A method for increasing the production of polyketides by splitting modular polyketide synthase genes

Abstract

This invention relates to a method for increasing the yield of polyketide compounds by splitting a modular polyketide synthase gene. (1) Constructing a RedEx gene cassette for splitting the modular polyketide synthase gene; (2) Constructing a target plasmid carrying an amp-ccdB gene selection expression cassette; (3) Constructing a target plasmid carrying an amp-ccdB gene selection expression cassette. C DD-TGA-RBS-ATG- N (3) Target plasmid encoding the DD coding sequence; (4) Construction of recombinant strains; (5) Fermentation culture to obtain polyketide compounds. This invention is the first to discover that the biosynthetic efficiency of host strains carrying the split PKS gene is much higher than that of host strains carrying the natural PKS gene, which can achieve the purpose of increasing the yield of polyketide compounds. Using this method of splitting modular polyketide synthase genes, high-yield butenyl spinosad genetically engineered bacteria and high-yield avermectin genetically engineered bacteria were successfully constructed, which effectively increased the yield of butenyl spinosad and avermectin. The yield of butenyl spinosad increased by a maximum of 30 times, and the yield of avermectin increased by a maximum of 5.8 times.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a method for increasing the yield of polyketide compounds by splitting modular polyketide synthase genes. Background Technology

[0002] Modular polyketide synthases (mPKSs) are giant synthases with catalytic domains, the largest proteins in bacteria, responsible for the biosynthesis of many important drug compounds, including antibiotics, immunosuppressants, pesticides, and anticancer drugs. mPKSs proteins are sequenced through docking domains, forming collinear giant multi-enzyme synthase complexes that function like an assembly line in biosynthesis. Each mPKSs protein consists of one or more modules, each module comprising a set of catalytic domains. Each module within an mPKSs catalyzes an assembly cycle in biosynthesis by adding an acyl-CoA (CoA) building block to the polyketide chain. Individual modules are typically larger than 150 kDa, and the molecular weight of an mPKSs protein is determined by the number of its modules and catalytic domains; thousands of mPKSs have been discovered to date. The biosynthetic efficiency of polyketide compounds has been improved through optimizing gene transcription or post-translational modifications, modifying regulatory factors, and increasing precursor supply. However, little is known about the transcription and translation of mPKS genes; therefore, research on mPKS genes will further promote the engineering of polyketide compound biosynthesis.

[0003] Truncated messenger RNAs (mRNAs) are produced by premature termination of transcription or degradation by ribonucleases. In bacteria, transcription and translation are coupled; both intact and truncated mRNAs are translated indiscriminately into polypeptides, and mRNAs with intact open reading frames (ORFs) are translated into functional PKS proteins. Translation is inhibited on mRNAs with truncated ORFs lacking stop codons because they cannot terminate translation correctly; the stalled ribosomes are subsequently rescued by the ribosome rescue system. The translation of truncated mRNAs into polypeptides lacking C-terminal catalytic domains leads to a waste of cellular resources. Furthermore, translating truncated mRNAs also reduces the number of active ribosomes. Genes encoding mPKSs are typically larger than 10 kb and form operons; therefore, their mRNAs are longer than those of ordinary genes. The longer the mRNA, the greater the risk of truncation; the translation of truncated mPKS mRNAs will produce non-functional PKS fragments that cannot be used for polyketide biosynthesis. Currently, no studies have assessed the impact of truncated mRNAs on mPKS translation and polyketide biosynthesis. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for increasing the yield of polyketide compounds by splitting modular polyketide synthase genes.

[0005] Terminology Explanation:

[0006] RedEx Method: The basic structure of the RedEx gene cassette consists of "linear loop recombination homologous arms (HA)". 50 )-Target DNA fragment-terminal circularized homologous arm (DR 20 - Single restriction enzyme site - Forward selection marker - Reverse selection marker - Restriction enzyme site - Linear circular recombination homologous arm (including terminal circularized homologous arm DR) 20 -HA 50 The method involves using a Redαβ homologous recombinase derived from λ phage to mediate loop homologous recombination, combined with CcdB reverse selection and exonuclease-mediated in vitro DNA annealing to perform seamless site-directed mutagenesis of the target gene. Simultaneously, the constructed engineered host strain GBred-gryA462 contains a Redαβ homologous recombinase controlled by the pBAD promoter and carries GyrA… Arg462Cys The mutation (the amino acid at position 462 of the GyrA subunit is changed from arginine to cysteine) has CcdB toxin protein tolerance and can carry plasmids containing the CcdB gene. Redα is a 5'-3' exonuclease, Redβ is a single-stranded DNA annealing protein, Redγ is a RecBCD inhibitor, and CcdB is a toxin that inhibits DNA helicase activity (see articles wang H, Bian X, Xia L, et al. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 2014 Mar; 42(5):e37. and Luan J, Song C, Liu Y, He R, Guo R, Cui Q, Jiang C, Li X, Hao K, Stewart AF, Fu J, Zhang Y, Wang H. Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method. Nat Protoc. 2024 Jul 15.).

[0007] The technical solution of the present invention is as follows:

[0008] A method for increasing the yield of polyketide compounds by disassembling modular polyketide synthase genes includes the following steps:

[0009] (1) Design several pairs of amplification primers according to the target modular polyketide synthase gene, and generate primers containing the target modular polyketide synthase gene for splitting by tandem PCR.C DD-TGA-RBS-ATG- N RedEx gene cassettes of DD coding sequences;

[0010] (2) The plasmid containing the target modular polyketide synthase gene cluster and the RedEx gene cassette obtained in step (1) were sequentially transformed into E. coli GBred-gryA462 that had been induced to express Redαβ recombinase. Through linear circular homologous recombination, the C-terminal docking domain in the RedEx gene cassette was transformed into the target modular polyketide synthase gene cluster. C DD) encoded sequence and N-end docking field ( N The linker sequence in the target modular polyketide synthase module coding sequence was replaced with the DD coding sequence. After transformation, the module was cultured, screened, and plasmids were extracted to obtain the target plasmid carrying the amp-ccdB gene selection expression cassette.

[0011] (3) The target plasmid carrying the amp-ccdB gene selection cassette obtained in step (2) was linearized and digested with enzymes. Then, the linearized plasmid was reacted in an exonuclease reaction system. After the reaction was completed, it was electroporated into Escherichia coli GB2005. After transformation, the plasmid was cultured, screened, and extracted to obtain the target plasmid carrying the amp-ccdB gene selection cassette. C DD-TGA-RBS-ATG- N The target plasmid for the DD coding sequence;

[0012] (4) The site-specific recombination mediated by phiC31 integrase is used to transfer the contents of step (3) to the site. C DD-TGA-RBS-ATG- N The target plasmid encoding the DD sequence was integrated into the phiC31 attB site on the host strain chromosome to obtain a recombinant strain;

[0013] (5) The recombinant strain obtained in fermentation culture step (4) is centrifuged to collect resin and bacterial cells, and after separation and purification, polyketide compound is obtained.

[0014] According to a preferred embodiment of the present invention, in step (1), the modular polyketide synthase gene is the modular polyketide synthase gene busA-E of butenyl spinosad or the modular polyketide synthase gene aveA1-4 of avermectin.

[0015] More preferably, the modular polyketide synthase gene is the modular polyketide synthase gene busA of butenyl spinosad or the modular polyketide synthase gene aveA2 of avermectin.

[0016] Among them, busA has a length of 13.0kb and GenBank accession number AX600586; aveA2 has a length of 18.7kb and GenBank accession number AB032367.1.

[0017] According to a preferred embodiment of the present invention, in step (1), the C DD-TGA-RBS-ATG- N DD encoded sequence is C DD, TGA, RBS, ATG and N DD are connected sequentially to form the following; C DD is the C-terminal docking domain coding sequence, TGA is the stop codon, RBS is the ribosome binding site, and ATG is the start codon. N DD is the N-end docking field encoding sequence.

[0018] Further preferably, the C DD is the coding sequence for the C-terminal docking domain of the slnA1 or slnA7 modular polyketide synthase gene salinomycin. N DD is the N-terminal docking domain coding sequence of the slnA2 or slnA8 modular polyketide synthase gene sln.

[0019] According to a preferred embodiment of the present invention, in step (2), the plasmid containing the target modular polyketide synthase gene cluster is either the pBAC-phiC31-bus plasmid or the pBAC-phiC31-avm plasmid.

[0020] According to a preferred embodiment of the present invention, in step (2), the information and construction method of the Escherichia coli GBred-gryA462 that has been induced to express Redαβ recombinase have been disclosed, and for details please refer to the article "Wang H, Bian X, Xia L, et al. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 2014 Mar; 42(5):e37."

[0021] According to a preferred embodiment of the present invention, in step (2), the amp-ccdB gene selection expression cassette includes an amp resistance selection marker and a ccdB toxicity gene (reverse selection marker). Information about the amp-ccdB gene expression cassette has been published; for details, please refer to the article "Wang H, Bian X, Xia L, et al. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 2014 Mar; 42(5):e37."

[0022] According to a preferred embodiment of the present invention, in step (3), the target plasmid carrying the amp-ccdB gene selection expression cassette is linearized by PacI enzyme digestion;

[0023] The exonuclease reaction system consists of 10 μL of 2×GibsonAssembly Master Mix, 5 pmol of the target plasmid to be reacted, and sterile deionized water to a final volume of 20 μL.

[0024] According to a preferred embodiment of the present invention, in step (4), the host strain is *Streptomyces albus* J1074 or *Streptomyces coelicolor* CH999.

[0025] According to a preferred embodiment of the present invention, the method for increasing the yield of polyketide compounds by splitting modular polyketide synthase genes is as follows:

[0026] (1) Using plasmid pBAC-sal as a template, with busA-1- C DDslnA1-F / R and busA-1- N Using DDslnA2-F / R as primers, PCR amplification was performed to obtain HAL- with overlapping sequences. C DDslnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 Fragments; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 The fragments are joined together to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 Fragment;

[0027] Using plasmid p15A-ccdB-amp as a template and busA-1-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL-C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-1;

[0028] (2) Using the HAL- obtained in step (1) C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 Using the fragment as a template, with busA-2-slnA1- C DD-F, busA-2-slnA2- N Using DD-R as primers, PCR amplification was performed to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 Fragment;

[0029] Using plasmid p15A-ccdB-amp as a template and busA-2-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-2;

[0030] (3) Using plasmid pBAC-sal as a template, and busA-3-slnA7- C DD-F, busA-3-slnA8- N Using DD-R as primers, PCR amplification was performed to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 Fragment;

[0031] Using plasmid p15A-ccdB-amp as a template and busA-3-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DD slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-3;

[0032] (4) Plasmid pBAC-phiC31-bus and HAL- C DD-TGA-RBS-ATG- N DD-HAR 20 -PacI-ampccdB-PacI-HAR cassette-1 was sequentially electroporated into *E. coli* GBred-gryA462, which had been induced to express Redαβ recombinase. Through linear-circular homologous recombination, HAL- C DD-TGA-RBS-ATG- N DD-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette salinomycin C DD sequences and N The DD sequence replaced the adapter sequence between busA-1-1 and busA-1-2; the transformed E. coli GBred-gryA462 was cultured and inoculated on LB solid medium supplemented with ampicillin (100 μg / mL), positive colonies were screened, and plasmids were extracted to obtain plasmid pBAC-phiC31-busA-1-ampccdB;

[0033] (5) Following the method described in step (4), make HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-2 salinomycin C DD sequences andN The DD sequence was used to replace the linker sequence between busA-2-1 and busA-2-2 to obtain plasmid pBAC-phiC31-busA-2-ampccdB.

[0034] Following the method described in step (4), HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-3 and HAL- C DDslnA7-TGA-RBS-ATG- N DD slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-2 salinomycin C DD sequences and N The DD sequence was used to replace the linker sequence between busA-3-1, busA-3-2 and busA-3-3 to obtain plasmid pBAC-phiC31-busA-3-ampccdB.

[0035] (6) The plasmids pBAC-phiC31-busA-1-ampccdB, pBAC-phiC31-busA-2-ampccdB, and pBAC-phiC31-busA-3-ampccdB were linearized by PacI enzyme digestion, respectively, to remove the forward and reverse selection marker amp-ccdB gene, exposing the circular homologous arm (DR) and obtaining the linearized target plasmid. The linearized plasmid was then reacted in an exonuclease reaction system, and the reaction mixture was transferred to a Millipore membrane filter for dialysis at room temperature for 30 minutes to remove salt. 20 μL of the dialysis reaction mixture was then electroporated into E. coli GB2005 cells. After transformation, the cells were cultured, screened, and the plasmid was extracted to obtain the target plasmid carrying the target gene. C DD-TGA-RBS-ATG- N The target plasmids for the DD coding sequences are pBAC-phiC31-busA-1, pBAC-phiC31-busA-2, and pBAC-phiC31-busA-3.

[0036] The exonuclease reaction system consists of 10 μL of 2×GibsonAssembly Master Mix, 5 pmol of the target plasmid to be reacted, and sterile deionized water to a final volume of 20 μL.

[0037] The exonuclease reaction conditions are: incubation at 50°C for 1 hour, followed by incubation at 4°C.

[0038] (7) pBAC-phiC31-busA-1, pBAC-phiC31-busA-2 and pBAC-phiC31-busA-3 were integrated into the phiC31 attB site on chromosome J1074 of Streptomyces albus, respectively, through site-specific recombination mediated by phiC31 integrase, to obtain butenyl spinosad genetically engineered bacteria busA-1, busA-2 and busA-3;

[0039] (8) Fermentation culture step (7) The butenyl spinosad genetically engineered bacteria busA-1, busA-2 and busA-3 obtained by fermentation culture step (7) were centrifuged to collect resin and bacterial cells, and after separation and purification, butenyl spinosad was obtained.

[0040] A genetically engineered bacterium that produces butenyl spinosad was constructed according to steps (1) to (7) of the above method.

[0041] According to a preferred embodiment of the present invention, the method for increasing the yield of polyketide compounds by splitting modular polyketide synthase genes is as follows:

[0042] 1) Following the steps (1) to (7) above, the genetically engineered bacteria busA-1, busA-2 and busA-3 that produce butenyl spinosad were constructed;

[0043] 2) The strong constitutive promoter KasOp* was inserted upstream of busA-1-2, busA-2-2, busA-3-2, busB, busC, busD, and busE, respectively, into the genetically engineered bacteria busA-1, busA-2, and busA-3 that produce butenyl spinosad, to obtain genetically engineered bacteria KbusA-1-2, KbusA-2-2, KbusA-3-2, KbusB, KbusC, KbusD, and KbusE that produce high levels of butenyl spinosad;

[0044] 3) The high-yield butenyl spinosad genetically engineered bacteria KbusA-1-2, KbusA-2-2, KbusA-3-2, KbusB, KbusC, KbusD, and KbusE obtained in step 2) were centrifuged to collect the resin and bacterial cells. After separation and purification, butenyl spinosad was obtained.

[0045] A genetically engineered bacterium that produces high levels of avermectin was constructed according to steps 1) to 2) of the above method.

[0046] According to a preferred embodiment of the present invention, the method for increasing the yield of polyketide compounds by splitting modular polyketide synthase genes is as follows:

[0047] a. Using plasmid pBAC-sal as a template, and aveA2-1-sln12-C DD-F / R and aveA2-1-sln12- N Using DD-F / R as primers, PCR amplification was performed to obtain HAL- with overlapping sequences. C DD-slnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 Fragments; then HAL- was separated using tandem PCR. C DD-slnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 The fragments are joined together to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 Fragment;

[0048] Using plasmid p15A-ccdB-amp as a template and aveA2-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD-slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette;

[0049] b. Using plasmid pBAC-sal as a template, and aveA2-1-sln78- C DD-F / R and aveA2-1-sln78- N Using DD-F / R as primers, PCR amplification was performed to obtain HAL- with overlapping sequences. C DD-slnA7-TGA-RBS fragment and ATG- N DDslnA8-HAR 20 Fragments; then HAL- was separated using tandem PCR. C DD-slnA7-TGA-RBS fragment and ATG- NDDslnA8-HAR 20 The fragments are joined together to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 Fragment;

[0050] Using plasmid p15A-ccdB-amp as a template and aveA2-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DD-slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette;

[0051] c. Plasmid pBAC-phiC31-avm and HAL- C DDslnA1-TGA-RBS-ATG- N DD-slnA2-HAR 20 The PacI-ampccdB-PacI-HAR gene cassette was sequentially electroporated into *E. coli* GBred-gryA462 cells induced to express Redαβ recombinase. Through linear-circular homologous recombination, HAL- C DDslnA1-TGA-RBS-ATG- N DD-slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette salinomycin C DD sequences and N The DD sequence replaced the adapter sequence between aveA2-1-1 and aveA2-1-2; the transformed E. coli GBred-gryA462 was cultured and inoculated on LB solid medium supplemented with ampicillin (100 μg / mL), positive colonies were screened, and plasmids were extracted to obtain plasmid pBAC-phiC31-avm-ampccdB-1;

[0052] d. Following the method described in step c, make HAL- C DDslnA7-TGA-RBS-ATG- N DD-slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette salinomycin C DD sequences and N The DD sequence was used to replace the linker sequence between aveA2-2-1 and aveA2-2-2 to obtain plasmid pBAC-phiC31-avm-ampccdB-2.

[0053] e. Using PacI enzyme, plasmids pBAC-phiC31-avm-ampccdB-1 and pBAC-phiC31-avm-ampccdB-2 were linearized by restriction enzyme digestion, removing the forward and reverse selection marker amp-ccdB gene, exposing the circular homologous arm (DR), and obtaining linearized target plasmids. The linearized plasmids were then reacted in an exonuclease reaction system. The reaction mixture was then transferred to a Millipore membrane filter and dialyzed to remove salts at room temperature for 30 minutes. 20 μL of the dialyzed reaction mixture was then electroporated into *E. coli* GB2005 cells. After transformation, the cells were cultured, screened, and plasmids were extracted to obtain plasmids carrying… C DD-TGA-RBS-ATG- N The target plasmids for the DD coding sequence were named pBAC-phiC31-aveA2-1 and pBAC-phiC31-aveA2-2, respectively.

[0054] The exonuclease reaction system consists of 10 μL of 2×GibsonAssembly Master Mix, 5 pmol of the target plasmid to be reacted, and sterile deionized water to a final volume of 20 μL.

[0055] The exonuclease reaction conditions are: incubation at 50°C for 1 hour, followed by incubation at 4°C.

[0056] f. By site-specific recombination mediated by phiC31 integrase, pBAC-phiC31-aveA2-1 and pBAC-phiC31-aveA2-2 were integrated into the phiC31 attB site of chromosome S. coelicolorCH999, respectively, to obtain the high-avermectin-producing genetically engineered bacteria aveA2-2-1 and aveA2-2-2;

[0057] g. The high-yield abamectin-producing genetically engineered bacteria aveA2-2-1 and aveA2-2-2 obtained in fermentation culture step f are centrifuged to collect resin and bacterial cells, and after separation and purification, abamectin is obtained.

[0058] A genetically engineered bacterium that produces high levels of avermectin was constructed according to steps a to f of the above method.

[0059] All steps not described in detail in this invention are performed in accordance with existing technology.

[0060] Technical features and beneficial effects of the present invention:

[0061] 1. The inventors of this application have discovered that during the expression of the ultra-large modular polyketide synthase gene (PKS), truncated mRNA constitutes the majority of the PKS mRNA, and the truncated mRNA leads to a higher abundance and production rate of proteins encoded by genes closer to the promoter in the operon. Therefore, the presence of truncated mRNAs results in a higher abundance and production rate of proteins encoded by genes closer to the promoter of the operon. Based on these findings, the applicant provides a novel method for increasing the yield of polyketide compounds by splitting the modular polyketide synthase gene. This method involves splitting the multi-module PKS protein into independently translatable single-module PKS subunits and adding a heterologous PKS docking domain to the end of the split PKS subunits to maintain the correct sequence of the multi-module PKS protein. This method can translate the truncated mRNA into functional PKS subunits and increases the concentration of PKS subunits encoded by sequences closer to the 5' end of the mRNA. Therefore, the biosynthetic efficiency of host strains carrying the split PKS gene is much higher than that of host strains carrying the natural PKS gene, thereby achieving the goal of increasing the yield of polyketide compounds.

[0062] 2. This invention successfully constructed high-yield spinosad and high-yield avermectin genetically engineered bacteria using the modular polyketide synthase gene splitting method, effectively increasing the yield of spinosad and avermectin. Compared with wild-type strains carrying natural PKS genes, the yield of spinosad by the genetically engineered bacteria was increased by up to 30 times, and the yield of avermectin was increased by up to 5.8 times. Attached Figure Description

[0063] Figure 1 A schematic diagram of three strategies for splitting the busA gene.

[0064] Figure 2 A schematic diagram of the insertion site of the strong constitutive promoter KasOp* and a bar chart of butenyl spinosad production in different strains.

[0065] In the figure, a represents the insertion site of KasOp*; b represents the butenyl spinosad production of genetically engineered bacteria busA-1 and KbusA-1-2; c represents the butenyl spinosad production of genetically engineered bacteria busA-2 and KbusA-2-2; and d represents the butenyl spinosad production of genetically engineered bacteria busA-3KbusA-1-2, KbusA-3-2, KbusB, KbusC, KbusD, and KbusE.

[0066] Figure 3 High-resolution mass spectrometry analysis of butenyl spinosad produced by different strains.

[0067] Figure 4 Comparison of butenyl spinosad production by different strains.

[0068] Figure 5 This diagram illustrates two strategies for splitting the aveA2 gene.

[0069] Figure 6 High-resolution mass spectrometry analysis of avermectin produced by different strains.

[0070] Figure 7 Comparison of avermectin production among different strains.

[0071] Figure 8 The results of qRT-PCR analysis of the transcriptional expression level of the gusA gene in different genetically engineered bacteria are presented.

[0072] Figure 9 The results show the β-glucuronidase activity in different genetically engineered bacteria.

[0073] Figure 10 A schematic diagram for detecting genetically engineered bacteria and the synthesis rate of gusA protein in different genetically engineered bacteria;

[0074] In the figure, a represents the position of the promoters of busA-gusA, busA-1-1-gusA, busA-2-1-gusA, and p-isopropylbenzoic acid (cumate); b represents the rate of gusA protein synthesis in genetically engineered bacteria BusA-1-1, BusA-2-1, and BusA. Detailed Implementation

[0075] To facilitate understanding of the present invention, a more detailed description is provided below with reference to the accompanying drawings and specific embodiments. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0076] The amp-ccdB gene expression cassette includes an amp resistance selection marker and a ccdB toxicity gene (reverse selection marker). Information about this amp-ccdB gene expression cassette has been published; please see the article "Wang H, Bian X, Xia L, et al. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 2014 Mar; 42(5):e37." for details.

[0077] Information and construction methods for Escherichia coli GBred-gryA462 that has been induced to express Redαβ recombinase have been published. For details, please refer to the article "Wang H, Bian X, Xia L, et al. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 2014 Mar; 42(5):e37."

[0078] Information and construction methods for Escherichia coli GB05-red that has been induced to express Redαβ recombinase have been published. For details, please refer to the article "Wang H, Bian X, Xia L, et al. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 2014 Mar; 42(5):e37."

[0079] The plasmid pBAC-sal contains the salinomycin biosynthetic gene cluster, and related information has been publicly available. For details, please refer to the article "Jiang, C., et al.,Establishing an efficient salinomycin biosynthetic pathwayin three heterologous Streptomyces hosts by constructing a 106-kb multioperonartificial gene cluster.Biotechnol.Bioeng.,2021.118(12):p.4668-4677.".

[0080] Information about plasmid p15A-ccdB-amp has been made public, see the article “Wang H, Bian X, Xia L, et al. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 2014 Mar; 42(5):e37.”.

[0081] The plasmid pBAC-phiC31-bus contains the butenyl spinosad biosynthesis gene cluster. Information about pBAC-phiC31-bus has been published, see the article “Song, C., et al., RedEx: a method for seamless DNA insertion and deletion in large multimodal polyketide synthase geneclusters. Nucleic Acids Res., 2020. 48(22): p.e130”.

[0082] Information and construction methods for plasmids pBR322-amp-tetR-tetO-hyg-ccdB and pR6K-TnpA-oriT-kan have been published. For details, please refer to the article "Wang, H. et al. RecET direct cloning and Redabrecombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. Nat. Protoc. 11, 1175-1190 (2016)".

[0083] Information about the strong constitutive promoter KasOp* has been publicly available. For details, please refer to the article “Wang W, Li X, Wang J, Xiang S, Feng X, Yang K. An engineered strong promoter for streptomycetes. Appl Environ Microbiol. 2013 Jul; 79(14):4484-92. doi:10.1128 / AEM.00985-13. Epub2013May 17. PMID:23686264;PMCID:PMC3697493”.

[0084] Information regarding plasmid pBAC-spnNE-cum-spnS has been publicly available; see the article “Li, X. et al. Improving spinosad production by tuning expressions of the forosaminemethyltransferase and the forosaminyl transferase to reduce undesired lessactive byproducts in the heterologous host Streptomyces albus J1074. Microb. Cell Fact. 22, 15 (2023)”.

[0085] The high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HPLC) methods for the analysis of butenyl spinosad and avermectin involved in the examples are as follows: High-resolution mass spectrometry analysis was performed using a standard electrospray ionization (ESI) source in positive ion scanning mode with a mass-to-charge ratio (m / z) of 100-1500, and in auto MS2 mode with a secondary mass spectrometer. Analysis was conducted on an Ultimate 3000U HPLC-DAD system (Thermo Fisher Scientific) equipped with an Acclaim RSLC 120C18 column (2.2 m, 2.1 x 100 mm, Thermo Scientific), at a flow rate of 0.3 mL / min, and with a UV wavelength of 200–600 nm. Mobile phase solvent A was 0.1% (v / v) formic acid aqueous solution. Mobile phase solvent B was 0.1% (v / v) formic acid acetonitrile. The HPLC elution program for butenyl spinosad is as follows: 0-5 min, 5%-55% B; 5-20 min, 55% B; 20-25 min, 55%-95% B; 25-30 min, 95% B; 30-35 min, 5% B. The HPLC elution program for abamectin is as follows: 0-5 min, 40% B; 5-6 min, 40%-70% B; 6-18 min, 70% B; 18-24 min, 70%-90% B; 24-28 min, 90% B; 28-31 min, 40% B.

[0086] In the examples, "%" refers to mass percentage.

[0087] Example 1: Decomposition and modification of busA in the butenyl spinosad modular polyketide synthase gene (PKS)

[0088] The inventors of this application selected the mPKS genes for the biosynthesis of butenyl spinosad as an example. The five PKS genes, busA-E, for the biosynthesis of butenyl spinosad range in length from 6.5kb to 16.8kb, and each PKS contains one to three modules. Genes busA, busB, and busC form the first operon, and genes busD and busE form the second operon. Since gene busA is closest to the operon promoter, and the number of modules in the busA protein is moderate for dissection, gene busA was selected as the gene to be dissected.

[0089] The gene busA has a molecular weight of 456 kDa and a length of 13.0 kb. Its GenBank accession number is AX600586. The expressed busA protein consists of three modules: LM, EM1, and EM2. Therefore, based on its module composition, busA can be split in three different ways: ① Two subunits (2+1 mode): the first two modules form the busA-1-1 subunit (8.1 kb), and the second module is the busA-1-2 subunit (4.9 kb); ② Two subunits (1+2 mode): the first module is the busA-2-1 subunit (2.9 kb), and the second two modules form the busA-2-2 subunit (10.1 kb); ③ Three subunits (1+1+1 mode): the first module is the busA-3-1 subunit (3.2 kb), the second module is the busA-3-2 subunit (5.5 kb), and the third module is the busA-3-2 (4.9 kb).

[0090] To ensure the correct order of the gene busA module orientation, the C-terminal docking domain in the salinomycin modular polyketide synthase gene is used. C DD) encoded sequence and N-end docking field ( N The DD coding sequence replaces the adapter sequence between adjacent modules of gene busA. Specifically, when using splitting strategies ① and ②, salinomycin PKS SlnA1 is used. C DD sequence and salinomycin PKS SlnA2 N The DD sequence replaces the connector sequences between busA-1-1 and busA-1-2, and between busA-2-1 and busA-2-2; when using splitting strategy ③, salinomycin PKS SlnA1 is used. C DD sequence and salinomycin PKS SlnA2 N The DD sequence replaces the connector sequence between busA-3-1 and busA-3-2, using salinomycin PKSSlnA7. C DD sequence and salinomycin PKSSlnA8 N The DD sequence replaces the connector sequence between busA-3-2 and busA-3-3.

[0091] Specific splitting and replacement, such as Figure 1 As shown, the specific methods for splitting and replacing are as follows:

[0092] (1) Using plasmid pBAC-sal as a template, with busA-1- C DDslnA1-F / R and busA-1- N Using DDslnA2-F / R as primers, PCR amplification was performed to obtain HAL- with overlapping sequences. C DDslnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 Fragments; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 The fragments are joined together to obtain HAL- C DD slnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 Fragment;

[0093] Using plasmid p15A-ccdB-amp as a template and busA-1-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene box-1, used to split mode ①, subsequently obtained busA-1-1 and busA-1-2;

[0094] (2) Using the HAL- obtained in step (1) C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 Using the fragment as a template, with busA-2-slnA1-C DD-F, busA-2-slnA2- N Using DD-R as primers, PCR amplification was performed to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 Fragment;

[0095] Using plasmid p15A-ccdB-amp as a template and busA-2-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene box-2, used to split mode ②, subsequently obtained busA-2-1 and busA-2-2;

[0096] (3) Using plasmid pBAC-sal as a template, and busA-3-slnA7- C DD-F, busA-3-slnA8- N Using DD-R as primers, PCR amplification was performed to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 Fragment;

[0097] Using plasmid p15A-ccdB-amp as a template and busA-3-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA7-TGA-RBS-ATG-N DDslnA8-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DD slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-3, used for resolving mode ③, followed by HAL- C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-2 yields busA-3-1, busA-3-2, and busA-3-3.

[0098] The primer busA-1- involved in this embodiment C DDslnA1-F / R、busA-1- N DDslnA2-F / R, busA-1-ampccdB-F / R, busA-1- N DDslnA2-F / R, busA-2-slnA1- C DD-F, busA-2-slnA2- N DD-R, busA-2-ampccdB-F / R, busA-3-slnA7- C DD-F, busA-3-slnA8- N The sequences DD-R and busA-3-ampccdB-F / R are shown in Table 1. Among them, the sequence information of busA-1-ampccdB-F / R and busA-3-ampccdB-F / R is the same.

[0099] Table 1

[0100]

[0101]

[0102] Example 2: Construction of Butenyl Spinosad Genetically Engineered Bacteria

[0103] 1. Plasmids pBAC-phiC31-bus and HAL- C DD-TGA-RBS-ATG- N DD-HAR 20-PacI-ampccdB-PacI-HAR cassette-1 was sequentially electroporated into *E. coli* GBred-gryA462, which had been induced to express Redαβ recombinase. Through linear-circular homologous recombination, HAL- C DD-TGA-RBS-ATG- N DD-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette salinomycin C DD sequences and N The DD sequence replaced the adapter sequence between busA-1-1 and busA-1-2; the transformed E. coli GBred-gryA462 was cultured and inoculated on LB solid medium supplemented with ampicillin (100 μg / mL). Positive colonies were screened, plasmids were extracted, and the target plasmid carrying the amp-ccdB gene selection expression cassette was obtained and sequenced to confirm that the amp-ccdB gene selection expression cassette was successfully cloned. The plasmid was named pBAC-phiC31-busA-1-ampccdB.

[0104] Using the same method, make HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-2 salinomycin C DD sequences and N The DD sequence replaced the linker sequence between busA-2-1 and busA-2-2, and the resulting plasmid was named pBAC-phiC31-busA-2-ampccdB.

[0105] Using the same method, make HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-2 and HAL- C DDslnA7-TGA-RBS-ATG- N DD slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-3 salinomycin C DD sequences and N The DD sequence replaced the linker sequence between busA-3-1, busA-3-2 and busA-3-3, and the resulting plasmid was named pBAC-phiC31-busA-3-ampccdB.

[0106] 2. The plasmids pBAC-phiC31-busA-1-ampccdB, pBAC-phiC31-busA-2-ampccdB, and pBAC-phiC31-busA-3-ampccdB were linearized using PacI enzyme digestion, excising the forward and reverse selection marker amp-ccdB gene and exposing the circular homologous arm (DR) to obtain linearized target plasmids. The linearized plasmids were then reacted in an exonuclease reaction system. The reaction mixture was then transferred to a Millipore membrane filter and dialyzed at room temperature for 30 minutes to remove salt. 20 μL of the dialyzed reaction mixture was then electroporated into *E. coli* GB2005 cells. After transformation, the cells were cultured, screened, and plasmids were extracted to obtain plasmids carrying… C DD-TGA-RBS-ATG- N The target plasmids for the DD coding sequence were named pBAC-phiC31-busA-1, pBAC-phiC31-busA-2, and pBAC-phiC31-busA-3, respectively.

[0107] The exonuclease reaction system consisted of 10 μL of 2×GibsonAssembly Master Mix, 5 pmol of the target plasmid to be reacted, and sterile deionized water to a final volume of 20 μL.

[0108] The exonuclease reaction conditions are: incubation at 50°C for 1 hour, followed by incubation at 4°C.

[0109] 3. pBAC-phiC31-busA-1, pBAC-phiC31-busA-2, and pBAC-phiC31-busA-3 were integrated into the phiC31 attB site on chromosome J1074 of Streptomyces albus, mediated by phiC31 integrase, to obtain butenyl spinosad genetically engineered bacteria busA-1, busA-2, and busA-3.

[0110] Example 3: Construction of a genetically engineered bacterium producing high levels of butenyl spinosad

[0111] Based on the butenyl spinosad genetically engineered bacteria constructed in Example 2, the strong constitutive promoter KasOp* from Streptomyces was inserted upstream of busA-1-2, busA-2-2, busA-3-2, busB, busC, busD, and busE, respectively, to construct high-yielding butenyl spinosad genetically engineered bacteria KbusA-1-2, KbusA-2-2, KbusA-3-2, KbusB, KbusC, KbusD, and KbusE.

[0112] The specific sequence of the strongly constitutive promoter KasOp* is as follows:

[0113] 5'-tgttcacattcgaaccgtctctgctttgacaacatgctgtgcggtgttgtaaagtcgtggcca-3'.

[0114] Specific insertion sites such as Figure 2 As shown in a, the specific construction method is as follows:

[0115] (1) Using plasmid p15A-ccdB-amp as a template and kasop-busB-1 and kasop-busB-2 as primers, PCR amplification was performed to obtain amp-kasOp-B gene cassette, which was used to construct the high-yield butenyl spinosad genetically engineered bacterium KbusB.

[0116] Using the amp-kasOp-B gene cassette as a template and kasop-busC-1 and kasop-busC-2 as primers, PCR amplification was performed to obtain the amp-kasOp-C gene cassette, which was used to construct the high-yield butenyl spinosad genetically engineered bacterium KbusC.

[0117] Using the amp-kasOp-B gene cassette as a template and kasop-busD-1 and kasop-busD-2 as primers, PCR amplification was performed to obtain the amp-kasOp-D gene cassette, which was used to construct the high-yield butenyl spinosad genetically engineered bacterium KbusD.

[0118] Using the amp-kasOp-B gene cassette as a template and kasop-busE-1 and kasop-busE-2 as primers, PCR amplification was performed to obtain the amp-kasOp-E gene cassette, which was used to construct the high-yield butenyl spinosad genetically engineered bacterium KbusE.

[0119] Using the amp-kasOp-B gene cassette as a template and busA-1 / 3-2-kasop-1 and busA-1 / 3-2-kasop-2 as primers, PCR amplification was performed to obtain the amp-kasOp-1-2 / 3-2 gene cassette, which was used to construct high-yield butenyl spinosad-producing genetically engineered bacteria KbusA-1-2 and KbusA-3-2.

[0120] Using the amp-kasOp-B gene cassette as a template and busA-2-2-kasop-1 and busA-2-2-kasop-2 as primers, PCR amplification was performed to obtain the amp-kasOp-2-2 gene cassette, which was used to construct the high-yield butenyl spinosad-producing genetically engineered bacterium KbusA-2-2.

[0121] The plasmid pBAC11-phiC31-busA and the amp-kasOp-B gene cassette were then electroporated into E. coli GB05-red that had been induced to express Redαβ recombinase. Through linear-circular homologous recombination, the strong constitutive promoter KasOp* sequence in the amp-kasOp-B gene cassette was inserted upstream of busB. The transformed E. coli GB05-red were cultured and inoculated on LB solid medium supplemented with ampicillin (100 μg / mL). Positive colonies were screened, and the plasmid was extracted to obtain the target plasmid carrying the amp-kasOp-B gene selection expression cassette. Sequencing confirmed that the amp-kasOp-B gene selection expression cassette was successfully cloned, and the plasmid was named pBAC-phiC31-KbusB.

[0122] Using the same method, the strong constitutive promoter KasOp* sequence in the amp-kasOp-C gene cassette was inserted upstream of busC to obtain the plasmid pBAC-phiC31-KbusC.

[0123] Using the same method, the strong constitutive promoter KasOp* sequence in the amp-kasOp-D gene cassette was inserted upstream of busD to obtain plasmid pBAC-phiC31-KbusD.

[0124] Using the same method, the strong constitutive promoter KasOp* sequence in the amp-kasOp-E gene cassette was inserted upstream of busE to obtain plasmid pBAC-phiC31-KbusE.

[0125] Using the same method, the strong constitutive promoter KasOp* sequence in the amp-kasOp-1-2 / 3-2 gene cassette was inserted upstream of bus-1-2 and downstream of bus-3-2, respectively, to obtain plasmids pBAC-phiC31-KbusA-1-2 and pBAC-phiC31-KbusA-3-2.

[0126] Using the same method, the strong constitutive promoter KasOp* sequence in the amp-kasOp-2-2 gene cassette was inserted upstream of bus-2-2 to obtain plasmid pBAC-phiC31-KbusA-2-2.

[0127] 3. pBAC-phiC31-KbusB* was integrated into the phiC31 attB site on chromosome J1074 of Streptomyces albus, mediated by phiC31 integrase, to obtain the butenyl spinosad genetically engineered strain KbusB.

[0128] Following the same method, plasmids pBAC-phiC31-KbusC, pBAC-phiC31-KbusD, pBAC-phiC31-KbusE, pBAC-phiC31-KbusA-1-2, pBAC-phiC31-KbusA-3-2, and pBAC-phiC31-KbusA-2-2 were integrated to obtain butenyl spinosad genetically engineered bacteria KbusA-1-2, KbusA-2-2, KbusA-3-2, KbusC, KbusD, and KbusE.

[0129] The sequences of primers kasop-busB-1, kasop-busB-2, kasop-busC-1, kasop-busC-2, kasop-busD-1, kasop-busD-2, kasop-busE-1, kasop-busE-2, busA-1 / 3-2-kasop-1, busA-1 / 3-2-kasop-2, busA-2-2-kasop-1, and busA-2-2-kasop-2 involved in this embodiment are shown in Table 2.

[0130] Table 2

[0131]

[0132] Example 4: Comparison of butenyl spinosad production from different strains

[0133] 1. The butenyl spinosad genetically engineered bacteria busA-1, busA-2, and busA-3 constructed in Example 2, and the high-yield butenyl spinosad genetically engineered bacteria KbusA-1-2, KbusA-2-2, KbusA-3-2, KbusB, KbusC, KbusD, and KbusE constructed in Example 3 were inoculated into 250mL Erlenmeyer flasks containing 30mL TSB liquid medium and cultured in a constant temperature shaker at 30℃ and 220rpm for 72h to obtain seed culture.

[0134] 1 mL of seed culture was transferred to a 25 mL Erlenmeyer flask containing 30 mL of fermentation medium and cultured for 7 days. Then, 600 μL (2%, v / v) of Amberlite XAD-16 adsorption resin was added, and the mixture was cultured for another 2 days to obtain a fermentation broth containing butenyl spinosad.

[0135] Meanwhile, using the white Streptomyces S. albus J1074 strain containing the wild-type busA gene cluster as the control strain busA, butenyl spinosad was prepared according to the above method.

[0136] The fermentation broth containing butenyl spinosad was centrifuged to collect the resin and bacterial cells. 40 mL of methanol was added, and the resin and precipitated bacterial cells were vortexed to resuspend them. The cells were then ultrasonically cleaned for 20 min to disrupt the cell structure. The suspension was transferred back to the original conical flask and shaken on a shaker at 30℃ and 220 rpm for 3 h. The methanol solution was filtered into a rotary evaporator and evaporated completely. The methanol was then redissolved in 1 mL of methanol. The yield of butenyl spinosad for each strain was compared by high-performance liquid chromatography (HPLC). The results are as follows: Figure 2 As shown in b to d.

[0137] The formula for TSB liquid culture medium is as follows: 30.0g of tryptic soybean peptone (TSB) culture medium solid powder is fully dissolved in 1L of distilled water, dispensed, and then autoclaved at 121℃ for 15min.

[0138] The fermentation medium was formulated as follows: 1% glycerol, 1.5% soybean peptone, 4% glucose, 3% soluble starch, 0.65% peptone, 1% beef extract, 0.1% magnesium sulfate, 0.05% yeast extract, 0.24% CaCO3, and 0.2% NaCl.

[0139] Depend on Figure 2 As shown in b to d, the butenyl spinosad-producing genetically engineered strains KbusA-1-2 and KbusA-2-2 produced higher levels of butenyl spinosad than the genetically engineered strains busA-1 and busA-2. Furthermore, the butenyl spinosad-producing genetically engineered strains KbusA-3-2, KbusB, KbusC, KbusD, and KbusE produced significantly higher levels of butenyl spinosad than the control strain busA. This clearly demonstrates that inserting the strong constitutive promoter KasOp* can effectively increase the yield of butenyl spinosad.

[0140] 2. High-resolution mass spectrometry analysis and yield comparison of butenyl spinosad produced by control strain busA and the butenyl spinosad-producing genetically engineered strains busA-1, busA-2, busA-3, KbusA-1-2, KbusA-2-2, KbusA-3-2, KbusB, KbusC, KbusD, and KbusE were performed on an Impact HD micro TFF-QIII mass spectrometer (Bruker Daltonics, Bremen, Germany). The results are as follows: Figures 3-4 As shown in Table 3.

[0141] Depend on Figure 3 It can be seen that the control strain busA, the butenyl spinosad genetically engineered strains busA-1, busA-2, busA-3, KbusA-1-2, KbusA-2-2, KbusA-3-2, KbusB, KbusC, KbusD and KbusE all successfully produced butenyl spinosad.

[0142] Table 3

[0143] strain busA busA-1 busA-2 kbusA-1-2 kbusA-2-2 busA-3 kbusA-3-2 kbusB kbusC kbusD kbusE relative output 0.75 6.01 5.91 13.13 13.02 13.16 16.89 30.71 27.39 21.94 18.22 relative output 1.33 5.14 6.32 11.76 11.13 12.51 20.36 24.09 33.27 22.36 21.43 relative output 0.93 4.59 5.54 15.54 9.29 14.21 18.99 22.84 28.42 28.02 25.90

[0144] Depend on Figure 2 , Figure 4 As shown in Table 3, the butenyl spinosad-producing genetically engineered strains busA-1, busA-2, and busA-3 all had significantly higher butenyl spinosad content than the control strain busA. Among them, busA-3 had the highest content, reaching 13.3 times that of the control strain busA. kbusC reached 30 times that of the control strain busA. Figure 4 The data in Table 3 show the relative yields of the genetically engineered bacteria and the control strain.

[0145] Example 5: Decomposition and modification of aveA2 in the avermectin modular polyketide synthase gene (PKS)

[0146] The inventors of this application continue to use the mPKS gene aveA in avermectin synthesis as an example. Avermectin biosynthesis includes four genes aveA1 to 4, and gene aveA2 was selected as the gene to be separated.

[0147] The aveA2 gene is 18.7 kb in length, with GenBank accession number AB032367.1. The expressed aveA2 protein consists of four modules: EM3, EM4, EM5, and EM6. Therefore, there is only one possible splitting method: combining the first two modules and then combining the last two. This splitting method corresponds to two strategies: ① using salinomycin PKS SlnA12. C DD sequences and N The DD sequence replaces the linker sequence between EM4 and EM5, dividing it into aveA2-1-1 and aveA2-1-2; ② Salinomycin PKS SlnA78 is used. C DD sequences and N The DD sequence replaces the connector sequence between EM4 and EM5, dividing it into aveA2-2-1 and aveA2-2-2.

[0148] Specific splitting and replacement, such as Figure 5 As shown, the specific methods for splitting and replacing are as follows:

[0149] (1) Using plasmid pBAC-sal as a template, and aveA2-1-sln12- C DD-F / R and aveA2-1-sln12- N Using DD-F / R as primers, PCR amplification was performed to obtain HAL- with overlapping sequences. C DD-slnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR20 Fragments; then, tandem PCR was used to ligate the two fragments together to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 Fragment;

[0150] Using plasmid p15A-ccdB-amp as a template and aveA2-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD-slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette was used to separate mode ①, which subsequently yielded aveA2-1-1 and aveA2-1-2;

[0151] (2) Using plasmid pBAC-sal as a template, and aveA2-1-sln78- C DD-F / R and aveA2-1-sln78- N Using DD-F / R as primers, PCR amplification was performed to obtain HAL- with overlapping sequences. C DD-slnA7-TGA-RBS fragment and ATG- N DDslnA8-HAR 20 Fragments; then, tandem PCR was used to ligate the two fragments together to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 Fragment;

[0152] Using plasmid p15A-ccdB-amp as a template and aveA2-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DD-slnA8-HAR 20 The -PacI-ampccdB-PacI-HAR gene cassette was used to split pattern ②, which subsequently yielded aveA2-2-1 and aveA2-2-2.

[0153] The primer involved in this embodiment is aveA2-1-sln12- C DD-F / R, aveA2-1-sln12- N DD-F / R, aveA2-ampccdB-F / R, aveA2-1-sln78- C DD-F / R, aveA2-1-sln78- N The sequences of DD-F / R are shown in Table 4.

[0154] Table 4

[0155]

[0156]

[0157] Example 6: Construction of a high-yield avermectin-producing genetically engineered bacterium

[0158] 1. Plasmid pBAC-phiC31-avm and HAL- C DDslnA1-TGA-RBS-ATG- N DD-slnA2-HAR 20 The PacI-ampccdB-PacI-HAR gene cassette was sequentially electroporated into *E. coli* GBred-gryA462 cells induced to express Redαβ recombinase. Through linear-circular homologous recombination, HAL- C DDslnA1-TGA-RBS-ATG- N DD-slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette salinomycin C DD sequences and NThe DD sequence replaced the adapter sequence between aveA2-1-1 and aveA2-1-2; the transformed E. coli GBred-gryA462 was cultured and inoculated on LB solid medium supplemented with ampicillin (100 μg / mL). Positive colonies were screened, plasmids were extracted, and the target plasmid carrying the amp-ccdB gene selection expression cassette was obtained and sequenced to confirm that the amp-ccdB gene selection expression cassette was successfully cloned. The plasmid was named pBAC-phiC31-avm-ampccdB-1.

[0159] Using the same method, make HAL- C DDslnA7-TGA-RBS-ATG- N DD-slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette salinomycin C DD sequences and N The DD sequence replaced the linker sequence between aveA2-2-1 and aveA2-2-2, and the resulting plasmid was named pBAC-phiC31-avm-ampccdB-2.

[0160] 2. PacI enzyme was used to linearize plasmids pBAC-phiC31-avm-ampccdB-1 and pBAC-phiC31-avm-ampccdB-2, respectively, removing the forward and reverse selection marker amp-ccdB gene and exposing the circular homologous arm (DR) to obtain linearized target plasmids. The linearized plasmids were then reacted in an exonuclease reaction system. The reaction mixture was then transferred to a Millipore membrane filter and dialyzed at room temperature for 30 minutes to remove salt. 20 μL of the dialyzed reaction mixture was then electroporated into *E. coli* GB2005 cells. After transformation, the cells were cultured, screened, and plasmids were extracted to obtain plasmids carrying… C DD-TGA-RBS-ATG- N The target plasmids for the DD coding sequence were named pBAC-phiC31-aveA2-1 and pBAC-phiC31-aveA2-2, respectively.

[0161] The exonuclease reaction system consisted of 10 μL of 2×GibsonAssembly Master Mix, 5 pmol of the target plasmid to be reacted, and sterile deionized water to a final volume of 20 μL.

[0162] The exonuclease reaction conditions are: incubation at 50°C for 1 hour, followed by incubation at 4°C.

[0163] The pBAC-phiC31-avm was obtained by cloning an 80kb avermectin gene cluster from the genome of *Streptomyces avermectin* DSM46492. The *Streptomyces avermectin* DSM46492 genome was digested with BglII to release a 73kb fragment, and digested with SnaBI+EcoRV to release a 15kb fragment. The 73kb aveR-aveA4 fragment was cloned into the pBeloBAC11 vector using the ExoCET method. The 15kb aveA4-aveB8 fragment was cloned into the pBBR322 vector using the ExoCET method. Recombinant engineering was used to insert the kanamycin resistance gene (kan), the homologous arm (HA) for splicing, and the EcoRV site. After SnaBI+EcoRV digestion, the 15kb aveA4-aveB8 fragment was inserted into pBAC-ave73kb using recombination engineering. Finally, the complete avermectin gene cluster was cloned into the pBeloBAC11 vector (pBAC-ave) to obtain the plasmid pBAC-phiC31-avm.

[0164] The specific method is as follows:

[0165] 1) Using plasmid pBAC-Sal as a template, and ave73-1 and ave73-2 as primers, the pBAC vector was amplified to clone the 73kb aveR-aveA4 fragment;

[0166] 2) Using pBR322-amp-tetR-tetO-hyg-ccdB plasmid as a template, amplify the pBR322 vector with ave15-1 and ave15-2 primers to clone the 15kb aveA4-aveB8 fragment;

[0167] 3) Using pR6K-TnpA-oriT-kan plasmid as a template, and kan-1 and kan-2 as primers, amplify the mycin resistance gene (kan), the homologous arm (HA) for splicing, and the EcoRV site to obtain plasmid pBAC-phiC31-avm.

[0168] 3. pBAC-phiC31-aveA2-1 and pBAC-phiC31-aveA2-2 were integrated into the phiC31 attB site on the chromosome of Streptomyces coelicolor CH999 via site-specific recombination mediated by phiC31 integrase, respectively, to obtain the high-avermectin-producing genetically engineered strains aveA2-2-1 and aveA2-2-2.

[0169] The sequences of primers ave73-1, ave73-2, ave15-1, ave15-2, kan-1, and kan-2 involved in this embodiment are shown in Table 5.

[0170] Table 5

[0171]

[0172] Example 7: Comparison of abamectin production among different strains

[0173] 1. The high-yield avermectin-producing genetically engineered bacteria aveA2-2-1 and aveA2-2-2 constructed in Example 6 were inoculated into 250 mL Erlenmeyer flasks containing 30 mL of seed culture medium A, and cultured in a constant temperature shaker at 30 °C and 220 rpm for 24 h to obtain seed liquid A.

[0174] 1 mL of seed culture A was transferred to a 250 mL Erlenmeyer flask containing 30 mL of seed culture medium B, and cultured in a constant temperature shaker at 30 °C and 220 rpm for 72 h to obtain seed culture B.

[0175] 1 mL of seed culture B was transferred to a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium and cultured for 6 days. Then, 600 μL (2%, v / v) of Amberlite XAD-16 adsorption resin was added and cultured for another 2 days to obtain a fermentation broth containing abamectin.

[0176] Centrifuge the fermentation broth containing abamectin to collect the resin and bacterial cells. Add 40 mL of methanol and vortex to resuspend the resin and precipitated bacterial cells. Use an ultrasonic cleaner to sonicate for 20 min to break the cells. Transfer the suspension back to the original conical flask and shake it in a constant temperature shaker at 30℃ and 220 rpm for 2-3 h. Filter the methanol solution into a rotary evaporator and evaporate it completely. Redissolve the methanol in 1 mL of methanol.

[0177] The seed culture medium A has the following formula: 1.5% soybean flour, 2.5% glucose, and 0.3% calcium carbonate.

[0178] The formula for seed culture medium B is: 1.5% soybean flour, 4% glucose, 1.5% skim milk powder, and 0.5% yeast extract.

[0179] The fermentation medium formula is: 3% glucose, 0.2% (NH4)2SO4, 0.5% CaCO3, 0.3% NaCl, 0.005% FeSO4, 0.005% MnSO4, and 0.01% MgSO4.

[0180] 2. Simultaneously, using the sky-blue Streptomyces S. coelicolorCH999 strain containing the wild-type aveA2 gene cluster as the control strain aveA2, avermectin was prepared according to the above method.

[0181] High-resolution mass spectrometry analysis and yield comparison of avermectin produced by control strain aveA2, high-avermectin-producing genetically engineered strains aveA2-2-1 and aveA2-2-2 were performed on an Impact HD micro TFF-QIII mass spectrometer (Bruker Daltonics, Bremen, Germany). The results are as follows: Figures 6-7 As shown in Table 6.

[0182] Depend on Figure 6 It can be seen that the control strain aveA2, the high-abamectin-producing genetically engineered strains aveA2-2-1 and aveA2-2-2 all successfully produced avemectin.

[0183] Table 6

[0184] strain aveA2 aveA2-1 aveA2-2 relative output 1.01 5.17 4.98 relative output 0.96 5.55 4.45 relative output 1.04 5.13 4.92

[0185] Depend on Figure 7 As shown in Table 6, the avermectin-producing genetically engineered strains aveA2-2-1 and aveA2-2-2 both produced significantly higher levels of avermectin than the control strain busA. Among them, aveA2-1 produced the highest level, reaching 5.8 times that of the control strain aveA2. Figure 7 The data in Table 6 show the relative yields of the genetically engineered bacteria and the control strain.

[0186] Example 8: Mechanism verification of the method to increase polyketide compound yield by splitting modular polyketide synthase genes.

[0187] The above examples demonstrate that dissecting the modular polyketide synthase (mPKSs) genes of butenyl spinosad and avermectin and then recombining and expressing them heterologously can effectively improve the biosynthesis of butenyl spinosad and avermectin. The inventors of this application have further elucidated the potential molecular mechanism and inferred that the method can be used to increase the yield of other polyketide compounds.

[0188] 1. Verification that the truncated mRNA constitutes the majority of busA PKS mRNA.

[0189] The gusA gene (β-glucuronidase) was used as a reporter gene. The mRNA and protein concentrations of the gusA gene were precisely quantified, and the transcriptional expression levels of the wild-type busA gene and the split-type busA gene in S. albus J1074 were measured. The GenBank accession number of the gusA gene is NC_000913.3.

[0190] The strategy was as follows: following the splitting methods ① and ② in Example 1, the gusA gene and adapter coding sequence were inserted downstream of wild-type busA, split-type busA-1-1, busA-1-2, busA-2-1, and busA-2-2, respectively. Then, the genetically engineered bacteria were constructed and verified according to the method described in Example 2, and the expression level of the gusA gene and the expression level of β-glucuronidase were detected. The amino acid sequence of the adapter coding sequence was: GGSGGGGGG.

[0191] The specific method is as follows:

[0192] (1) Using the artificially synthesized gusA gene as a template and busA-1-1-gusA-F / R as primers, PCR amplification was performed to obtain the gusA-1-1 gene sequence;

[0193] Using plasmid p15A-ccdB-amp as a template and busA-1-1-gusA-amp-F / R as primers, PCR amplification was performed to obtain the amp resistance gene;

[0194] Then, tandem PCR was used to link the gusA-1-1 gene, the adapter coding sequence, and the amp resistance gene together to obtain the busA-1-1-gusA-amp gene cassette, which was used to obtain busA-1-1-gusA;

[0195] (2) Using the busA-1-1-gusA-amp gene cassette as a template and busA-2-1-gusA-F / R as primers, PCR amplification was performed to obtain the gusA-2-1 gene sequence;

[0196] Using plasmid p15A-ccdB-amp as a template and busA-2-1-gusA-amp-F / R as primers, PCR amplification was performed to obtain the amp resistance gene;

[0197] Then, tandem PCR was used to link the gusA-2-1 gene, the adapter coding sequence, and the amp resistance gene together to obtain the busA-2-1-gusA-amp gene cassette, which was used to obtain busA-2-1-gusA;

[0198] (3) Using the busA-1-1-gusA-amp gene cassette as a template and busA-(1 / 2-2)-gusA-F / R as primers, PCR amplification was performed to obtain the gusA-(1 / 2-2) gene sequence;

[0199] Using plasmid p15A-ccdB-amp as a template and busA-(1 / 2-2)-gusA-amp-F / R as primers, PCR amplification was performed to obtain the amp resistance gene;

[0200] Then, tandem PCR was used to link the gusA-(1 / 2-2) gene, the adapter coding sequence, and the amp resistance gene together to obtain the busA-(1 / 2-2)-gusA-amp gene cassette, which was used to obtain busA-gusA, busA-1-2-gusA, and busA-2-1-gusA.

[0201] (4) The plasmid pBAC-phiC31-bus and the busA-1-1-gusA-amp gene cassette were electroporated into Escherichia coli GB05-red that had been induced to express Redαβ recombinase. The gusA gene in the busA-1-1-gusA-amp gene cassette was inserted downstream of busA-1-1 by linear-circular homologous recombination. The transformed Escherichia coli GB05-red was cultured and inoculated on LB solid medium supplemented with ampicillin (100 μg / mL). Positive colonies were screened, and the plasmid was extracted to obtain the target plasmid pBAC-phiC31-busA-1-1-gusA carrying the gusA gene coding sequence.

[0202] (5) The target plasmid pBAC-phiC31-busA-1-1-gusA carrying the gusA gene coding sequence was integrated into the phiC31 attB site on the chromosome of Streptomyces albus J1074 by site-specific recombination mediated by phiC31 integrase, and the genetically engineered strain busA-1-1 was obtained.

[0203] (6) Following the method described in steps (4) to (5), the genetically engineered bacteria busA, busA-1-2, busA-2-1, and busA-2-2 were constructed using the busA-2-1-gusA-amp gene cassette and the busA-(1 / 2-2)-gusA-amp gene cassette.

[0204] The sequences of the primers busA-1-1-gusA-F / R, busA-1-1-gusA-amp-F / R, busA-2-1-gusA-F / R, busA-2-1-gusA-amp-F / R, busA-(1 / 2-2)-gusA-F / R, and busA-(1 / 2-2)-gusA-amp-F / R involved in this embodiment are shown in Table 7.

[0205] Table 7

[0206]

[0207]

[0208] Next, the obtained verification genetically engineered bacteria busA, busA-1-1, busA-1-2, busA-2-1, and busA-2-2 were cultured according to the method described in Example 4, and the bacterial culture was collected. The transcriptional expression level of the gusA gene in the bacterial culture was analyzed by real-time quantitative PCR (qRT-PCR), and the results are as follows: Figure 8 As shown.

[0209] Depend on Figure 8 It was found that the transcriptional levels of the gusA gene in the genetically engineered bacteria busA-gusA, busA-1-2-gusA, and busA-2-2-gusA were identical. This finding indicates that BusA splitting does not affect the transcription of the busA-busC operon, and the same distance from the start to the end of the operon results in the same mRNA abundance. Overall, the 5.0 kb busA-2-1-gusA showed the highest transcriptional level, while the 14.8 kb busA-gusA showed the lowest. More specifically, the transcriptional levels of the 5.0 kb busA-2-1-gusA and 10.2 kb busA-1-1-gusA were 14.8 and 6.5 times higher than those of busA-gusA, respectively. Since all fusion genes are located in the busA-busC operon and are controlled by the SA15p promoter, the above transcriptional analysis shows that a considerable number of truncated PKS mRNAs were generated in S. albus J1074. For the 14.8 kb busA-gusA gene ORF, the intact mRNA accounts for only 6.8% (1 / 14.8) of the total transcribed mRNA. This observation is consistent with the view that, due to mRNA truncation, mRNA sequences closer to the operon start position are more abundant than those farther from the start position.

[0210] 2. mRNA truncation produces more PKS protein.

[0211] It has been demonstrated that truncated mRNA constitutes the majority of mPKS mRNA, and due to mRNA truncation, the mRNA sequence near the operon start is more abundant than the mRNA sequence far from the start. Next, the inventors of this application tested the β-glucuronidase activity in the bacterial cultures of the verification genetically engineered bacteria busA, busA-1-2, busA-2-1, and busA-2-2 from step 1 to determine the abundance of gusA protein. The results are as follows... Figure 9 As shown.

[0212] The specific detection method was as follows: The genetically engineered bacteria busA, busA-1-2, busA-2-1, and busA-2-2 were inoculated into 250 mL Erlenmeyer flasks containing 30 mL of TSB liquid medium, respectively, and incubated at 30 °C and 220 rpm for 72 h. The optical density (OD6000) of the culture at 600 nm was measured, and the culture was diluted to an OD of 1.0. 2 mL of the fermentation broth was centrifuged at 12000 rpm for 1 min, and the supernatant was discarded. 900 μL of gusA Buffer 2 was added to resuspend the cells. Cell lysis was performed at 37 °C for 20 min, and 900 μL of dilution buffer (50 mM phosphate buffer, pH 7.0, 0.1% Triton X-100, 5 mM DTT solution) was added to the lysis buffer. The diluted lysis buffer was centrifuged at 16400 × g for 10 min at 4 °C. Then, 100 μL of supernatant was added to a 96-well plate and mixed with 100 μL of dilution buffer containing 2 mM p-nitrophenyl-β-d-glucuronide. Glucuronase activity was measured at 415 nm for 90 minutes using an automated microplate reader. A unit of glucuronase activity refers to the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol per minute. Glucuronase activity is calculated per gram of dry biomass. 100 μL of lysis buffer was mixed with 100 μL of dilution buffer as a control.

[0213] The formulation of gusA Buffer 2 is as follows: 50 mM phosphate buffer (pH = 7.0), 0.1% Triton X-100, 5 mM MTT solution, 1 mg / mL lysozyme.

[0214] Depend on Figure 9 It was found that the expression levels of the genetically engineered bacteria BusA-GusA, BusA-1-2-GusA, and BusA-2-2-GusA were identical, consistent with their mRNA abundance; the same mRNA concentration led to the same protein yield. Since the transcription levels of these three genes were identical, inserting RBSs upstream of the busA1-2-gusA and busA2-2-gusA genes did not increase their yield. Meanwhile, the β-glucuronidase activity of BusA-2-1-GusA was 3.9 times higher than that of BusA-GusA. The β-glucuronidase activity of BusA-1-1-GusA was 2.5 times that of BusA-GusA, consistent with its mRNA abundance; higher mRNA concentration resulted in higher protein yield. Therefore, the abundance of PKS protein in S. albus J1074 mainly depends on its mRNA concentration. The β-glucuronidase activity assay of the above GusA fusion proteins showed that the N-terminus of the BusA polyketide synthase protein was more abundant than the C-terminus.

[0215] 3. Detection of increased PKS protein synthesis rate due to mRNA truncation

[0216] The synthesis rate of gusA protein in genetically engineered bacteria BusA-1-1, BusA-2-1, and BusA was measured.

[0217] The specific method is as follows:

[0218] (1) Using plasmid pBAC-spnNE-cum-spnS as a template and cum-F / R as primers, PCR amplification was performed to obtain the p-isopropylbenzoic acid (cumate) gene cassette.

[0219] cum-F:

[0220] 5'-TCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACTCACCGCTTGAACTTGGCGTA-3',

[0221] cum-R:

[0222] 5'-AGGCGGCAGGACAGTCCGATGACGGCTATCAGGTTCCCGGCTTCGCTCATGTCCGTACCTCCGTTGCTCG-3';

[0223] (2) The plasmid pBAC-phiC31-busA-1-1-gusA and the cumate gene cassette were electroporated into Escherichia coli GB05-red that had been induced to express Redαβ recombinase. The cumate gene cassette was inserted upstream of busA-1-1-gusA through linear-circular homologous recombination. The transformed Escherichia coli GB05-red was cultured and inoculated on LB solid medium supplemented with ampicillin (100 μg / mL). Positive colonies were screened, and the plasmid was extracted to obtain the target plasmid pBAC-phiC31-busA-1-1-gusA-cumate carrying the cumate gene cassette coding sequence.

[0224] The positions of the busA-gusA, busA-1-1-gusA, busA-2-1-gusA, and p-isopropylbenzoic acid (cumate) inducible promoters are as follows: Figure 10 As shown in a;

[0225] (3) Then, obtain the target plasmids pBAC-phiC31-busA-1-1-gusA-cumate, pBAC-phiC31-busA-2-1-gusA-cumate, and pBAC-phiC31-busA-gusA-cumate using the above method;

[0226] (4) The target plasmids pBAC-phiC31-busA-1-1-gusA-cumate, pBAC-phiC31-busA-2-1-gusA-cumate, and pBAC-phiC31-busA-gusA-cumate were integrated into the phiC31 attB site on the chromosome of Streptomyces albus J1074 to obtain the genetically engineered bacteria BusA-1-1, BusA-2-1, and BusA.

[0227] The genetically engineered bacteria BusA-1-1, BusA-2-1, and BusA were cultured to the exponential growth phase, and p-isopropylbenzoic acid was added to induce gusA protein synthesis. 1 ml of the induced culture was collected after induction for 10, 20, 30, 40, 60, 80, 100, 120, 140, and 160 minutes. A protein termination solution of 3 μg / mL chloramphenicol and 1.4 mM erythromycin was added to the collected culture to stop protein synthesis. β-glucuronidase activity was then measured, and the results are as follows: Figure 10 As shown in b.

[0228] Depend on Figure 10 b shows that β-glucuronidase activity of BusA-2-1 and BusA-1-1 was detected after 20 min of induction; however, β-glucuronidase activity of BusA was only detected after 60 min of induction. This result is consistent with the length of these fusion proteins. After 40–80 min of induction, the synthesis rate of GusA protein from BusA-2-1 (7.0 × 10⁻⁶) was [not specified in the original text]. -3 units g -1 min -1 The rate of GusA protein synthesis of BusA-1-1 is 3.1 × 10⁻⁶. -3 units g -1 min -1 It is 2.3 times faster. After induction for 80-160 min, the protein synthesis rate of GusA in BusA-2-1 is 13.0 × 10⁻⁶. -3 units g -1 min -1 The rate of GusA protein synthesis of BusA-1-1 (4.7 × 10⁻⁶) and BusA-1-1 (4.7 × 10⁻⁶) -3 units g -1 min -1 ) are BusA(2.1×10 -3 units g -1 min -1The concentrations of GusA protein in BusA, BusA-1-1, and BusA-2-1 were 6.2-fold and 2.2-fold higher, respectively. The differences in GusA protein concentration and synthesis rate among BusA, BusA-1-1, and BusA-2-1 were consistent with their differences in mRNA abundance. Within 160 min, the GusA protein concentration in BusA-2-1 was higher than that in BusA-1-1, and higher than that in BusA.

[0229] In summary, dissecting large PKS genes by inserting coding sequences between modular coding sequences can rescue the translation of truncated mRNA and allow for the generation of functional PKS subunits. Since the truncated mRNA constitutes the majority of the transcribed mRNA, this method generates numerous functional PKS subunits encoded by sequences near the 5' end of the mRNA. Dissecting the PKS gene does not affect operon transcription; therefore, the concentration of C-terminal subunits generated by the dissected PKS gene is the same as the protein concentration generated by the native, undissected full-length PKS gene. The PKS subunits generated by the dissected gene form a functional PKS complex through protein interactions mediated by the terminal docking domain. PKS gene dissecting leads to a significant increase in the concentration of N-terminal PKS subunits; therefore, the biosynthetic efficiency of the giant modular PKS polyketide synthase assembly line is significantly improved.

[0230] The method for increasing polyketide compound yield by disassembling modular polyketide synthase genes provided in this invention enriches the toolbox of PKS pathway engineering. This method can be combined with known engineering strategies to improve the biosynthetic efficiency of polyketide natural products. Furthermore, this method can be applied to other PKSs and other large proteins composed of multiple modules or domains to enhance their function.

Claims

1. A method for increasing the yield of polyketide compounds by disassembling modular polyketide synthase genes, characterized in that, The steps include the following: (1) Design several pairs of amplification primers according to the target modular polyketide synthase gene, and generate primers containing the target modular polyketide synthase gene for splitting by tandem PCR. C DD-TGA-RBS-ATG- N RedEx gene cassettes of DD coding sequences; (2) The plasmid containing the target modular polyketide synthase gene cluster and the RedEx gene cassette obtained in step (1) were sequentially transformed into Escherichia coli GBred-gryA462 that had been induced to express Redαβ recombinase. Through linear-circular homologous recombination, the C-terminal docking domain coding sequence and the N-terminal docking domain coding sequence in the RedEx gene cassette replaced the adapter sequence in the coding sequence of the target modular polyketide synthase module. After transformation, the cells were cultured, screened, and the plasmid was extracted to obtain the target plasmid carrying the amp-ccdB gene selection expression cassette. (3) The target plasmid carrying the amp-ccdB gene selection cassette obtained in step (2) was linearized and digested with enzymes. Then, the linearized plasmid was reacted in an exonuclease reaction system. After the reaction was completed, it was electroporated into Escherichia coli GB2005. After transformation, the plasmid was cultured, screened, and extracted to obtain the target plasmid carrying the amp-ccdB gene selection cassette. C DD-TGA-RBS-ATG- N The target plasmid for the DD coding sequence; (4) The site-specific recombination mediated by phiC31 integrase is used to transfer the contents of step (3) to the site. C DD-TGA-RBS-ATG- N The target plasmid encoding the DD sequence was integrated into the phiC31 attB site on the host strain chromosome to obtain a recombinant strain; (5) The recombinant strain obtained in fermentation culture step (4) is centrifuged to collect resin and bacterial cells, and after separation and purification, polyketide compound is obtained.

2. The method as described in claim 1, characterized in that, In step (1), the modular polyketide synthase gene is the modular polyketide synthase gene busA-E of butenyl spinosad or the modular polyketide synthase gene aveA1-4 of avermectin. More preferably, the modular polyketide synthase gene is the modular polyketide synthase gene busA of butenyl spinosad or the modular polyketide synthase gene aveA2 of avermectin. Among them, busA is 13.0kb in length and has GenBank accession number AX600586; aveA2 is 18.7kb in length and has NCBI accession number and GenBank accession number AB032367.

1.

3. The method as described in claim 1, characterized in that, In step (1), the C DD-TGA-RBS-ATG- N DD encoded sequence is C DD, TGA, RBS, ATG and N DD are connected sequentially to form the following; C DD is the C-terminal docking domain coding sequence, TGA is the stop codon, RBS is the ribosome binding site, and ATG is the start codon. N DD is the N-end docking field encoding sequence; Further preferably, the C DD is the coding sequence for the C-terminal docking domain of the slnA1 or slnA7 modular polyketide synthase gene salinomycin. N DD is the N-terminal docking domain coding sequence of the slnA2 or slnA8 modular polyketide synthase gene sln.

4. The method as described in claim 1, characterized in that, In step (2), the plasmid containing the target modular polyketide synthase gene cluster is either pBAC-phiC31-bus or pBAC-phiC31-avm. In step (3), the target plasmid carrying the amp-ccdB gene selection expression cassette is linearized and digested using PacI enzyme; The exonuclease reaction system consists of: 10 μL of 2×GibsonAssembly Master Mix, 5 pmol of the target plasmid to be reacted, and sterile deionized water to a final volume of 20 μL. In step (4), the host strain is either *Streptomyces albus* J1074 or *Streptomyces coelicolor* CH999.

5. The method as described in claim 1, characterized in that, The specific steps are as follows: (1) Using plasmid pBAC-sal as a template, with busA-1- C DDslnA1-F / R and busA-1- N Using DDslnA2-F / R as primers, PCR amplification was performed to obtain HAL- with overlapping sequences. C DDslnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 Fragments; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 The fragments are joined together to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 Fragment; Using plasmid p15A-ccdB-amp as a template and busA-1-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-1; (2) Using the HAL- obtained in step (1) C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 Using the fragment as a template, with busA-2-slnA1- C DD-F, busA-2-slnA2- N Using DD-R as primers, PCR amplification was performed to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 Fragment; Using plasmid p15A-ccdB-amp as a template and busA-2-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-2; (3) Using plasmid pBAC-sal as a template, and busA-3-slnA7- C DD-F, busA-3-slnA8- N Using DD-R as primers, PCR amplification was performed to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 Fragment; Using plasmid p15A-ccdB-amp as a template and busA-3-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DD slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-3; (4) Plasmid pBAC-phiC31-bus and HAL- C DD-TGA-RBS-ATG- N DD-HAR 20 -PacI-ampccdB-PacI-HAR cassette-1 was sequentially electroporated into *E. coli* GBred-gryA462, which had been induced to express Redαβ recombinase. Through linear-circular homologous recombination, HAL- C DD-TGA-RBS-ATG- N DD-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette salinomycin C DD sequences and N The DD sequence replaced the adapter sequence between busA-1-1 and busA-1-2; the transformed E. coli GBred-gryA462 was cultured and positive colonies were screened out. Plasmids were extracted to obtain plasmid pBAC-phiC31-busA-1-ampccdB. (5) Following the method described in step (4), make HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-2 salinomycin C DD sequences and N The DD sequence was used to replace the linker sequence between busA-2-1 and busA-2-2 to obtain plasmid pBAC-phiC31-busA-2-ampccdB. Following the method described in step (4), HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-3 and HAL- C DDslnA7-TGA-RBS-ATG- N DD slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette-2 salinomycin C DD sequences and N The DD sequence was used to replace the linker sequence between busA-3-1, busA-3-2 and busA-3-3 to obtain plasmid pBAC-phiC31-busA-3-ampccdB. (6) The plasmids pBAC-phiC31-busA-1-ampccdB, pBAC-phiC31-busA-2-ampccdB, and pBAC-phiC31-busA-3-ampccdB were linearized using PacI enzyme to obtain linearized target plasmids. The linearized plasmids were then reacted in an exonuclease reaction system, and the reaction mixture was dialyzed at room temperature for 30 minutes to remove salts. The dialyzed reaction mixture was then electroporated into E. coli GB2005 cells. After transformation, the cells were cultured, screened, and plasmids were extracted to obtain the target plasmids carrying the target plasmids. C DD-TGA-RBS-ATG- N The target plasmids for the DD coding sequences are pBAC-phiC31-busA-1, pBAC-phiC31-busA-2, and pBAC-phiC31-busA-3. The exonuclease reaction system consists of 10 μL of 2×GibsonAssembly Master Mix, 5 pmol of the target plasmid to be reacted, and sterile deionized water to a final volume of 20 μL. The exonuclease reaction conditions are: incubation at 50°C for 1 hour, followed by incubation at 4°C. (7) pBAC-phiC31-busA-1, pBAC-phiC31-busA-2 and pBAC-phiC31-busA-3 were integrated into the phiC31attB site on chromosome J1074 of Streptomyces albus, respectively, through site-specific recombination mediated by phiC31 integrase, to obtain butenyl spinosad genetically engineered bacteria busA-1, busA-2 and busA-3; (8) Fermentation culture step (7) The butenyl spinosad genetically engineered bacteria busA-1, busA-2 and busA-3 obtained by fermentation culture step (7) were centrifuged to collect resin and bacterial cells, and after separation and purification, butenyl spinosad was obtained.

6. A genetically engineered bacterium for producing butenyl spinosad, characterized in that, The sample is constructed according to steps (1) to (7) of claim 5.

7. The method as described in claim 1, characterized in that, The specific steps are as follows: 1) Construct butenyl spinosad genetically engineered bacteria busA-1, busA-2 and busA-3 according to steps (1) to (7) of claim 5; 2) The strong constitutive promoter KasOp* was inserted upstream of busA-1-2, busA-2-2, busA-3-2, busB, busC, busD, and busE, respectively, into the genetically engineered bacteria busA-1, busA-2, and busA-3 that produce butenyl spinosad, to obtain genetically engineered bacteria KbusA-1-2, KbusA-2-2, KbusA-3-2, KbusB, KbusC, KbusD, and KbusE that produce high levels of butenyl spinosad; 3) The high-yield butenyl spinosad genetically engineered bacteria KbusA-1-2, KbusA-2-2, KbusA-3-2, KbusB, KbusC, KbusD, and KbusE obtained in step 2) were centrifuged to collect the resin and bacterial cells. After separation and purification, butenyl spinosad was obtained.

8. A genetically engineered bacterium that produces high levels of avermectin, constructed according to steps 1) to 2) of claim 7.

9. The method as described in claim 1, characterized in that, The specific steps are as follows: a. Using plasmid pBAC-sal as a template, and aveA2-1-sln12- C DD-F / R and aveA2-1-sln12- N Using DD-F / R as primers, PCR amplification was performed to obtain HAL- with overlapping sequences. C DD-slnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 Fragments; then HAL- was separated using tandem PCR. C DD-slnA1-TGA-RBS fragments and ATG- N DDslnA2-HAR 20 The fragments are joined together to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD slnA2-HAR 20 Fragment; Using plasmid p15A-ccdB-amp as a template and aveA2-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA1-TGA-RBS-ATG- N DDslnA2-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA1-TGA-RBS-ATG- N DD-slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette; b. Using plasmid pBAC-sal as a template, and aveA2-1-sln78- C DD-F / R and aveA2-1-sln78- N Using DD-F / R as primers, PCR amplification was performed to obtain HAL- with overlapping sequences. C DD-slnA7-TGA-RBS fragment and ATG- N DDslnA8-HAR 20 Fragments; then HAL- was separated using tandem PCR. C DD-slnA7-TGA-RBS fragment and ATG- N DDslnA8-HAR 20 The fragments are joined together to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 Fragment; Using plasmid p15A-ccdB-amp as a template and aveA2-ampccdB-F / R as primers, PCR amplification was performed to obtain plasmids similar to HAL-. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The PacI-ampccdB-PacI-HAR fragments overlapped; then HAL- was separated using tandem PCR. C DDslnA7-TGA-RBS-ATG- N DDslnA8-HAR 20 The fragment is concatenated with the PacI-ampccdB-PacI-HAR fragment to obtain HAL- C DDslnA7-TGA-RBS-ATG- N DD-slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette; c. Plasmid pBAC-phiC31-avm and HAL- C DDslnA1-TGA-RBS-ATG- N DD-slnA2-HAR 20 The PacI-ampccdB-PacI-HAR gene cassette was sequentially electroporated into *E. coli* GBred-gryA462 cells induced to express Redαβ recombinase. Through linear-circular homologous recombination, HAL- C DDslnA1-TGA-RBS-ATG- N DD-slnA2-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette salinomycin C DD sequences and N The DD sequence replaced the adapter sequence between aveA2-1-1 and aveA2-1-2; the transformed E. coli GBred-gryA462 was cultured and positive colonies were screened out. The plasmid was extracted to obtain plasmid pBAC-phiC31-avm-ampccdB-1. d. Following the method described in step b, make HAL- C DDslnA7-TGA-RBS-ATG- N DD-slnA8-HAR 20 -PacI-ampccdB-PacI-HAR gene cassette salinomycin C DD sequences and N The DD sequence was used to replace the linker sequence between aveA2-2-1 and aveA2-2-2 to obtain plasmid pBAC-phiC31-avm-ampccdB-2. e. Using PacI enzyme, plasmids pBAC-phiC31-avm-ampccdB-1 and pBAC-phiC31-avm-ampccdB-2 were linearized to obtain linearized target plasmids. The linearized plasmids were then reacted in an exonuclease reaction system, and the reaction mixture was dialyzed at room temperature for 30 minutes to remove salts. The dialyzed reaction mixture was then electroporated into *E. coli* GB2005 cells. After transformation, the cells were cultured, screened, and plasmids were extracted to obtain plasmids carrying… C DD-TGA-RBS-ATG- N The target plasmids for the DD coding sequence were named pBAC-phiC31-aveA2-1 and pBAC-phiC31-aveA2-2, respectively. The exonuclease reaction system consists of 10 μL of 2×GibsonAssembly Master Mix, 5 pmol of the target plasmid to be reacted, and sterile deionized water to a final volume of 20 μL. The exonuclease reaction conditions are: incubation at 50°C for 1 hour, followed by incubation at 4°C. f. By integrating pBAC-phiC31-aveA2-1 and pBAC-phiC31-aveA2-2 into the phiC31 attB site of chromosome S. coelicolorCH999 through site-specific recombination mediated by phiC31 integrase, the genetically engineered bacteria aveA2-2-1 and aveA2-2-2 with high abamectin production were obtained. g. The high-abamectin-producing genetically engineered bacteria aveA2-2-1 and aveA2-2-2 obtained from fermentation culture step f are centrifuged to collect resin and bacterial cells, and after separation and purification, avermectin is obtained.

10. A genetically engineered bacterium that produces high levels of avermectin, constructed according to steps a to f of claim 9.

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