002] Butenylspinosad is produced by Saccharopolyspora pogona (S. pogona). Its structure is similar to spinosad and has activities such as insecticidal, acaricidal, and anthelmintic. Compared with spinosad, butenylspinosad has a wider insecticidal spectrum and is currently the only biopesticide that can be applied to grain storage.

[0003] Microbial natural products and their derivatives are important sources of bioactive molecules such as antibiotics, antiparasitic drugs, and pesticides. These molecules are of great significance in medicine, agriculture, and animal husbandry. However, these natural products are usually sufficient for actinomycetes under laboratory conditions or field conditions, but cannot meet the economic requirements under industrial production conditions.

[0004] With the development of gene manipulation techniques, metabolic engineering transformation provides a feasible strategy for further increasing the yield of target secondary metabolites. Many high-yield strains of natural products for industrial large-scale fermentation are obtained by random mutagenesis. Random mutagenesis can not only increase the yield of secondary metabolites but also provide rich materials for subsequent research. Currently, the yield of butenylspinosad fermented and synthesized by wild-type S. pogona is extremely low, and genetic manipulation is difficult, which cannot meet the growing demand for industrial production. Therefore, it is of great significance to increase the yield of butenylspinosad. Summary of the Invention

[0005] To solve the above problems and technical deficiencies, the following technical solutions are adopted in this application. A method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora pogona includes:

[0006] Using the genomic DNA of the mutant strain S. pogona aG6 as a template, five overexpression plasmids are transferred into the mutant strain S. pogona aG6 by conjugation transfer. The overexpression mutant strains are respectively named spO2611, spO1943, spO6746, spO4102, and spO1322;

[0007] According to the experimental results, it is judged that the overexpression of sp1322 and sp6746 can increase the yield of butenylspinosad. The co-overexpression of sp1322 and sp6746 is carried out to construct the engineered strain O1322-6746.

[0008] Preferably, the mutant strain S. pogona aG6 is derived from the wild strain S. pogona WT, and random mutagenesis, screening, and stability testing are carried out using ARTP / UV mutagenesis combined with ribosome engineering to obtain the high-yield mutant strain S. pogona aG6.

[0009] Furthermore, the 5 overexpression plasmids include 4 genes with non-synonymous mutations and 1 gene with a frameshift mutation;

[0010] The 4 genes with non-synonymous mutations include:

[0011] sp2611 encodes a class I SAM-dependent methyltransferase;

[0012] sp1943 encodes a response-regulating transcription factor;

[0013] sp4102 encodes an NAD-binding domain oxidoreductase;

[0014] sp6746 encodes dTDP-glucose 4,6-dehydrogenase;

[0015] The 1 gene with a frameshift mutation includes:

[0016] sp1322 encodes NAD-glutamate dehydrogenase.

[0017] Preferably, the sequences of sp2611, sp1943, sp1322, sp4102, and sp6746 are amplified using primers pIB2611F / pIB2611R, pIB1943F / pIB1943R, pIB1322-F / pIB1322-R, pIB4102-F / pIB4102-R, and pIB6746-F / pIB6746-R, respectively.

[0018] Furthermore, after obtaining the high-yield mutant strain S. pogona aG6, targeted metabolomics analysis is also required for the mutant strain S. pogona aG6 and the starting strain S. pogona WT to identify the mutant gene loci and key metabolic pathways related to the increased production of butenylspinosyn biosynthesis, and the mutant genes are used as targets to carry out metabolic engineering transformation on the mutant strain S. pogona aG6 based on the analysis of the mutant gene loci and key metabolic pathways.

[0019] Still further, for conjugation transfer, a conjugation transfer system for Saccharopolyspora pogona needs to be established, including:

[0020] Systematically optimizing the key parameters and selecting and determining the conjugation transfer medium, including: the time of spore culture, the concentration of MgCl2, the heat shock temperature, the donor-recipient ratio, and the covering resistance time;

[0021] Scrape the spores of the wild strain S. pogona WT and the mutant strain S. pogona aG6 respectively and inoculate them into the sporulation liquid medium.

[0022] Preferably, the process of genome sequencing is as follows:

[0023] Use the PacBio Sequel system and the Illumina NovaSeq PE150 sequencing platform;

[0024] Randomly fragment the DNA samples qualified by electrophoresis into fragments with a length of 350 bp using a Covaris ultrasonic disruptor;

[0025] After the treatment of the DNA fragments, end repair, A-tailing, addition of sequencing adapters, purification, and PCR amplification are carried out to complete the preparation of the entire library.

[0026] Use Qubit 2.0 to preliminarily quantify the constructed library, dilute the library to 2 ng / μL, and use Agilent 2100 to detect the insert fragment size of the library;

[0027] After the insert fragment size meets the requirements, determine the effective concentration of the library by qPCR method, and perform sequencing after passing the library inspection;

[0028] Assemble the genomic sequences using SOAP, Spades, and Abyss software, and integrate them using CISA software;

[0029] Use Genemark software to predict coding sequences, use IslandPath-DIOMB software to predict gene islands, use CRISPRdigger to predict CRISPR sequences in the genome, and perform functional annotation and pathway analysis through GO, KEGG, COG, NR, Pfam, TCDB, and Swiss-Prot databases.

[0030] Furthermore, the specific steps of functional annotation include:

[0031] Perform Diamond alignment of the protein sequences of the predicted genes with each functional database,

[0032] Select the result with the highest score and meeting the threshold in the alignment results of each sequence for annotation;

[0033] Use MUMmer software to perform sequence alignment to detect variant genes.

[0034] Preferably, the process of plasmid construction and engineering strain construction includes:

[0035] Recover each amplified fragment by gel extraction method;

[0036] Use NdeI and EcoRI for double digestion of the pIB139 plasmid to obtain a linearized plasmid, and purify the double-digested vector by gel extraction method;

[0037] Connect and transform the overexpressed gene fragment with the linearized vector through one-step cloning enzyme to obtain the corresponding recombinant plasmid;

[0038] Use primers pIB6746-F2 / pIB6746-R to amplify the coding sequence of G6.63.6_GM006746;

[0039] The amplified sequence and sp1322 are ligated to the NdeI and EcoRI double-digested linearized pIB139 vector using one-step cloning enzyme;

[0040] Transform the above-constructed recombinant plasmid into ET12567 / pUZ8002 for demethylation, and finally transfer it into the S. pogonaaG6 strain by the method of conjugation transfer;

[0041] Perform primary screening of resistance and PCR identification of conjugation transfer transformants to obtain the corresponding engineering strains with correct bands.

[0042] Preferably, after constructing the engineering strain O1322-6746, it is necessary to verify the engineering strain O1322-6746, and measure and compare the engineering strain O1322-6746 with the mutant S. pogona aG6 control strain respectively, including:

[0043] Perform bioreactor fermentation verification and judge the fermentation characteristics according to the production of butenylspinosyn;

[0044] Perform intracellular metabolite determination, and compare and analyze the intracellular amino acid content, intracellular organic acids, phosphosugar content, intracellular coenzymes and energy substance content.

[0045] Compared with the prior art, the beneficial effects of this application are:

[0046] This application applies the DBTL method to the strain metabolic engineering research of the mutant strain S. pogona aG6. First, through comparative genomic analysis of the high-yield mutant strain S. pogona aG6 obtained by random mutagenesis in Chapter 4 and the parental strain S. pogona WT, the mined mutant gene loci were used for the rational design of the mutant strain S. pogona aG6. An integration plasmid was used to overexpress the target gene to construct an engineered strain, and the obtained engineered strain was tested. Physiological parameter measurements were carried out in shake flasks and bioreactors, and intracellular metabolite concentration measurements were performed to study the adaptive changes of intracellular metabolites caused by metabolic pathway modification. Finally, in the learning stage, by analyzing potential modification targets to guide the construction of the next round of strains, a feasible scheme for rational design and improvement of the butenylspinosyn yield was provided by constructing an ideal phenotype in the target strain. Description of the Drawings

[0047] In the drawings:

[0048] Figure 1 Table of strains and plasmids used in the examples of this application;

[0049] Figure 2 Table of primers used in the examples of this application;

[0050] Figure 3 Table of growth conditions of conjugation transfer transformants in the examples of this application;

[0051] Figure 4 Graph showing the effect of spore culture time on conjugation transfer in the examples of this application;

[0052] Figure 5 Graph showing the effect of MgCl2 addition concentration on conjugation transfer efficiency in the examples of this application;

[0053] Figure 6 Graph showing the effect of different heat shock temperatures on conjugation transfer efficiency in the examples of this application;

[0054] Figure 7 Graph showing the effect of donor-recipient ratio on conjugation transfer efficiency in the examples of this application;

[0055] Figure 8 Graph showing the effect of different resistance coverage times on conjugation transfer in the examples of this application;

[0056] Figure 9 Table of general characteristics of the genomes of S. pogona WT and aG6 strains in the examples of this application;

[0057] Figure 10 Table of the number of genes affected by different SNPs and InDels in aG6 in the examples of this application;

[0058] Figure 11 Table of mutant genes with SNPs and Indels in the S.pogonaaG6 coding region of this application example;

[0059] Figure 12 Table of mutant proteins in S.pogonaaG6 of this application example;

[0060] Figure 13 Genomic analysis diagram of aG6 and wild-type strain WT in this application example;

[0061] Figure 14 Circular diagram of differentially expressed genes in comparative genomics of this application example;

[0062] Figure 15 Schematic diagram of combined overexpression target pathways in this application example;

[0063] Figure 16 Schematic diagram of overexpression plasmid construction in this application example;

[0064] Figure 17 Fermentation level diagram of overexpressed engineering strain butenyl polyoxin in this application example;

[0065] Figure 18 Yield diagram of verifying O1322 - 6746 in a 5L bioreactor in this application example;

[0066] Figure 19 Comparison diagram of intracellular amino acid metabolism of the strain in this application example;

[0067] Figure 20 Network diagram of intracellular phosphosugar and organic acid metabolism of the strain in this application example;

[0068] Figure 21 Relative concentration diagram of intracellular coenzyme A, redox cofactors, and energy metabolism substances in this application example;

[0069] Figure 22 Analysis diagram of the high-yield mechanism of strain O1322 - 6746 in this application example. Detailed implementation manners

[0070] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are some, but not all, of the embodiments of this application. Usually, the components of the embodiments of this application described and illustrated in the accompanying drawings here can be arranged and designed in various different configurations.

[0071] Example 1

[0072] Using S. pogona WT wild bacteria as the starting strain, a well plate screening model for mutant strains was established and optimized. Random mutagenesis and screening of WT were carried out by ARTP / UV mutagenesis combined with ribosome engineering, and the high-yield strains were subjected to stability tests to obtain a mutant strain S. pogona aG6 with good genetic stability and high spinosad production.

[0073] The specific process is as follows:

[0074] The 24-well plate was used for primary screening culture. By investigating the effects of liquid loading volume and shear force on well plate culture, a liquid loading volume of 1.6 mL and adding 2 glass beads per well were determined as the optimal culture conditions for the 24-well plate of S. pogona, and a 24-well plate primary screening culture system was established.

[0075] The seed medium was optimized. The original primary seed transferred to secondary seed was optimized into a single seed medium, and the seed culture time was shortened from 96 h to 64 h, improving the subsequent strain screening efficiency.

[0076] Through ARTP / UV mutagenesis combined with streptomycin pressure screening, primary screening and secondary screening were carried out in well plates and shake flasks respectively to obtain a mutagenesis library of 1006 mutant strains. Subsequently, through 2 rounds of shake flask secondary screening and genetic stability tests, a high spinosad-producing strain aG6 was successfully screened, providing a strain basis for the research.

[0077] Example 2

[0078] Using the high spinosad-producing mutant strain S. pogona aG6 successfully obtained by random mutagenesis as the research object, the physiological characteristics differences between aG6 and the starting strain WT were investigated. First, the differences in morphological development characteristics between the two strains were revealed through phenotypic comparison, and then the preliminary fermentation characteristics at the shake flask level were studied. The macroscopic metabolic parameters of the high-yield mutant strain aG6 and the starting strain WT were compared and analyzed through 5 L bioreactor fermentation experiments. Finally, combined with targeted metabolomics, the effects of random mutagenesis on the key metabolic pathways of spinosad biosynthesis in strain aG6 were deeply analyzed to clarify the possible mechanism of high yield of the mutant strain.

[0079] The main process is as follows:

[0080] Phenotypic analysis was carried out on the wild-type strain WT of S. pogona and the high spinosad-producing mutant strain aG6, and significant differences were found in the spore state and hyphal morphology between the two.

[0081] The growth and fermentation characteristics of the mutant high-yield strain and the wild strain were investigated at the shake flask level. The results showed that the spinosad yield reached 122.5 mg / L at 120 h, which was 6.4 times that of the wild-type strain.

[0082] The physiological and metabolic characteristics of aG6 were investigated at the laboratory scale in a 5 L bioreactor. The spinosyn J production of the mutant strain aG6 reached 130 mg / L at 120 h, which was 6.4 times that of the parental strain WT, indicating that the aG6 strain maintained stable high-yield characteristics during the fermentation scale-up process.

[0083] Analysis of its physiological and metabolic parameters revealed that the aG6 strain could utilize glucose faster than the WT strain, with higher growth rate and metabolic intensity. It was speculated that these fermentation characteristics provided more substrates and energy support for the biosynthesis of spinosyn J, thus promoting the increase in spinosyn J production.

[0084] Targeted intracellular metabolomics analysis showed that the increased contents of metabolic intermediates such as G6P, F6P, succinyl-CoA, and propionyl-CoA provided more abundant precursor substances for the biosynthesis of spinosyn J, which was beneficial to the efficient synthesis of spinosyn J by the aG6 strain.

[0085] Example 3

[0086] Genome sequencing can provide a deeper understanding of the growth, phenotypic changes, or target product changes of strains. The application of comparative genomics in reverse metabolic engineering provides an effective way to optimize the product synthesis of strains.

[0087] By comparing and analyzing the genomes of the wild-type strain and high-yield strain of S. lincolnensis, several targets were verified by molecular manipulation to significantly increase the lincomycin production.

[0088] In addition, 133 mutation sites were identified by comparative genomic analysis of the high-yield mutant strain and the parental strain. Overexpression or downregulation of three mutant genes led to an increase in the milbemycin A3 / A4 production. Therefore, it is necessary to combine genomic analysis with metabolic engineering to further increase the spinosyn J production.

[0089] The Design-Build-Test-Learn (DBTL) cycle is an iterative optimization method widely used in the fields of synthetic biology and metabolic engineering. In this example, the DBTL method was applied to the metabolic engineering research of the aG6 strain.

[0090] First, comparative genomic analysis was performed on the high-yield strain aG6 and WT obtained by random mutagenesis in Chapter 4.

[0091] The mined mutant gene loci were used for the rational design of the aG6 strain, and an integrated plasmid was used to overexpress the target gene to construct an engineered strain.

[0092] Then, the obtained engineered strains were tested, including determination of physiological parameters in shake flasks and 5-L bioreactors, and determination of intracellular metabolite concentrations to study the adaptive changes of intracellular metabolites caused by metabolic pathway modification;

[0093] Finally, in the learning stage, potential modification targets were analyzed to guide the construction of the next round of strains.

[0094] The plasmids and strains used in the experiments of this example are as Figure 1 shown, and the primers used are as Figure 2 shown. Figure 2 The restriction enzyme cutting sites in the primers are underlined.

[0095] The plasmid construction and engineered strain construction processes include:

[0096] Using the aG6 strain genome as a template for PCR amplification of overexpression gene fragments. The primers pIB2611F / pIB2611R, pIB1943F / pIB1943R, pIB1322-F / pIB1322-R, pIB4102-F / pIB4102-R, and pIB6746-F / pIB6746-R were used for the amplification of sp2611, sp1943, sp1322, sp4102, and sp6746 sequences respectively;

[0097] Each amplified fragment was recovered by the method of gel extraction;

[0098] NdeI and EcoRI were used for double digestion of the pIB139 plasmid to obtain a linearized plasmid, and the double-digested vector was purified by the method of gel extraction;

[0099] The overexpression gene fragment was ligated and transformed with the linearized vector by one-step cloning enzyme to obtain the corresponding recombinant plasmid;

[0100] For the construction of the recombinant plasmid co-overexpressing sp1322 and sp6746, the primer pIB6746-F2 / pIB6746-R was used to amplify the G6.63.6_GM006746 coding sequence. The amplified sequence and sp1322 were ligated to the NdeI and EcoRI double-digested linearized pIB139 vector by one-step cloning enzyme;

[0101] The above-constructed recombinant plasmids were transformed into ET12567 / pUZ8002 for demethylation, and finally transferred into the S. pogona aG6 strain by the method of conjugation transfer. The corresponding engineered strains with correct bands were obtained by primary screening with resistance and PCR identification of the conjugation transfer transformants.

[0102] Example 4

[0103] The components of the culture medium used for genomic sampling include: 20.0 g / L of glucose, 25.0 g / L of dry corn steep liquor, 1.0 g / L of magnesium sulfate heptahydrate, 1.0 g / L of peptone, 5.0 g / L of calcium carbonate, 2.0 g / L of sodium chloride, the pH is adjusted to 7.2, and it is sterilized at 115 °C for 25 min.

[0104] Spores of WT and aG6 were separately scraped and inoculated into the sporulation liquid medium. After culturing for 72 h, 2 mL of the fermentation broth was taken from each strain and three biological replicates were set up.

[0105] After centrifuging the cells, the supernatant was discarded, and they were washed three times with PBS buffer, then quickly placed in liquid nitrogen for quick freezing and stored at -80 °C for sequencing analysis.

[0106] Example 5

[0107] The process of S. pogona genome sequencing and data analysis includes:

[0108] The genome sequencing of the aG6 mutant strain and the WT wild-type strain was completed by Beijing Novogene Bioinformatics Technology Co., Ltd., using the PacBio Sequel system and the Illumina NovaSeq PE150 sequencing platform;

[0109] The DNA samples qualified by electrophoresis were randomly fragmented into fragments with a length of about 350 bp using a Covaris ultrasonic disruptor. After the treatment of the DNA fragments, the whole library preparation was completed through steps such as end repair, adding A tails, adding sequencing adapters, purification, and PCR amplification;

[0110] The constructed library was preliminarily quantified using Qubit 2.0, diluted to 2 ng / μL, and then the insert fragment size of the library was detected using Agilent 2100;

[0111] After the insert fragment size met the requirements, the effective concentration of the library was determined by qPCR method, and after passing the library inspection, sequencing was carried out.

[0112] The genomic sequences were assembled using SOAP, Spades, and Abyss software, and finally integrated using CISA software. The coding sequences (CDS) were predicted using Genemark software, gene islands were predicted using IslandPath-DIOMB software, CRISPR sequences in the genome were predicted using CRISPRdigger, and functional annotation and pathway analysis were carried out through databases such as GO, KEGG, COG, NR, Pfam, TCDB, and Swiss-Prot.

[0113] The specific steps of functional annotation include:

[0114] The protein sequences of the predicted genes were aligned with various functional databases using Diamond (E-value ≤ 1e-5).

[0115] And for each sequence alignment result, the result with the highest score (score) and meeting the thresholds (identity ≥ 40%, coverage ≥ 40%) was selected for annotation;

[0116] The MUMmer software was used for sequence alignment to detect mutant genes.

[0117] Example 6

[0118] A conjugation transfer system of Saccharopolyspora pogona was established, and its key parameters were systematically optimized.

[0119] Determination of the conjugation transfer medium:

[0120] The effects of four media, ISP4, ISP2, MS, and SFM, on the conjugation transfer efficiency were tested. The results are as Figure 3 shown. No conjugants grew on SFM and ISP2 media, a small amount of conjugants grew on MS media, and among them, the number of conjugants was the largest on ISP4 media. Based on the above results, ISP4 media was determined to be used for subsequent conjugation transfer experiments.

[0121] Determination of the spore culture days:

[0122] During the process of collecting spores, it was found that the amount of spores formed on the culture dish was significantly correlated with the spore age. To explore the optimal spore age, different culture times were set for investigation. The results are as Figure 4 shown. S. pogona cultured at 30°C for 10 days could produce an appropriate amount of spores and obtain a relatively high conjugation transfer efficiency. Based on this, the optimal spore culture time was determined to be 10 days.

[0123] Optimization of the MgCl2 concentration:

[0124] An appropriate amount of MgCl2 was added to the conjugation transfer medium to promote the transfer of recombinant plasmids between donor and recipient strains. To explore the effect of the MgCl2 concentration on the conjugation transfer efficiency, a series of MgCl2 concentration gradients (15, 20, 30, and 40 mM) were set;

[0125] The experimental results are as Figure 5 shown. When the MgCl2 concentration was too low or too high, the number of transformants decreased significantly. In the range of 20 - 30 mM, the number of transformants increased significantly, and the efficiency was the highest at 30 mM. Based on this, the addition concentration of MgCl2 in subsequent experiments was determined to be 30 mM.

[0126] Optimization of the heat shock temperature:

[0127] To promote spore germination, the spores were subjected to appropriate heat shock treatment. If the heat shock temperature is too high, the spore survival rate will decrease; if the heat shock temperature is too low, the effect of promoting spore germination cannot be achieved. To determine the optimal heat shock temperature, the conjugation efficiency at four temperature gradients of 35, 40, 50, and 55 °C was compared;

[0128] The results are as Figure 6 shown. The largest number of zygotes was obtained under the condition of 50 °C, and based on this, it was determined as the heat shock temperature.

[0129] Optimization of the donor-recipient ratio:

[0130] The number of donor Escherichia coli spores was fixed at 1 × 106, and the donor-recipient ratio was changed by adjusting the number of recipient S. pogona spores. Five donor-recipient ratios (10:1, 100:1, 1:1, 1:10, 1:100) were set to investigate the effect of different ratios on the conjugation transfer efficiency. The results are as Figure 7 shown. No transformants grew at the ratio of 100:1, a small number of transformants grew at the ratios of 1:1 and 10:1, and the largest number of transformants grew at the ratio of 1:10. Based on this, the donor-recipient ratio for subsequent experiments was determined to be 1:10.

[0131] Optimization of the coverage resistance time:

[0132] The growth rates of different actinomycetes on the conjugation transfer medium are different, and the optimal coverage resistance time is also different. Too short a culture time is difficult to ensure sufficient plasmid transfer, while too long a time may lead to excessive growth of Escherichia coli, affecting subsequent purification;

[0133] To optimize the coverage resistance time of S. pogona, four time points of 17, 19, 21, and 23 h were investigated. The results are as Figure 8 shown. The conjugation efficiency was the highest at 19 h. Too short (17 h) or too long (21 - 23 h) time both led to a significant decrease in efficiency. When the coverage time exceeded 23 h, the antibiotic could not effectively inhibit the growth of Escherichia coli.

[0134] In summary, by optimizing the spore culture time, donor-recipient ratio, heat shock conditions, MgCl2 concentration, types of conjugation transfer media, and coverage resistance time, a conjugation transfer system of Escherichia coli - Saccharopolyspora pogona was established.

[0135] The specific conditions are as follows: spores were cultured for 10 days, the donor-recipient ratio was 1:10, heat shock at 50 °C for 10 min, the MgCl2 concentration in ISP4 medium was 30 mM, and the coverage resistance time was 19 h.

[0136] Example 7

[0137] Whole-genome sequencing and analysis of WT and aG6 included:

[0138] The whole genome of aG6 and WT was sequenced by combining Illumina and PacBio sequencing technologies. The main features of the whole genome sequences of the high-yield strain aG6 and the wild-type strain WT are as follows Figure 9 As shown, the genome of aG6 is 431.158 kb shorter than that of the wild-type strain WT, and aG6 has the same non-coding RNA (ncRNA) profile as the wild-type strain WT, indicating that random mutagenesis has not affected its transcriptional regulatory conservation.

[0139] The whole genomes of aG6 and WT were compared and analyzed to explore the genetic basis of the high-yield phenotype of aG6. The genomes of WT and aG6 showed relatively conservative gene order, and there was no chromosomal translocation or inversion.

[0140] like Figure 10 As shown, compared with the wild-type strain WT, a total of 29 single nucleotide polymorphisms (SNPs) and 69 insertion / deletion variations (InDels) were detected in the aG6 genome.

[0141] like Figure 11 As shown, 97 mutations were detected in the CDS region, including 17 non-synonymous mutations, which resulted in changes in the amino acid sequence and may affect the function of the corresponding protein.

[0142] like Figure 12 As shown, the coding sequences of 38 genes were affected, including 3 regulatory factors, 5 transposases, 24 enzymes and 6 proteins of unknown function. In addition, there was 1 SNP and 26 Indels located in the intergenic region in aG6.

[0143] The mutated coding sequences (CDS) were classified based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) and COG (Clusters of Orthologous Groups) databases.

[0144] KEGG pathway enrichment analysis results are as follows Figure 13 As shown in A, the mutated CDSs are mainly enriched in pathways such as amino acid metabolism and carbohydrate metabolism;

[0145] The results of COG database analysis are as follows Figure 13 As shown in B, the mutant genes are mainly concentrated in pathways such as amino acid transport and metabolism, lipid transport and metabolism, carbohydrate transport and metabolism, and coenzyme transport and metabolism.

[0146] Moreover, there is a SNP mutation in the coding sequence of dTDP-glucose 4,6-dehydratase, which is responsible for catalyzing the dehydration of dTDP-glucose to produce dTDP-4-keto-6-deoxyglucose. The end products of this pathway, dTDP-rhamnose and dTDP-fucosamine, are ultimately linked to the aglycone of butenylspinosyns. Rhamnose not only participates in the biosynthesis of butenylspinosyns but is also a major component of the cell wall.

[0147] Meanwhile, mutations occurred in the genes encoding transposases, such as 1_orf05513 (encoding a transposase of the IS1380 family), 1_orf06188 (encoding a transposase of the IS110 family), and 1_orf10840 (encoding a transposase of the IS1182 family).

[0148] Transposase is an enzyme that can catalyze transposition reactions and plays a key role in the movement and rearrangement of DNA or RNA sequences within and outside the genome. It is speculated that these mutations improve genome stability, thus being beneficial to the high-yield performance of the strain.

[0149] By systematically comparing the genomes of WT and aG6, key genes and metabolic pathways related to the increased production of butenylspinosyns were identified. This method not only helps to reveal the genetic differences of the high-yield strain aG6 at the gene level but also provides valuable targets for the metabolic engineering transformation of the aG6 strain. Based on this, the mutant genes obtained from comparative genomic analysis were selected as targets for the metabolic engineering transformation of the aG6 strain.

[0150] Through the above comparative genomic analysis, 17 non-synonymous mutations and 18 frameshift mutations were identified between WT and aG6.

[0151] Both non-synonymous mutations and frameshift mutations can affect the structure and function of proteins: non-synonymous mutations change the amino acid sequence, which may affect cellular physiological processes, while frameshift mutations lead to a complete change in the codon sequence after the mutation site.

[0152] Here, 4 genes with non-synonymous mutations and 1 gene with a frameshift mutation were selected for individual overexpression. The positions of these 5 genes in the genome are as Figure 14 shown.

[0153] These genes encode various functional proteins, among which:

[0154] sp2611 (G6.63.6_GM002611) encodes a class I SAM-dependent methyltransferase;

[0155] sp1943 (G6.63.6_GM001943) encodes a response-regulating transcription factor;

[0156] sp6746 (G6.63.6_GM006746) encodes dTDP-glucose 4,6-dehydrogenase;

[0157] sp4102 (G6.63.6_GM004102) encodes NAD-binding domain oxidoreductase;

[0158] sp1322 (G6.63.6_GM001322) encodes NAD-glutamate dehydrogenase.

[0159] The flow chart of overexpression plasmid construction is as Figure 16 shown. Five overexpression plasmids were transferred into strain aG6 by conjugation, and the overexpression mutants of these genes were named O2611, O1943, O6746, O4102, and O1322, respectively.

[0160] The combined overexpression target pathway is as Figure 15 shown; the process of overexpression plasmid construction is as Figure 16 shown.

[0161] As Figure 17 shown in C, the butenyl polyketide spinosyn production of engineered strain O1322 was increased by 27.5% compared with that of aG6.

[0162] The mutation site of sp1322 is as Figure 17 shown in A. In aG6, GAC at positions 1359 - 1361 was replaced by AAC, resulting in the mutation of aspartic acid to asparagine.

[0163] As Figure 17 shown in C, the butenyl polyketide spinosyn production of engineered strain O6746 was increased by 30.8% compared with that of aG6.

[0164] The mutation site of sp6746 is as Figure 17 shown in B. In aG6, TCC at positions 233 - 235 was replaced by TTT, resulting in the mutation of serine to phenylalanine.

[0165] In contrast, overexpression of the other three genes had no significant effect on the production of butenyl polyketide spinosyn.

[0166] As a key metabolic enzyme, glutamate dehydrogenase catalyzes the oxidative deamination of glutamate to produce α-ketoglutaric acid. The ammonia and NAD(P)H produced play important roles in key pathways such as amino acid metabolism, energy metabolism, and nitrogen metabolism. In addition to central carbon metabolism, glycosylation modification also plays an important role in natural product biosynthesis.

[0167] In the biosynthetic process of butenylspinosyn, forosamine and 3-O-methylrhamnose are respectively linked to the glycosyl moiety through dTDP-formosamine sugar and dTDP-rhamnose. dTDP-glucose 4,6-dehydrogenase catalyzes the conversion of TDP-D-glucose to TDP-4-keto-6-deoxy-D-glucose, which serves as a precursor for the biosynthesis of dTDP-rhamnose and dTDP-forosamine.

[0168] To investigate the effect of the combined overexpression of sp1322 and sp6746 on the yield of butenylspinosyn, a co-overexpression strain O1322-6746 of the two genes was constructed.

[0169] The results of determining the butenylspinosyn yield of the engineered strain O1322-6746 at the shake flask level are as Figure 17 shown in D of the figure. Compared with the individual overexpression of sp1322 and sp6746, the co-expression further increased the butenylspinosyn yield, which was 40% higher than that of aG6.

[0170] Example 8

[0171] Fermentation verification of the engineered strain O1322-6746 in a 5L bioreactor was carried out. Batch fermentation in a 5L fermenter was performed on it and the control strain aG6 respectively. The dynamic changes of the physiological parameters during the fermentation process are as Figure 18 shown.

[0172] The overall change trends of the PMV of the two strains were similar. Starting from 20h, the PMV of the two strains increased rapidly. The growth rate of aG6 was slightly faster than that of O1322-6746. The PMV of the two strains reached the peak at 80h, with aG6 being about 24% and O1322-6746 being about 22%. After 80h, the PMV of the two strains decreased sharply. By the end of the fermentation at 120h, the PMV dropped to about 10%.

[0173] During the fermentation process, the PMV of aG6 was slightly higher than that of O1322-6746, indicating that the overexpression of the target gene might have had a slight impact on the growth of the strain, but the overall impact was not obvious. The online monitored CER parameter also confirmed this trend.

[0174] It can be seen from the CER curve that the respiratory metabolism of the O1322-6746 strain was basically consistent with the change trend of aG6, and the impact of the metabolic pathway modification on the respiratory metabolism was relatively small.

[0175] In the early stage of fermentation, the respiratory intensity of aG6 was similar to that of O1322-6746. In the exponential growth phase, the CER peak value of the genetically engineered strain O1322-6746 was less than that of aG6. As the cells grew and metabolized, the pH value underwent a series of changes, which to a certain extent reflected its metabolic state. In the early stage of fermentation, the physiologically basic amino acids were rapidly consumed, resulting in a gradual increase in the pH of the fermentation system.

[0176] In the middle stage of fermentation, the continuous consumption of glucose to produce organic acids led to a decrease in pH.

[0177] In the late stage of fermentation, due to the exhaustion of glucose, the pH began to rise.

[0178] The pH of O1322-6746 also showed a trend of first rising, then falling, and finally rising during fermentation. However, since the glucose consumption rate of the O1322-6746 strain was higher than that of aG6, the pH of the O1322-6746 fermentation broth showed a downward trend earlier in the early stage of fermentation. Although aG6 had a higher biomass, the biosynthesis ability of butenylspinosyn was lower than that of O1322-6746.

[0179] From the spinosyn production curve, it can be seen that O1322-6746 showed a higher accumulation rate of butenylspinosyn during the whole fermentation process. At the end of fermentation, the butenylspinosyn yield of the O1322-6746 strain reached 210 mg / L, which was 75% higher than that of the control strain aG6.

[0180] Example 9

[0181] The metabolic mechanism of the engineered strain O1322-6746 during fermentation was analyzed. The intracellular metabolites of the engineered strain O1322-6746 and the control strain aG6 were determined. A total of 40 intracellular metabolites were identified, including 17 amino acids, 10 phosphosugars, 5 organic acids, 4 coenzymes, and 4 energy substances.

[0182] The analysis results of amino acid concentrations are as Figure 19 shown. The contents of glutamate and glutamine in the O1322-6746 strain were significantly reduced. It was speculated that the overexpression of NAD-glutamate dehydrogenase (sp1322) promoted the conversion of glutamate to α-ketoglutaric acid, resulting in a significant reduction in the contents of glutamate and glutamine.

[0183] Moreover, the methionine concentration of the O1322-6746 strain was significantly lower than that of aG6. After butenylspinosyn formed a macrolide structure under the catalysis of type I polyketide synthase (PKSI), it needed to be methylated at multiple positions in a S-adenosylmethionine (SAM)-dependent manner. This process increased the synthesis demand for SAM, thus consuming more methionine.

[0184] Since the butenylspinosyn production of strain O1322-6746 is significantly higher than that of strain aG6, it is speculated that the increased methylation process leads to a decrease in the methionine content of strain O1322-6746. The serine content in strain O1322-6746 is significantly decreased, presumably because more serine is converted into acetyl-CoA via the pyruvate pathway, thus providing precursor substances for the synthesis of butenylspinosyn.

[0185] The leucine content in strain O1322-6746 is significantly decreased, presumably because precursor substances such as pyruvate, acetyl-CoA, and NADPH required for leucine synthesis flow more towards the synthesis of butenylspinosyn, resulting in the inhibition of leucine synthesis.

[0186] The histidine content in strain O1322-6746 is significantly increased. Its biosynthetic starting substance, 5-phosphoribosyl-1-pyrophosphate, is an important product of the pentose phosphate pathway. Therefore, the change in the pentose phosphate pathway may promote the synthesis of histidine.

[0187] The analysis results of the intracellular organic acids and phosphosugars are as Figure 20 shown. Among the 15 detected phosphosugars and organic acids, the concentrations of 5 metabolites have changed significantly. Among them, the accumulation amount of α-ketoglutaric acid in O1322-6746 is significantly higher than that of the control strain, which corresponds to the significant decrease in the glutamate content, presumably related to the overexpression of NAD-glutamate dehydrogenase.

[0188] At the same time, the contents of metabolites involved in the TCA cycle, such as succinic acid, fumaric acid, and malic acid, in O1322-6746 are significantly decreased, while the content of succinyl-CoA is significantly higher than that of the control strain.

[0189] Succinyl-CoA is mainly metabolized through the following pathways: on the one hand, it can enter the TCA cycle after being oxidized by succinyl-CoA dehydrogenase; on the other hand, it can be reversibly isomerized into methylmalonyl-CoA, a precursor for the synthesis of butenylspinosyn, under the catalysis of methylmalonyl-CoA mutase (MCM). It is speculated that the conversion of α-ketoglutaric acid to succinyl-CoA in strain O1322-6746 is enhanced, thereby increasing the metabolic flux of succinyl-CoA to methylmalonyl-CoA, which is beneficial to the synthesis of butenylspinosyn precursors.

[0190] Moreover, in strain O1322-6746, the xylitol content is significantly decreased. Xylitol is oxidized to xylose under the catalysis of xylitol dehydrogenase, and xylose enters glycolysis through the non-oxidative pentose phosphate pathway. It is speculated that the enhanced sugar metabolism ability of strain O1322-6746 promotes the metabolism of xylitol, resulting in a decrease in its content.

[0191] Four coenzyme substances related to the synthesis of butenyl spinosyn precursors, including succinyl coenzyme A, acetyl coenzyme A, propionyl coenzyme A, and methylmalonyl coenzyme A, were measured for O1322 - 6746 and the aG6 mutant strain, and the results are as Figure 21 shown in A of

[0192] Succinyl coenzyme A plays an important role in the TCA cycle. The content of succinyl coenzyme A increases in the O1322 - 6746 strain, which may be caused by an increased metabolic flux from α - ketoglutaric acid to succinyl coenzyme A;

[0193] Higher contents of acetyl coenzyme A, propionyl coenzyme A, and methylmalonyl coenzyme A may be due to the increased production of butenyl spinosyn in the O1322 - 6746 strain requiring more precursor supply.

[0194] The concentrations of NADP+ and NAD+ in aG6 and O1322 - 6746 were measured, and the results are as Figure 21 shown in B of

[0195] The content of NADP+ decreases in O1322 - 6746. As the oxidized form of nicotinamide adenine dinucleotide phosphate, NADP+ plays an important biological role in cells, especially in redox reactions and metabolic processes.

[0196] The decrease in the content of NADP+ in O1322 - 6746 indicates that it has stronger reducing ability and antioxidant defense ability to support cell growth and proliferation.

[0197] As an important coenzyme in cell energy metabolism, NAD+ participates in key metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation.

[0198] At the same time, the increase in the content of NAD+ in O1322 - 6746 indicates that it has more active energy metabolism and can efficiently synthesize ATP to meet the energy requirements of cells.

[0199] The levels of ADP and AMP both increase in the O1322 - 6746 strain, and the results are as Figure 21 shown in C of, indicating that the cells are rapidly consuming ATP, resulting in the hydrolysis of ATP to ADP and further hydrolysis to AMP, which may be due to the increased energy demand for the precursors of butenyl spinosyn synthesis.

[0200] The development of genome sequencing technology has made it possible to systematically analyze the mutation sites in high - yield strains obtained by mutagenic breeding. In this example, functional analysis of the mutant genes found that many genes located in the primary metabolic pathways have mutated.

[0201] For example, a SNP mutation occurred in the alcohol dehydrogenase gene (1_orf03210), while frameshift mutations occurred in the GDP-mannose dehydrogenase gene (1_orf00692), serine-pyruvate aminotransferase gene (1_orf11087), and formyl-CoA transferase gene (1_orf10170). Statistical analysis showed that the gene mutations related to primary metabolism accounted for 51.3% ( Figure 13 A in

[0202] ), indicating that the differential genes of primary metabolism play an important regulatory role in the high-yield phenotype of the aG6 strain. The alcohol dehydrogenase gene catalyzes the reversible oxidation-reduction reaction between ethanol and acetaldehyde. It has been reported that knocking out the alcohol dehydrogenase gene helps increase the intracellular content of acetyl-CoA and redirects the metabolic flux to the TCA cycle, thereby promoting the growth of the strain. Therefore, it is speculated that the mutation of G to S at position 291 of the alcohol dehydrogenase gene in aG6 leads to a change in the catalytic function of alcohol dehydrogenase, thus affecting the intracellular content of acetyl-CoA. In the aG6 strain, a frameshift mutation occurred in the serine-pyruvate aminotransferase gene. The results of intracellular metabolite analysis showed that the intracellular serine concentration in the aG6 strain also decreased significantly. It is speculated that the frameshift mutation leads to a decrease in its functional activity, blocking the synthesis pathway of converting pyruvate to serine, and ultimately causing a decrease in the intracellular serine concentration in the aG6 strain.

[0203] Moreover, GDP-mannose dehydrogenase catalyzes the oxidation of GDP-mannose to GDP-mannuronic acid, and formyl-CoA transferase catalyzes the formyl-CoA transfer reaction. However, their direct effects on the synthesis of secondary metabolites have not been reported, which may be potential targets for future research.

[0204] These findings emphasize the importance of primary metabolic pathways as potential targets for secondary metabolite-producing strains and provide valuable information for metabolic engineering strategies to further improve strain yields.

[0205] In this example, through systematic comparative genomics analysis, the genomic differences between the high-yield butenylspinosyn strain and the parental strain were revealed, and the genes encoding dTDP-glucose 4,6-dehydratase and NAD-glutamate dehydrogenase were successfully identified as key targets for increasing the yield of butenylspinosyn.

[0206] This result indicates that comparative genomics-guided metabolic engineering is an effective strategy for obtaining high-yield butenylspinosyn strains, and the mutation sites analyzed in this example will also provide a reference for subsequent research.

[0207] The content and supply of precursor substances are crucial for the biosynthesis of secondary metabolites. Primary metabolism is the direct or indirect source of these precursor substances. Therefore, it is particularly important to optimize the availability of precursor substances.

[0208] The biosynthesis of secondary metabolites usually depends on the supply of a variety of precursor substances. Therefore, the precursor engineering strategy of target combination is a feasible strategy to improve the yield of secondary metabolites.

[0209] Co-overexpression of asm13-17 and asmUdpg in Actinosynnema pretiosum led to an increase in the yield of P-3. Deletion of repressors and overexpression of activators could improve the yield of the bioherbicide thaxtomin. In this example, by simultaneously increasing the supply of two precursor substances: (1) overexpressing sp6746 to enhance the supply of TDP-4-keto-6-deoxy-D-glucose; (2) sp1322 to enhance the metabolic flux of succinyl-CoA to methylmalonyl-CoA, it was demonstrated that regulating different precursor substances in S. pogona was more conducive to the precursor supply for the synthesis of butenylspinosyn.

[0210] The biosynthetic pathways of rhamnose and forosamine play a key role in the formation of butenylspinosyn. In addition, rhamnose not only serves as the sugar group of butenylspinosyn but also plays an important role in primary metabolism. The common intermediate in the biosynthesis of these two sugar groups is TDP-4-keto-6-deoxy-D-glucose, and the dTDP-glucose 4,6-dehydratase encoded by sp6746 participates in the conversion step of glucose-1-phosphate to TDP-4-keto-6-deoxy-D-glucose.

[0211] Therefore, it is speculated that overexpressing sp6746 can enhance the metabolic flux of glucose-1-phosphate to TDP-4-keto-6-deoxy-D-glucose, ultimately promoting the biosynthesis of the two sugar groups. Succinyl-CoA can either participate in the TCA cycle through oxidation by succinyl-CoA dehydrogenase or undergo reversible isomerization with methylmalonyl-CoA.

[0212] Overexpression of glutamate dehydrogenase may promote the metabolic flux of succinyl-CoA to the direct precursor pathway of butenylspinosyn, thereby promoting the biosynthesis of butenylspinosyn. This also indicates that regulating the metabolic flux of indirect precursors by controlling key enzymes is beneficial to the accumulation of the direct precursors of the target secondary metabolites.

[0213] Co-overexpression of sp1322 and sp6746 further increased the yield of butenylspinosyn. This result indicates that the combined optimization of the expression levels of genes related to primary metabolism can improve the yield of secondary metabolites.

[0214] Example 10

[0215] In this example, whole-genome sequencing and comparative genomics analysis were performed on wild-type S. pogona WT and mutagenized high-yield strain aG6, and key mutation sites were verified using metabolic engineering strategies. The specific process is as follows:

[0216] Combining the PacBio Sequel and Illumina NovaSeq PE150 systems, whole-genome sequencing of wild-type S. pogona WT and mutagenized high-yield strain aG6 was completed, and the main characteristics of the two genomes were compared. The results showed that the genome of aG6 was 431.158 kb shorter than that of the wild-type strain WT. aG6 had the same non-coding RNA profile as the wild-type strain WT;

[0217] Functional analysis of mutant genes in the genomes of WT and aG6 was carried out. The results of functional classification and annotation in the KEGG and COG databases showed that the types of differential gene mutations in the two genomes included 29 SNPs and 69 Indels. Among them, 97 mutations were detected in the CDS region, including 17 non-synonymous mutations, and the coding sequences of 38 genes were affected. There was also 1 SNP and 26 Indels in the intergenic region;

[0218] Five mutant genes were selected for overexpression verification in aG6. The results showed that overexpression of sp1322 and sp6746 could increase the production of butenylspinosyn. Further, sp1322 and sp6746 were co-overexpressed to construct the engineering strain O1322-6746. Fermentation was carried out on a 5 L bioreactor scale, and the butenylspinosyn production of O1322-6746 was increased by 75% compared with aG6;

[0219] Intracellular metabolite analysis was performed on strains aG6 and O1322-6746. The results showed that overexpression of NAD-glutamate dehydrogenase (sp1322) increased the conversion flux of glutamate to α-ketoglutarate, and at the same time increased the metabolic flux of succinyl-CoA to methylmalonyl-CoA, promoting the accumulation of precursors for butenylspinosyn synthesis;

[0220] Overexpression of the dTDP-glucose 4,6-dehydratase-encoding gene (sp6746) was beneficial for the synthesis of the butenylspinosyn glycosyl-rhamnose and forosamine. Changes in the metabolite pools of key amino acids such as glutamate, glutamine, and methionine provided favorable conditions for the efficient synthesis of butenylspinosyn.

[0221] In addition, the active states of intracellular energy substances ADP, AMP, and NAD+ met the energy requirements of the engineering strain O1322-6746, as Figure 22 shown.

[0222] The above-described embodiments merely represent the preferred embodiments of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications, improvements, and substitutions can be made, and these all fall within the protection scope of the present application.

Claims

1. A method for constructing a high-yield genetically engineered bacterium of Saccharopolysaccharide, characterized in that: include: Using the genome of mutant S. pogona aG6 as a template, five overexpression plasmids were transferred into mutant S. pogona aG6 by conjugation transfer. The overexpression mutants were named spO2611, spO1943, spO6746, spO4102, and spO1322, respectively. According to the experimental results, it was determined that overexpression of sp1322 and sp6746 could increase the yield of butenyl spinosad, so sp1322 and sp6746 were co-overexpressed to construct the engineered strain O1322-6746.

2. The method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora aspergillus according to claim 1, characterized in that: The mutant strain S.pogona aG6 is based on the wild strain S.pogona WT. ARTP / UV mutagenesis combined with ribosome engineering is used for random mutation, screening and stability testing to obtain a high-yield mutant strain S.pogona aG6.

3. The method for constructing a high-yield genetically engineered bacterium of Saccharopolysaccharides according to claim 2, characterized in that: The five overexpression plasmids included four genes with nonsynonymous mutations and one gene with a frameshift mutation; The 4 genes with non-synonymous mutations include: sp2611 encodes a class I SAM-dependent methyltransferase; sp1943 encodes a response-regulatory transcription factor; sp4102 encodes an NAD-binding domain oxidoreductase; sp6746 encodes dTDP-glucose 4,6-dehydrogenase; 1 gene with a frameshift mutation includes: sp1322 encodes NAD-glutamate dehydrogenase.

4. The method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora aspergillus according to claim 3, characterized in that: The sp2611, sp1943, sp1322, sp4102 and sp6746 sequences were amplified using primers pIB2611F / pIB2611R, pIB1943F / pIB1943R, pIB1322-F / pIB1322-R, pIB4102-F / pIB4102-R, and pIB6746-F / pIB6746-R, respectively.

5. The method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora aspergillus according to claim 1, characterized in that: After obtaining the high-yield mutant strain S.pogona aG6, it is also necessary to conduct targeted metabolomics analysis on the mutant strain S.pogona aG6 and the starting strain S.pogona WT to identify the mutant gene sites and key metabolic pathways related to the increased biosynthesis yield of butenyl spinosad, and use the mutant genes obtained based on the analysis of the mutant gene sites and key metabolic pathways as targets for metabolic engineering of the mutant strain S.pogona aG6.

6. The method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora aspergillus according to claim 1, characterized in that: Conjugative transfer requires the establishment of a Saccharopolysaccharides conjugative transfer system, including: Systematic optimization of key parameters to select and determine the conjugation transfer medium, including: spore culture time, MgCl2 concentration, heat shock temperature, donor-receptor ratio, and covering resistance time; Then, spores of wild-type S. pogona WT and mutant S. pogona aG6 were scraped and inoculated into spore-producing liquid culture medium.

7. The method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora aspergillus according to claim 1, characterized in that: The process of genome sequencing is as follows: The PacBio Sequel system and the Illumina NovaSeqPE150 sequencing platform were used; DNA samples that passed the electrophoresis test were randomly broken into fragments of 350 bp in length using a Covaris ultrasonic disruptor; After the treatment, the DNA fragments are repaired at the end, A-tailed, sequenced adapters are added, purified, and PCR amplified to complete the entire library preparation. The constructed library was preliminarily quantified using Qubit 2.0, the library was diluted to 2 ng / μL, and the insert size of the library was detected using Agilent2100; After the insert size meets the requirements, the effective concentration of the library is determined by qPCR, and sequencing is performed after the library is qualified; The genome sequence was assembled using SOAP, Spades, and Abyss software and integrated using CISA software; Genemark software was used to predict coding sequences, IslandPath-DIOMB software was used to predict gene islands, CRISPRdigger was used to predict CRISPR sequences in the genome, and functional annotation and pathway analysis were performed using the GO, KEGG, COG, NR, Pfam, TCDB, and Swiss-Prot databases.

8. The method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora aspergillus according to claim 7, characterized in that: The specific steps of functional annotation include: The protein sequence of the predicted gene was compared with various functional databases by Diamond. The result with the highest score and meeting the threshold in each sequence alignment result is selected for annotation; MUMmer software was used for sequence alignment and detection of variant genes.

9. The method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora aspergillus according to claim 1, characterized in that: The process of plasmid construction and engineering strain construction includes: The amplified fragments were recovered by gel recovery method; The pIB139 plasmid was double-digested with NdeI and EcoRI to obtain a linearized plasmid, and the double-digested vector was purified by gel recovery method; The overexpressed gene fragment was connected and transformed with the linearized vector by a one-step cloning enzyme to obtain the corresponding recombinant plasmid; The G6.63.6_GM006746 coding sequence was amplified using primers pIB6746-F2 / pIB6746-R; The amplified sequence and sp1322 were ligated into the pIB139 linearized vector double-digested with NdeI and EcoRI using a one-step cloning enzyme; The recombinant plasmid constructed above was transformed into ET12567 / pUZ8002 for demethylation, and finally transferred into S.pogonaaG6 strain by conjugation transfer method; Primary resistance screening and PCR identification of conjugative transfer transformants were performed to obtain the corresponding engineering strains with correct bands.

10. The method for constructing a high-yield genetically engineered bacterium of Saccharopolyspora aspergillus according to claim 1, characterized in that: After constructing the engineered strain O1322-6746, it is necessary to verify the engineered strain O1322-6746 by comparing the engineered strain O1322-6746 with the mutant strain S.pogona aG6 control strain, including: Conduct bioreactor fermentation validation and judge the fermentation characteristics based on the production of butenyl spinosad; Intracellular metabolites were determined, and the contents of intracellular amino acids, organic acids, phosphate sugars, coenzymes and energy substances were compared and analyzed.

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