An engineered strain with high production of spinosad by optimizing the mannose metabolic pathway

Abstract

An engineered strain that optimizes the mannose metabolic pathway and produces high amounts of spinosad is developed. The strain is based on Saccharopolyspora spinosa. manB Genes and leuA Gene blocking, and aceF Gene overexpression is obtained; manB The gene refers to the phosphomannose mutase gene; leuA The gene refers to the 2-isopropylmalate synthase gene; aceF The gene refers to the pyruvate dehydrogenase gene. The present invention starts with the precursor supply pathway of Saccharopolyspora spinosa, takes the mannose metabolic pathway as the optimization object, reduces the competitive utilization of the substrate glucose, and promotes more glucose metabolic flow to the pyruvate metabolic pathway; then reduces the competitive utilization of acetyl-CoA, improves the ability to synthesize acetyl-CoA from pyruvate, increases the accumulation of the precursor substance acetyl-CoA, and finally achieves the promotion of the efficient biosynthesis of spinosad in the engineered strain.

Description

Technical Field

[0001] The invention relates to an engineering strain with high spinosyn production, in particular to an engineering strain with high spinosyn production and optimized metabolic pathway. Background Art

[0002] Spinosyn is a new type of microbial insecticide with contact and feeding toxins produced by aerobic fermentation of Saccharopolyspora spinosa. It is one of the most promising new biopesticides in the world. Although spinosyn has significant insecticidal effects and its synthesis pathway has been clearly described, the production and application of spinosyn are seriously restricted by the low and unstable production of wild-type strains and the long fermentation cycle.

[0003] In recent years, in the study of targeted editing of the Saccharopolyspora spinosa genome to improve the production of spinosad, the main focus is on single gene knockout and overexpression of important functional genes involved in precursor supply and regulation, such as overexpression of S-adenosylmethionine synthetase gene (metK), transhydrogenase gene (pntAB), acetoin utilization protein gene (acuC), and knockout of genes pepM and manB that competitively utilize glucose metabolic flow. Although the modification of a single functional gene can promote the biosynthesis of spinosad to a certain extent, the biosynthetic pathway of spinosad is very complex and is jointly controlled by multiple functional genes. Modification of only a single functional gene may have limited impact on the entire synthesis pathway, resulting in difficulty in significantly increasing the yield. Therefore, how to adopt effective strategies to systematically optimize the biosynthetic pathway of spinosad to improve its yield has become one of the current research hotspots.

[0004] In many studies on increasing the yield of natural products, it was found that the use of combinatorial metabolic engineering strategies can greatly increase the yield of target natural products. Commonly used technical means include using strong promoters to regulate the expression of genes in the synthesis pathway, knocking out key genes in the byproduct synthesis pathway, and constructing energy metabolism regeneration systems to optimize the intracellular metabolic network to achieve high yield, high production intensity and high conversion rate. In the study of promoting the yield of N-acetylglucosamine, by increasing the expression of positive regulatory transcription factor RamAM, interfering with the expression of negative regulatory transcription factors (RamB, LldR, FruR and AmtR) through the CRISPRi system, site-directed mutation of glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase genes, and fermentation tank amplification production, the titer can reach 117.1g / L, which is 6.62 times that of the control group. Similarly, by knocking out the genes tyrR, ptsG, crr, pykF and pheA in Escherichia coli BL21 (DE3), overexpressing galP, glk, tktA and ppsA, and directed evolution of HpaB, the fermentation yield of L-DOPA finally reached 25.53 g / L. The above studies show that the use of combinatorial metabolic engineering strategies can effectively promote the biosynthesis of target natural products based on the rational design and reconstruction of intracellular metabolic networks. It is necessary to apply this strategy to optimize the metabolic network of Saccharopolyspora spinosa to increase the production of spinosad.

[0005] The synthetic substrates of spinosad include acetyl-CoA, malonyl-CoA, methylmalonyl-CoA, rhamnose, forosamine and S-adenosyl-methionine, etc., which are relatively complex. At present, no relevant research has been reported. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide an engineered strain that optimizes the mannose metabolic pathway and produces high amounts of spinosad.

[0007] The technical solution adopted by the present invention to solve the technical problem is as follows: an engineered strain for high-yield spinosad that optimizes the mannose metabolic pathway is obtained by blocking the manB gene and leuA gene of the spinosad and overexpressing the aceF gene, using Saccharopolyspora spinosa as the starting strain;

[0008] The manB gene refers to the phosphomannose mutase gene;

[0009] The leuA gene refers to the 2-isopropylmalate synthase gene;

[0010] The aceF gene refers to the pyruvate dehydrogenase gene.

[0011] Preferably, the construction method comprises the following steps:

[0012] (1) Using Saccharopolyspora spinosa as the starting strain, the CRISPR / Cas9 technology was used to knock out the manB gene in the fructose and mannose metabolic pathways to obtain the mutant strain -ΔmanB;

[0013] (2) Using CRISPR / Cas9 technology to knock out the leuA gene in the leucine biosynthesis pathway of the mutant strain -ΔmanB, the mutant strain -ΔmanBΔleuA was obtained;

[0014] (3) The aceF gene was overexpressed in the mutant strain -ΔmanBΔleuA to obtain an engineered strain with high production of spinosad.

[0015] Preferably, the knockout is achieved by fusing the upstream homology arm, the downstream homology arm and the sgRNA of the gene, subcloning them into a vector, and transferring the vector into Saccharopolyspora spinosa by protoplast transformation, thereby achieving gene knockout.

[0016] Preferably, the overexpression is to introduce the gene into a vector, and transfer the vector into Saccharopolyspora spinosa by protoplast transformation, thereby achieving overexpression of the gene.

[0017] Preferably, the nucleotide sequence of the upstream homologous arm of the manB gene used for knockout is as shown in SEQ ID NO.1.

[0018] Preferably, the nucleotide sequence of the downstream homologous arm of the manB gene used for knockout is as shown in SEQ ID NO.2.

[0019] Preferably, the nucleotide sequence of the upstream homology arm of the leuA gene used for knockout is as shown in SEQ ID NO.3.

[0020] Preferably, the nucleotide sequence of the downstream homology arm of the leuA gene used for knockout is as shown in SEQ ID NO.4.

[0021] Preferably, the nucleotide sequence of the aceF gene sequence used for overexpression is shown as SEQ ID NO.5.

[0022] Preferably, the nucleotide sequence of the sgRNA sequence of the manB gene used for knockout is as shown in SEQ ID NO.6.

[0023] Preferably, the nucleotide sequence of the sgRNA sequence of the leuA gene used for knockout is shown in SEQ ID NO.7.

[0024] Preferably, the manB gene knockout method comprises the following steps:

[0025] (1.1) Using the genome of Saccharopolyspora spinosa as a template, according to the sequence of the manB gene, the upstream and downstream homology arm primer pairs manB-Up-F / manB-Up-R and manB-Down-F / manB-Down-R were designed at both ends of the gene, respectively, and the upstream and downstream homology arms of the manB gene were obtained by PCR amplification after synthesis; the sgRNA sequence of the manB gene and its primer pair manB-sgRNA-F / manB-sgRNA-R were designed and synthesized, and PCR amplification was performed using the vector pKCcas9dO as a template to obtain the sgRNA of the manB gene;

[0026] (1.2) Fusion PCR was used to fuse sgRNA with the upstream and downstream homology arms to obtain the fusion fragment sgRNA-Up manB -Down manB , and double-digested it with Spe I and Hind III; the fusion fragment sgRNA-Up manB -Down manB The linearized vector pKCcas9dO was connected to the vector under the catalysis of T4 DNA ligase at 16°C overnight. The ligation product was heat-shocked into competent E. coli DH5α and cultured on a LB solid plate coated with apramycin at 37°C and 150 rpm for 16 h. 8-10 transformants were randomly selected and placed in a 1.5 mL Eppendorf tube and cultured at 37°C and 850 rpm for 12 h. The plasmid was extracted and the gene manB knockout vector pKCcas9d-manB was obtained after double enzyme digestion verification and sequencing to confirm the absence of base mutations.

[0027] (1.3) The knockout vector pKCcas9d-manB constructed successfully was transferred into the protoplasts of Saccharopolyspora spinosa by protoplast transformation and spread on R6 solid plates. After inverted culture in a 30°C incubator for 16-20 hours, apramycin coverage screening was performed and culture was continued for 3-7 days until white transformants appeared. 8-10 transformants were randomly selected and streaked on TSB solid plates. The transformants were further purified by inverted culture in a 30°C incubator for 2-3 days. Some of the grown bacteria were selected and cultured in CSM liquid culture medium at 30°C and 150 rpm for 2 days. The genome was extracted and PCR verification was performed using the primer pair manB-Up-F / manB-Down-R;

[0028] (1.4) The successfully verified mutant strain is placed in culture at 37°C for plasmid elimination, and the mutant strain in which the recombinant plasmid pKCcas9d-manB is eliminated is obtained through screening.

[0029] The nucleotide sequence of each primer is recorded in Table 1. The design of each primer pair and sgRNA can be completed by existing technologies, such as using the website https: / / portals.broadinstitute.org / gpp / public / for corresponding design.

[0030] Preferably, the leuA gene knockout method comprises the following steps:

[0031] (2.1) Using the genome of Saccharopolyspora spinosa as a template, according to the leuA gene sequence, the upstream and downstream homology arm primer pairs leuA-Up-F / leuA-Up-R and leuA-Down-F / leuA-Down-R were designed at both ends of the gene, respectively, and the upstream and downstream homology arms of the gene leuA were obtained by PCR amplification after synthesis; the sgRNA sequence of the gene leuA and its primer pair leuA-sgRNA-F / leuA-sgRNA-R were designed and synthesized, and PCR amplification was performed using the vector pKCcas9dO as a template to obtain the sgRNA of the leuA gene;

[0032] (2.2) Fusion PCR was used to fuse sgRNA with the upstream and downstream homology arms to obtain the fusion fragment sgRNA-Up leuA -Down leuA , and double-digested it with Spe I and Hind III; the fusion fragment sgRNA-Up leuA- -Down leuA- The linearized vector pKCcas9dO was connected to the vector under the catalysis of T4 DNA ligase at 16°C overnight. The ligation product was heat-shocked into competent E. coli DH5α and cultured on a LB solid plate coated with apramycin at 37°C and 150 rpm for 16 h. 8-10 transformants were randomly selected and placed in a 1.5 mL Eppendorf tube and cultured at 37°C and 850 rpm for 12 h. The plasmid was extracted and the gene leuA knockout vector pKCcas9d-leuA was obtained after double enzyme digestion verification and sequencing to confirm the absence of base mutations.

[0033] (2.3) The knockout vector pKCcas9d-leuA constructed successfully was transferred into the protoplasts of Saccharopolyspora spinosa by protoplast transformation and spread on R6 solid plates. After inverted culture in a 30°C incubator for 16-20 hours, apramycin coverage screening was performed and culture was continued for 3-7 days until white transformants appeared. 8-10 transformants were randomly selected and streaked on TSB solid plates. The transformants were further purified by inverted culture in a 30°C incubator for 2-3 days. Some of the grown bacteria were selected and cultured in CSM liquid culture medium at 30°C and 150 rpm for 2 days. The genome was extracted and PCR verification was performed using primers leuA-Up-F / leuA-Down-R;

[0034] (2.4) The successfully verified mutant strain was placed in culture at 37°C for plasmid elimination, and the mutant strain in which the recombinant plasmid pKCcas9d-ΔleuA was eliminated was obtained through screening.

[0035] The nucleotide sequence of each primer is recorded in Table 1. The design of each primer pair and sgRNA can be completed by existing technologies, such as using the website https: / / portals.broadinstitute.org / gpp / public / for corresponding design.

[0036] Preferably, the aceF gene overexpression method comprises the following steps:

[0037] (3.1) Using the genome of Saccharopolyspora spinosa as a template and according to the aceF gene sequence, a primer pair aceF-F / aceF-R was designed to amplify the open reading frame of the aceF gene. After synthesis, the aceF gene was amplified by PCR and double-digested with Xba I and EcoR I;

[0038] (3.2) The aceF gene after restriction digestion was connected to the linearized vector pIB139 under the catalysis of T4 DNA ligase at 16°C overnight. The ligation product was heat-shocked into competent E. coli DH5α and cultured on a LB solid plate coated with apramycin at 37°C and 150 rpm for 16 h. 8-10 transformants were randomly selected and placed in a 1.5 mL Eppendorf tube and cultured at 37°C and 850 rpm for 12 h. The plasmid was extracted and the aceF gene overexpression vector pIB139-aceF was obtained after double restriction digestion verification and sequencing to confirm the absence of base mutations;

[0039] (3.3) The successfully constructed overexpression vector pIB139-aceF was transferred into the protoplasts of Saccharopolyspora spinosa through protoplast transformation and spread on R6 solid plates. After inverted culture in a 30°C incubator for 16-20 hours, apramycin coverage screening was performed and culture was continued for 3-7 days until white transformants appeared. 8-10 transformants were randomly picked and streaked on TSB solid plates. The transformants were further purified by inverted culture in a 30°C incubator for 2-3 days. Some of the grown bacteria were picked and cultured in CSM liquid culture medium at 30°C and 150 rpm for 2 days. The genome was extracted and PCR verification was performed using primers for apr-F / apr-R to obtain the gene aceF overexpression strain.

[0040] The nucleotide sequences of the primers are shown in Table 1.

[0041] Preferably, the starting strain is Saccharopolyspora spinosa LJ-1 or wild-type Saccharopolyspora spinosa.

[0042] Preferably, the manB gene, leuA gene and aceF gene are from the genome of Saccharopolyspora spinosa LJ-1 or the genome of wild-type Saccharopolyspora spinosa.

[0043] Saccharopolyspora spinosa generally have similar metabolic pathways, so the method of the present invention can be used for various Saccharopolyspora spinosa strains. Using a Saccharopolyspora spinosa strain that originally has a strong ability to synthesize spinosad as a starting strain is conducive to ultimately obtaining a better overall effect; using a common strain, such as a wild-type Saccharopolyspora spinosa as a starting strain is also feasible.

[0044] Preferably, the engineered strain that optimizes the mannose metabolic pathway and produces high amounts of spinosad is classified and named Saccharopolyspora spinosa XLJ-1, and was deposited in the China Center for Type Culture Collection on November 10, 2023, with a deposit number of CCTCC NO: M 20232185.

[0045] The invention discloses an application of the high-yield spinosyn engineering strain with optimized mannose metabolic pathway in the biosynthesis of spinosyn.

[0046] The inventors believe that the synthetic substrates of spinosad include acetyl-CoA, malonyl-CoA, methylmalonyl-CoA, rhamnose, forosamine and S-adenosyl-methionine, etc., which come directly or indirectly from the primary metabolic pathway. Since the biosynthesis of spinosad mainly exists in the stable growth period of the bacteria, but the primary metabolic capacity of the strain has decreased at this time, the synthesis of the polyketide skeleton of spinosad will be limited by the availability of these necessary synthetic substrates. Therefore, redesigning and / or optimizing the substrate supply pathway can be used as a preferred strategy to promote the biosynthesis of spinosad. Since the mannose metabolic pathway is an important pathway for the synthesis of acetyl-CoA (the detailed metabolic pathway for the production of acetyl-CoA from glucose in Saccharopolyspora spinosa can be referred to Figure 1 The main synthetic pathways of spinosad can be found in Figure 2 ), the construction of the high-yield engineered bacteria of the present invention is to take the mannose metabolic pathway as the research object in Saccharopolyspora spinosa, knock out the genes manB and leuA that competitively utilize the glucose metabolic flow, and overexpress the acetyl-CoA synthesis-related gene aceF to enhance the acetyl-CoA synthesis ability of the bacteria, so as to promote the efficient biosynthesis of spinosad.

[0047] Saccharopolyspora spinosa LJ-1 is an excellent variant strain of Saccharopolyspora spinosa obtained by room temperature atmospheric pressure plasma (ARTP) mutagenesis combined with ultraviolet mutagenesis from the Hunan Provincial Key Laboratory of Microbial Molecular Biology of Hunan Normal University, the applicant. On this basis, by optimizing the ability of strain LJ-1 to synthesize acetyl-CoA through the mannose metabolic pathway, including knocking out the phosphomannose mutase gene manB in the fructose and mannose metabolic pathways to reduce the competitive utilization of the substrate glucose, knocking out the 2-isopropylmalate synthase gene leuA in the leucine synthesis pathway to reduce the competitive utilization of the synthetic precursor acetyl-CoA, and overexpressing the pyruvate dehydrogenase gene aceF to enhance the acetyl-CoA synthesis ability, it is beneficial to efficiently promote the biosynthesis of polymyxins. Finally, the obtained engineered bacteria with efficient biosynthesis of polymyxins has a polymyxin yield that is 2.02 times higher than the starting strain LJ-1 and 4.9 times higher than the wild-type polymyxins, and has certain potential for application in the industrial production of polymyxins.

[0048] Beneficial effects of the present invention:

[0049] The present invention starts with the metabolic pathway of Saccharopolyspora spinosa, takes the mannose metabolic pathway as the optimization object, and knocks out the phosphomannose mutase gene manB screened out in the previous research work, reduces the competitive utilization of the substrate glucose, and promotes the glucose metabolic flow to flow more to the pyruvate metabolic pathway. Subsequently, by knocking out the 2-isopropylmalate synthase gene leuA and overexpressing the pyruvate dehydrogenase gene aceF, the competitive utilization of acetyl-CoA is reduced and the ability to synthesize acetyl-CoA from pyruvate is improved, the accumulation of the precursor substance acetyl-CoA is increased, and finally the efficient biosynthesis of spinosad is achieved.

[0050] Description of Microbiological Deposit

[0051] The engineered strain of the present invention that optimizes the mannose metabolic pathway and produces high amounts of spinosad was deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, Wuhan, China) on November 10, 2023, with a deposit number of CCTCCNO: M 20232185; the classification name is: Saccharopolyspora spinosa XLJ-1; the Latin scientific name is: Saccharopolyspora spinosaXLJ-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Metabolic network for glucose production of acetyl-CoA in Saccharopolyspora spinosa.

[0053] Figure 2 It is the main synthesis pathway of spinosad in Saccharopolyspora spinosa.

[0054] Figure 3 This is the PCR verification of the mutant strain LJ-1-ΔmanB in the example of the present invention.

[0055] Figure 4 This is the PCR verification of the mutant strain LJ-1-ΔmanBΔleuA in the example of the present invention.

[0056] Figure 5 This is the PCR verification of the engineering strain XLJ-1 in the embodiment of the present invention.

[0057] Figure 6 This is the qRT-PCR analysis of the engineered strain XLJ-1 in the example of the present invention.

[0058] Figure 7 This is an analysis of the spinosad production among the engineered strain XLJ-1, the starting strain LJ-1 and the wild-type strain in the examples of the present invention.

[0059] Figure 8 This is the mass spectrometry identification of spinosad: the upper figure is the primary mass spectrum of the spinosad molecular identification, and the lower figure is the secondary mass spectrum of the spinosad molecular identification.

[0060] Fig. 9 This is an analysis of the insecticidal activity of the engineered strain XLJ-1, the starting strain LJ-1 and the wild-type strain in the examples of the present invention.

[0061] Fig.10 This is an analysis of the acetyl-CoA content of the engineering strain XLJ-1 and the starting strain LJ-1 in the examples of the present invention. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with the embodiments and drawings. The following embodiments are descriptive rather than restrictive and are not intended to limit the scope of protection of the present invention.

[0063] The raw materials used in the present invention, unless otherwise specified, are all conventional commercially available products. The methods used in the present invention, unless otherwise specified, are all conventional methods in the art. The concentrations of the various substances used in the present invention are all conventionally used concentrations.

[0064] Example

[0065] The strains, plasmids and primers involved in this example are shown in Table 1.

[0066] This example optimizes the pathway of acetyl-CoA synthesis by mannose metabolism in Saccharopolyspora spinosa LJ-1 through combinatorial metabolic engineering technology to increase the production of spinosad, involving molecular manipulation of three-gene combination modification: blocking the negative regulatory genes manB and leuA and overexpressing the positive regulatory gene aceF.

[0067] Saccharopolyspora spinosa LJ-1 is derived from an excellent variant strain of Saccharopolyspora spinosa obtained by combining room temperature and atmospheric pressure plasma (ARTP) mutagenesis with ultraviolet mutagenesis in the Hunan Provincial Key Laboratory of Microbial Molecular Biology of Hunan Normal University, the applicant.

[0068] The method for constructing the genetically engineered bacteria of Saccharopolyspora spinosa with high spinosyn production described in this embodiment comprises the following steps:

[0069] (1) Using Saccharopolyspora spinosa LJ-1 as the starting strain, the CRISPR / Cas9 technology was used to knock out the phosphomannose mutase gene manB in the fructose and mannose metabolic pathway to obtain the mutant strain LJ-1-ΔmanB;

[0070] (2) Using CRISPR / Cas9 technology to knock out the 2-isopropylmalate synthase gene leuA in the leucine synthesis pathway of the mutant strain LJ-1-ΔmanB, the mutant strain LJ-1-ΔmanBΔleuA was obtained;

[0071] (3) The pyruvate dehydrogenase gene aceF was overexpressed in the mutant strain LJ-1-ΔmanBΔleuA to obtain the genetically engineered strain LJ-1-ΔmanBΔleuA::aceF (XLJ-1) with high spinosad production.

[0072] The specific operations are as follows:

[0073] (1) Knockout of the phosphomannose mutase gene manB

[0074] (1.1) Using the genome of Saccharopolyspora spinosa LJ-1 as a template, the upstream and downstream homology arm primer pairs manB-Up-F / R and manB-Down-F / R were designed at both ends of the gene according to the manB gene sequence. After primer synthesis, PCR amplification was performed to obtain the upstream and downstream homology arms of the gene manB; the sgRNA sequence of the gene manB and its primer pair manB-sgRNA-F / R were designed and synthesized using the website https: / / portals.broadinstitute.org / gpp / public / , and PCR amplification was performed using the vector pKCcas9dO as a template to obtain the fragment sgRNA (manB).

[0075] (1.2) Use fusion PCR to fuse sgRNA with the upstream and downstream homology arms to obtain the fusion fragment sgRNA-Up manB -Down manB , and double-digested it with Spe I and Hind III; the fusion fragment sgRNA-Up manB -Down manB The linearized vector pKCcas9dO was ligated at 16°C overnight under the catalysis of T4 DNA ligase. The ligation product was heat-shocked into competent E. coli DH5α and cultured on a LB solid plate coated with apramycin at 37°C and 150 rpm for 16 h. Eight transformants were randomly selected and placed in a 1.5 mL Eppendorf tube and cultured at 37°C and 850 rpm for 12 h. The plasmid was extracted and the gene manB knockout vector pKCcas9d-manB was obtained after double enzyme digestion verification and sequencing to confirm the absence of base mutations.

[0076] (1.3) The successfully constructed knockout vector pKCcas9d-manB was transferred into the protoplasts of Saccharopolyspora spinosa LJ-1 through protoplast transformation and spread on R6 solid plates. After inverted culture in a 30°C incubator for 16 hours, apramycin coverage screening was performed and culture was continued for 5 days. White transformants appeared. Eight transformants were randomly picked and streaked on TSB solid plates. Inverted culture was carried out in a 30°C incubator for 3 days to further purify the transformants. Some of the grown bacteria were picked and cultured in CSM liquid culture medium at 30°C and 150 rpm for 2 days. The genome was extracted and PCR verification was performed using primers manB-F / manB-R to obtain the gene manB knockout bacteria LJ-1-ΔmanB. PCR verification of manB knockout mutants is shown in Figure 3 .

[0077] (1.4) The successfully verified mutant strain LJ-1-ΔmanB was placed under 37°C culture conditions for plasmid elimination. After multiple screenings, the mutant strain LJ-1-ΔmanB with the recombinant plasmid pKCcas9d-manB eliminated was obtained.

[0078] (2) Knockout of 2-isopropylmalate synthase gene leuA

[0079] (2.1) Using the genome of Saccharopolyspora spinosa LJ-1 as a template, according to the leuA gene sequence, the upstream and downstream homology arm primer pairs leuA-Up-F / R and leuA-Down-F / R were designed at both ends of the gene, respectively. After primer synthesis, PCR amplification was performed to obtain the upstream and downstream homology arms of the gene leuA. The sgRNA sequence of the gene leuA and its primer pair leuA-sgRNA-F / R were designed and synthesized using the website https: / / portals.broadinstitute.org / gpp / public / , and PCR amplification was performed using the vector pKCcas9dO as a template to obtain the fragment sgRNA (leuA).

[0080] (2.2) Use fusion PCR to fuse sgRNA with the upstream and downstream homology arms to obtain the fusion fragment sgRNA-Up leuA -Down leuA , and double-digested it with Spe I and Hind III; the fusion fragment sgRNA-Up leuA- -Down leuA- The linearized vector pKCcas9dO was ligated at 16°C overnight under the catalysis of T4 DNA ligase, and the ligation product was heat-shocked into competent E. coli DH5α and cultured on a LB solid plate coated with apramycin at 37°C and 150 rpm for 16 h. Eight transformants were randomly selected and placed in a 1.5 mL Eppendorf tube and cultured at 37°C and 850 rpm for 12 h. The plasmid was extracted and the gene leuA knockout vector pKCcas9d-leuA was obtained after double enzyme digestion verification and sequencing to confirm the absence of base mutations.

[0081] (2.3) The successfully constructed knockout vector pKCcas9d-leuA was transferred into the mutant strain LJ-1-ΔmanB protoplasts through protoplast transformation and spread on R6 solid plates. After inverted culture in a 30°C incubator for 16-20 hours, apramycin coverage screening was performed and culture was continued for 6 days until white transformants appeared. Eight transformants were randomly selected and streaked on TSB solid plates. The transformants were further purified by inverted culture in a 30°C incubator for 3 days. Some of the grown bacteria were selected and cultured in CSM liquid culture medium at 30°C and 150 rpm for 2 days. The genome was extracted and PCR verification was performed using primers for leuA-F / leuA-R to obtain the gene leuA knockout bacteria LJ-1-ΔmanBΔleuA. The leuA knockout mutant strain PCR verification is shown in Figure 4 .

[0082] (2.4) The successfully verified mutant strain LJ-1-ΔmanBΔleuA was placed under 37°C culture conditions for plasmid elimination. After multiple screenings, the mutant strain LJ-1-ΔmanBΔleuA eliminated by the recombinant plasmid pKCcas9d-ΔleuA was obtained.

[0083] (3) Overexpression of pyruvate dehydrogenase gene aceF

[0084] (3.1) Using the genome of Saccharopolyspora spinosa LJ-1 as a template and according to the aceF gene sequence, a primer pair aceF-F / R was designed to amplify the open reading frame of the aceF gene. After primer synthesis, the aceF gene was amplified by PCR and double-digested with Xba I and EcoR I.

[0085] (3.2) The aceF gene after restriction digestion was ligated with the linearized vector pIB139 at 16°C overnight under the catalysis of T4 DNA ligase. The ligation product was heat-shocked into competent E. coli DH5α and cultured on a LB solid plate coated with apramycin at 37°C and 150 rpm for 16 h. Eight transformants were randomly selected and placed in a 1.5 mL Eppendorf tube and cultured at 37°C and 850 rpm for 12 h. The plasmid was extracted and the aceF gene overexpression vector pIB139-aceF was obtained after double restriction digestion verification and sequencing to confirm the absence of base mutations.

[0086] (3.3) The successfully constructed overexpression vector pIB139-aceF was transferred into the mutant strain LJ-1-ΔmanBΔleuA protoplasts through protoplast transformation and spread on R6 solid plates. After inverted culture in a 30°C incubator for 16 hours, apramycin coverage screening was performed and culture was continued for 5 days until white transformants appeared. Eight transformants were randomly picked and streaked on TSB solid plates. The transformants were further purified by inverted culture in a 30°C incubator for 3 days. Some of the grown bacteria were picked and cultured in CSM liquid culture medium at 30°C and 150rmp / min for 2 days. The genome was extracted and PCR verification of apr-F / R was performed using primers to obtain the gene aceF overexpression engineered strain XLJ-1, which is an engineered strain with efficient biosynthesis of polymyxins. PCR verification of aceF overexpression mutant strains is shown in Figure 5 .

[0087] The nucleotide sequence of the upstream homologous arm of the manB gene used for knockout is shown in SEQ ID NO.1.

[0088] The nucleotide sequence of the downstream homologous arm of the manB gene used for knockout is shown in SEQ ID NO.2.

[0089] The nucleotide sequence of the upstream homology arm of the leuA gene used for knockout is shown in SEQ ID NO.3.

[0090] The nucleotide sequence of the downstream homology arm of the leuA gene used for knockout is shown in SEQ ID NO.4.

[0091] The nucleotide sequence of the aceF gene sequence used for overexpression is shown in SEQ ID NO.5.

[0092] The nucleotide sequence of the sgRNA sequence of the manB gene used for knockout is shown in SEQ ID NO.6.

[0093] The nucleotide sequence of the sgRNA sequence of the leuA gene used for knockout is shown in SEQ ID NO.7.

[0094] Detection and analysis

[0095] 1. Real-time fluorescence quantitative PCR detection

[0096] In order to verify whether the transcription level of the modified genes in the engineered strain XLJ-1 has changed at the transcriptional level, the strain was detected and analyzed using the qRT-PCR method. First, the engineered strain XLJ-1 and the starting strain LJ-1 activated in the CSM activation medium were transferred to the synthetic fermentation medium in equal amounts for shake flask culture (30°C, 250r / min), and the fermentation broth on the second and fourth days was used for total RNA extraction. The RNA extraction method used the TRIzol kit method (Sangon) and strictly followed the instructions. In order to exclude the influence of genomic DNA on the experimental results, 16SrRNA was used as the internal reference, and the primer pairs manB-F / R, leuA-F / R and aceF-1-F / R were used to compare and analyze the mRNA expression levels of manB, leuA and aceF genes in the engineered strain XLJ-1 and the starting strain LJ-1, respectively. The ratio of manB, leuA and aceF gene mRNA in the starting strain LJ-1 to its internal reference mRNA was set to 1, and 4 replicates were set for each sample. Figure 6 It can be seen that compared with the starting strain LJ-1, the expression of manB and leuA was not detected at the mRNA level in the engineered strain XLJ-1, while the mRNA level of the aceF gene was significantly upregulated, and its transcription level increased by 3.7 times and 2.9 times on the 2nd and 4th days, respectively. This result shows that the engineering strains with manB and leuA knockout and aceF overexpression were successfully constructed.

[0097] 2. Comparison of Spinosad Yield and Insecticidal Effect

[0098] The activated engineered strain XLJ-1, the starting strain LJ-1 and the wild-type strain S. spinosa were transferred into the synthetic fermentation medium in equal amounts, and the fermentation broth on the 12th day was used for the production detection of spinosad and the insecticidal toxicity analysis against cotton bollworm.

[0099] The method for detecting the production of spinosad is as follows:

[0100] Take 500 μL of the fermentation broth on the 12th day of fermentation in a 2.0 mL Ep tube, add 100 μL of lysozyme at a concentration of 20 mg / L, treat for half an hour, and add methanol at a ratio of 1:2 to mix. After standing for 20 minutes, centrifuge at 10000×g for 10 minutes and retain the supernatant. The supernatant can be analyzed by a high-performance liquid chromatography system (HPLC1290, Agilent, USA) after filtering with a 0.22 μm filter membrane. Detection conditions: chromatographic column Zorbax SB-C18, 1.0 mm×150 mm; mobile phase A: 10% acetonitrile aqueous solution, mobile phase B: 100% acetonitrile; flow rate 1.0 mL / min; detection wavelength set to 250 nm; injection volume 20 μL, gradient elution. The elution procedure is as follows.

[0101]

[0102] The spinosad peak area was calculated using the instrument's own analysis software, and the peak was identified by mass spectrometry using LC-MS / MS to determine whether it was spinosad.

[0103] HPLC1290 test results are as follows Figure 7 As shown in the figure, there is a chromatographic peak with a significant change in peak area at around 17.9 min and the maximum absorption wavelength is 250 nm. The mass spectrometry identification results further confirmed that the chromatographic peak is spinosad ( Figure 8 ). After software analysis, the average peak area of ​​the engineered strain XLJ-1 at 17.9 min was the highest, which was 48349±216.88, the average peak area of ​​the starting strain LJ-1 at 17.9 min was 23882.57±256.88, and the average peak area of ​​the wild-type strain was 9872±186.13. Finally, the production of spinosad in the engineered strain XLJ-1 was 2.02 times higher than that of the starting strain LJ-1, and 4.9 times higher than that of the wild-type strain S. spinosa.

[0104] Analysis of insecticidal toxicity against cotton bollworm:

[0105] Take 1mL of fermentation liquid of the engineered strain XLJ-1, the starting strain LJ-1 and the wild-type strain S.spinosa, add it to 20mL of feed, stir evenly, and add it evenly to a 24-well culture plate. After the feed cools down, inoculate one cotton bollworm in each well. Culture at 28℃, and record the number of cotton bollworms surviving on the culture plate every 24 hours for a total of 7 days. Among them, the fermentation liquid of the starting strain LJ-1 was set as positive control 1, the fermentation liquid of the wild-type strain S.spinosa was set as positive control 2, and the culture medium was set as negative control. The results are as follows Fig. 9 The results showed that the insecticidal toxicity of the engineered strain XLJ-1 against cotton bollworm was significantly higher than that of other strains.

[0106] 3. Analysis of the content of synthetic precursor acetyl-CoA

[0107] The genes selected for modification in the present invention are all related to the accumulation and synthesis of acetyl-CoA. In order to evaluate whether the multi-gene modification promotes the biosynthesis of spinosad by improving the ability of the bacteria to synthesize acetyl-CoA, the acetyl-CoA content of all mutant strains constructed above was detected. The activated engineered strain XLJ-1 and the starting strain LJ-1 were transferred into synthetic fermentation medium in equal amounts, and the fermentation broth cultured on the 2nd and 4th days was taken for the acetyl-CoA content detection. The detection method was strictly carried out in accordance with the instructions of the kit. The results showed that the acetyl-CoA content of the engineered bacteria after the optimization of the mannose metabolic pathway was higher than that of other strains on the 2nd and 4th days, indicating that manB and leuA blocking and aceF overexpression can promote the biosynthesis of spinosad by increasing the supply of intracellular acetyl-CoA ( Fig.10 ).

[0108] Table 1 Information on strains, plasmids and primers involved in the embodiments of the present invention

[0109]

[0110]

Claims

1. An engineered strain for high-yield spinosad by optimizing the mannose metabolic pathway, characterized in that: Its taxonomic name is Saccharopolyspora spinosa ( Saccharopolyspora spinosa )XLJ-1, deposited in the China Center for Type Culture Collection on November 10, 2023, with the deposit number CCTCC NO: M 20232185.

2. Use of the engineered strain with high spinosyn production by optimizing the mannose metabolic pathway as claimed in claim 1 in the biosynthesis of spinosyn.

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