CN111909946A - Transposable plasmid for saccharopolyspora and application thereof - Google Patents

Abstract

The invention discloses a transposable plasmid for saccharopolyspora and application thereof; the transposition plasmid is Tn5 transposition plasmid-pJTn 1-pJTn 6, and the transposition plasmids can perform high-efficiency transposition in saccharopolyspora erythraea, wherein pJTn1 and pJTn5 successfully perform in vivo transposition in the saccharopolyspora erythraea to obtain 31 transposition mutants with different insertion sites. The fermentation of the transposition mutator and the detection of the yield of the spinosad in the fermentation liquid find that the insertion of the transposition has obvious influence on the yield of the spinosad. The transposition system has important significance for the biosynthesis regulation of spinosad in saccharopolyspora spinosa.

Description

Transposable plasmid for saccharopolyspora and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a transposable plasmid for saccharopolyspora and application thereof.

Background

Spinosad, which is mainly composed of two organic compounds, spinosad a and spinosad D, with a complex structure, is a natural product-like biopesticide, is effective against a variety of insects, and is commonly used to control a variety of pests, including thrips, liriomyza, spider mites, mosquitoes, ants, fruit flies, and the like. Since the registration of spinosyns for use as pesticides by the united states Environmental Protection Agency (EPA) in 1997, spinosyns have been used in agricultural production for controlling pests in many countries.

Spinosyns are produced by fermentation of the actinomycete Saccharopolyspora spinosa (Saccharopolyspora spinosa) isolated from soil. The fermentation titer of the spinosad of the wild type saccharopolyspora spinosa is very low, and the fermentation titer of the spinosad can be effectively improved by the traditional mutation breeding, genetic engineering breeding, culture medium optimization and other methods, so that the saccharopolyspora spinosa is suitable for industrial scale production of the spinosad. The conventional high-yield strain breeding method in factories is multi-round random mutagenesis screening, a wild strain is treated by a physical, chemical or biological mutagen to generate a plurality of random mutant strains, the strains with improved yield are screened from a large number of mutant strains, and then the next round of mutagenesis-screening circulation is carried out until the high-yield strain meeting the requirements is obtained. Genetic engineering is a new technology, and the biosynthesis amount of a target compound can be effectively improved, namely the fermentation titer is improved, by modifying a pathway specific regulatory gene, a pleiotropic regulatory gene, a resistance gene and a transporter gene for secondary metabolite synthesis, eliminating a substrate competitive branch pathway and the like. However, genetically engineered modified species must be based on the identification of effective candidate genes of interest. Transposition mutagenesis is a valuable technique in biological research, and can create a large number of random mutant strains; secondly, the mutation site generated by transposition mutagenesis has the advantages of easy identification and positioning, and can conveniently and quickly associate excellent mutation characters with the site generating mutation. Once determining multiple gene mutations related to high-yield traits, the high-yield mutation sites are hopefully recombined into one strain purposefully by means of a genetic engineering technology to quickly obtain a high-yield strain, and the defects that multiple rounds of mutagenesis and multiple rounds of mass screening are needed in conventional mutagenesis are overcome.

Transposons (transposons) were first discovered by Barbara McClintock in studying corn kernel color variation in the fifties of the last century, allowing positional changes in DNA sequences within and between genomes without relying on homologous recombination, and are therefore referred to as mobile elements or skipping genes. Taking Tn5 as an example, the transposition process in organisms is as follows: transposase gene expression, transposase identification and combination of the reverse repeat sequences specific to the two ends of the transposon, cutting out the DNA fragment containing the reverse repeat sequences and the middle part of the reverse repeat sequences and inserting the DNA fragment into a new gene locus, and completing the transposition process. In the process, insertion mutation of a new gene site is caused, and the method is a biological mutagenesis system. Transposons found naturally are the result of long-term evolutionary selection and have a low frequency of transposition.

The transposition efficiency can be greatly improved by improving the catalysis efficiency of the transposase; by constructing mini-Tn5(mini-Tn5, namely, transposase genes are not contained in DNA fragments subjected to transposition movement), after transposition is carried out under the action of external transposase, the transferred mini transposon cannot express the transposase, so that transposition ectopy does not occur any more, and the genetic stability of the mutant strain is ensured. Random and saturation mutation can be realized in gram-negative bacteria such as escherichia coli by using the high-efficiency transposition system. Gram-positive bacteria have thick cell walls, and therefore this system cannot be used directly in gram-positive bacteria such as actinomycetes. Mark Weber et al utilize circuitous In Vitro (In Vitro) transposition mutagenesis method, construct the genomic cosmid library of Saccharopolyspora erythraea with colibacillus as cloning host first, carry on transposition mutagenesis to colibacillus containing candidate cosmid separately, the sequencing positions the mutation site, obtain and carry transposon and insert the mutation site mutation cosmid library of mutation, introduce the cosmid In the mutation library into Saccharopolyspora erythraea separately through the protoplast transformation method, with the help of homologous recombination In vivo, exchange the mutant allele to Saccharopolyspora erythraea chromosome and construct and get the mutant strain, found 15 genotypes are correlated with yield of erythromycin, including influencing the mutation of primary metabolism and regulatory gene, hydrolase, peptidase, glycosyltransferase and unknown functional gene. This study showed that random transposition mutagenesis revealed genes associated with strain improvement, potentially useful for high-yield breeding. However, one of the disadvantages of this method is that it is cumbersome and heavy: constructing cosmid library using colibacillus as host, transposing and mutating colibacillus to construct cosmid mutant library, introducing mutated cosmid to saccharopolyspora erythraea, screening double-exchange strain, and screening mutant strain with changed phenotype. Since it cannot be directly transposed in saccharopolyspora erythraea cells, it is difficult to obtain a sufficient number of random mutants.

At present, there is no transposon which can directly function In Vivo (In Vivo) In Saccharopolyspora, and there is no In Vivo transposition mutagenesis system available In Saccharopolyspora spinosa. The construction of a transposition system capable of working in saccharopolyspora and the random insertion mutation of the genome have important value for researching the biosynthesis and metabolic regulation of compounds including spinosad and obtaining high-yield strains.

Disclosure of Invention

The invention aims to provide a transposable plasmid for saccharopolyspora and application thereof; also relates to a gene mutation method of important industrial microorganism strains for producing antibiotics and biological pesticides.

The invention realizes the in vivo transposition mutagenesis of saccharopolyspora spinosa by constructing the transposons capable of working in the saccharopolyspora spinosa, and searches for the genes related to the yield of spinosad, thereby laying a foundation for the transformation of high-yield strains through genetic engineering.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the present invention relates to a transposable plasmid for saccharopolyspora comprising the following elements in order: mini-Tn5, transposase gene tnp (5), ampicillin resistance gene bla, conjugation transfer initiation site oriT; the mini-Tn5 sequentially comprises a transposon terminal sequence ME, a specific DNA recombination site attB phi C31, a specific DNA recombination site attB phi BT1, a lox71 site, a replicon ori-pUC, an apramycin resistance gene aac (3) IV, a lox66 site, a specific DNA recombination site attB phi Rv1, a specific DNA recombination site attB phi Joe, a specific DNA recombination site attB phi Tp901-1 and a transposon terminal sequence ME.

In a second aspect, the present invention relates to the use of the above-described transposable plasmid for saccharopolyspora for transposable mutagenesis of saccharopolyspora in vivo.

Preferably, the saccharopolyspora spinosa comprises saccharopolyspora spinosa and saccharopolyspora erythraea.

In a third aspect, the present invention relates to a method for constructing the transposable plasmid for saccharopolyspora, comprising the following steps:

s1, synthesizing a DNA fragment 1 sequentially comprising a transposon terminal sequence ME, a specific DNA recombination site attB phi C31, an attB phi BT1 and a lox71 site, and connecting to a plasmid pHL734 after enzyme digestion to obtain a plasmid pHHB 735;

s2, synthesizing a DNA fragment 2 sequentially comprising a 3 'end part sequence of an apramycin resistance gene, a lox66 site, a specific DNA recombination site attB phi Rv1, attB phi Joe, attB phi Tp901-1, a transposon terminal sequence ME, a promoter KasOP and a transposase gene 5' end part sequence, and connecting to a plasmid pHHB735 after enzyme digestion to obtain a plasmid pHHB 736;

s3, respectively synthesizing a segment SEG-pro1, SEG-pro2, SEG-pro3, SEG-pro4, SEG-pro5 or SEG-pro6 containing Promoter Promoter1, Promoter2, Promoter3, Promoter4, Promoter5 or Promoter6DNA sequence through gene digestion, and connecting the segment SEG-pro1, SEG-pro2 or SEG-pro6 to a plasmid pHHB736 to obtain plasmids pJTn1, pJTn2, pJTn3, pJTn4, pJTn5 or pJTn 6;

the plasmid pHHB736, the plasmid pJTn1, the plasmid pJTn2, the plasmid pJTn3, the plasmid pJTn4, the plasmid pJTn5 and the plasmid pJTn6 are the transposable plasmids.

In a fourth aspect, the present invention relates to a host strain providing the above-described transposable plasmid for saccharopolyspora, the transposable plasmid is transformed into e.coli ET12567 by an electrotransformation method, and the obtained positive transformant is the host strain.

In a fifth aspect, the present invention relates to a host strain providing the above-mentioned transposable plasmid for saccharopolyspora, which has a accession number of CGMCC No.17351, CGMCC No.17352, CGMCC No.17353, CGMCC No.17421, CGMCC No.17422 or CGMCC No. 17423.

In a sixth aspect, the present invention relates to a transposable mutant strain obtained by transposon mutagenesis using the aforementioned transposable plasmid for saccharopolyspora.

In a seventh aspect, the present invention relates to the use of the above transposable mutant strain for the production of spinosad.

In an eighth aspect, the present invention relates to a method for transposon mutagenesis in saccharopolyspora using the aforementioned transposable plasmid for saccharopolyspora, the method comprising the steps of:

a1, transferring the transposition plasmid into E.coli DH10B/pUZ8002 by an electrotransformation method to obtain donor bacteria,

a2, conjugative transfer was performed using saccharopolyspora as a recipient bacterium.

In a ninth aspect, the present invention relates to a method for identifying a mutation site in the genome of saccharopolyspora spinosa, which is associated with spinosyn biosynthesis, using the aforementioned transposable plasmid for saccharopolyspora spinosa, the method comprising the steps of:

b1, transferring the transposition plasmid into E.coli DH10B/pUZ8002 by an electrotransformation method to obtain a donor bacterium, performing conjugal transfer by taking saccharopolyspora erythraea as a recipient bacterium, and determining the transposition plasmid with high transposition efficiency according to the number of obtained conjugars;

b2, transferring the transposition plasmid with high transposition efficiency obtained in the step B1 into E.coli DH10B/pUZ8002 by an electrotransformation method to obtain a donor bacterium, performing conjugation transfer by using saccharopolyspora spinosa as a recipient bacterium, and obtaining a saccharopolyspora spinosa transposition mutant strain with high transposition efficiency according to the number of obtained zygotes;

b3, designing PCR primers by taking a mini-Tn5DNA sequence as a template, determining a transposon insertion site of each saccharopolyspora spinosa transposition mutant strain by a transposon rescue method, and determining a mutated gene;

b4, fermenting the wild type saccharopolyspora spinosa strain and the saccharopolyspora spinosa transposition mutant strain to produce the spinosad, detecting the yield of the spinosad by an HPLC method, analyzing the association of gene mutation caused by transposition and spinosad biosynthesis, and determining the mutation site of the high-yield spinosad.

In the tenth aspect, the invention relates to a saccharopolyspora spinosa genetic engineering strain, and the mutant site of the high-yield spinosad determined by the method is recombined into the saccharopolyspora spinosa genetic engineering strain to obtain the saccharopolyspora spinosa genetic engineering strain.

In an eleventh aspect, the invention relates to the use of the saccharopolyspora spinosa genetic engineering strain in the production of spinosad.

The host strain of the transposition plasmid for saccharopolyspora of the invention has been submitted to CGMCC (China general microbiological culture Collection center) general microbiological culture Collection center for 18 days in 3 months and 21 months in 2019, respectively, the preservation address is No.3 of Xilu No.1 of the morning district of Beijing city, and the microbial research institute of the Chinese academy of sciences, the strains are respectively: e.coli ET12567/pJTn1 (with the strain preservation number of CGMCC NO.17351, the preservation date of 2019, 3 and 18 months), E.coli ET12567/pJTn2 (with the strain preservation number of CGMCC NO.17352, the preservation date of 2019, 3 and 18 months), E.coli ET12567/pJTn3 (with the strain preservation number of CGMCC NO.17353, the preservation date of 2019, 3 and 18 months), E.coli ET12567/pJTn4 (with the strain preservation number of CGMCC NO.17421, the preservation date of 2019, 3 and 21 months), E.coli ET12567/pJTn5 (with the strain preservation number of CGMCC NO.17422, the preservation date of 2019, 3 and 21 months), E.coli ET12567/pJTn6 (with the strain preservation number of CGMCC NO.17423, the preservation date of 2019, 3 and 21 months); the biological material samples are all classified and named as Escherichia coli.

Compared with the prior art, the invention has the following beneficial effects:

1) the transposon in the invention mutates the transposition in the saccharopolyspora, so that the saturation mutagenesis effect can be achieved, and the gene related to biosynthesis and metabolic regulation of a specific compound can be accurately confirmed by combining the fermentation experiment of a mutant strain, thereby laying a foundation for searching a target gene to be reformed and laying a foundation for the reformation of a high-yield strain; because the transposable peptide is directly transposed in vivo and randomly mutated, the in vivo situation can be truly reflected; the in vitro method involves a large investment in time and effort to create mutants, and the in vivo transposition method is simple, efficient and straightforward.

2) The invention constructs a series of Tn5 transposable plasmids, transposable plasmids with higher transposable efficiency are selected to transpose in saccharopolyspora spinosa by comparing the transposable efficiency of the transposable plasmids in the saccharopolyspora erythraea, the transposable conditions in the saccharopolyspora spinosa are searched, transposable mutants are successfully obtained, and the occurrence of transposition affects the capacity of the saccharopolyspora spinosa to generate spinosad; this is a successful in vivo transposition in Saccharopolyspora spinosa for the first time, and the method has important significance for the yield control of spinosad in Saccharopolyspora spinosa.

3) The transposition plasmid constructed by the invention has high transposition efficiency in saccharopolyspora erythraea, and is an effective tool for relevant research of saccharopolyspora erythraea.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 shows the construction process of plasmid pJTnX, the PCR-verified product of pJTnX, and the agarose gel electrophoresis pattern of the enzyme digestion-verified product; wherein, A is a construction flow diagram of a plasmid pJTnX, B is a PCR verification gel diagram of a plasmid pHHB736 seg1 (segment 1), C is an enzyme digestion verification gel diagram of a plasmid pHHB736 seg2 (segment 2), and D is a PCR verification gel diagram of transposase promoter parts of plasmids pJTn 1-pJTn 6;

FIG. 2 is a plasmid map of plasmid pJTnX;

FIG. 3 is a graph showing the comparison of the effect of the junction transfer experiment;

FIG. 4 is an HPLC chromatogram of spinosyn fractions A and D corresponding to the transposable mutant of Saccharopolyspora spinosa and the wild type strain NRRL 18395;

FIG. 5 is a graph showing a comparison of spinosad production by a Saccharopolyspora spinosa transposition mutant strain and a wild strain NRRL 18395.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention. The experimental methods not specifically described in the following examples are all conventional methods, and materials and reagents not specifically described are commercially available.

The terms involved in the present invention are explained as follows:

transposon (transpososon): transposons are DNA fragments that can move positions (i.e., transpositions) within a genome or between a genome, phage, and plasmid, and transpositions inserted into new chromosomal locations can mutate, e.g., inactivate genes, and alter genome size. Also known as a skipping gene.

Saturation mutation (Satured mutation): saturation mutation is a form of random mutagenesis that we attempt to generate mutations in all (or as many) genes in the genome; or to generate all possible (or as close as possible) mutations at specific sites or genes on the chromosome. This is a commonly used technique in directed evolution of bacteria or enzymes.

Bonding and transferring: temporary association of two bacteria or unicellular organisms for the exchange of genetic material, or fusion of two gametes. In genetic manipulation of microorganisms, one of the techniques for introducing a DNA fragment, a plasmid, or the like from a donor bacterium into a target host bacterium can be used as conveniently as transformation, transfection, or the like.

Saccharopolyspora: saccharopolyspora is a microorganism belonging to actinomycetes, belonging to one genus of the family of prokaryotic microorganisms actinomycetes in a classified position, and one of the species is more studied for its ability to produce the biopesticide spinosad, Saccharopolyspora spinosa. Another well-known species of Saccharopolyspora erythraea is Saccharopolyspora erythraea, which produces erythromycin.

Biosynthesis (biosynthesis): the enzymatic reaction carried out in vivo is a general term for the production of a certain chemical substance active in cells. Genes responsible for the synthesis of specific metabolites in an organism are collectively referred to as biosynthetic genes. Genes involved in the biosynthesis of a compound in microbial cells, such as chromatin of actinomycetes, include: structural genes, transporter genes, resistance genes, pathway-specific regulatory genes, etc., are often arranged together in clusters, which are referred to as biosynthetic gene clusters.

Transposon mutagenesis is a very valuable technique in biology for creating random mutations that are easy to identify and localize. Transposons are widely applied in transgenic research of animals and plants and genetic modification of microorganisms, and as a mutation introducing method, transposition mutation has the advantages of random transposition sites, convenience in positioning, capability of obtaining a large number of different transposition mutant strains at one time and the like.

In the present invention, a tool plasmid that can transpose in Saccharopolyspora spinosa (s. spinosa) and Saccharopolyspora erythraea (saccharomyces erythraea) was constructed, and a method for transposon mutagenesis in Saccharopolyspora spinosa and Saccharopolyspora erythraea was proposed. Because the transposon tool in vivo is not suitable for most actinomycetes, for the purpose of convenient use, a series of transposons are reconstructed and constructed on the basis of Tn5 transposon, and the transposon is placed on a plasmid which can autonomously replicate in Escherichia coli (Escherichia coli) and can shuttle between Escherichia coli and Saccharopolyspora sinensis. The transposon of the invention is used for transposable mutagenesis of saccharopolyspora spinosa, so that a large number of genes highly related to spinosad biosynthesis and metabolic regulation can be found, and the related genes are improved through genetic engineering, so that the transposon has important value for high-yield breeding.

The transposable plasmid that can be transposed in vivo in Saccharopolyspora is pJTnX (wherein X denotes Arabic numerals 1, 2, 3, 4, 5, 6), and is composed of mini-Tn5, conjugation transfer initiation site oriT (SEQ ID NO.29), screening marker gene bla (ampicillin resistance gene, "Zhong Xu, Yeast Wang, Keith F. Chater, Hong-Yu Ou, H.Howard Xu, Zixin Deng, Meifeng Tao; Large-Scale transfer Mutagenesis Mugnetisis of Streptomyces coelicolor identities Hundrednesses of Genes Influencing applied. Environ Microbiol.2017Mar 15; 83(6) (SEQ ID NO. 02889-16), which is publicly available from the public transportation synthesis). Mini-Tn5 is composed of replicon ori-pUC (SEQ ID NO.5) capable of replicating in Escherichia coli, a selection marker gene aac (3) IV (apramycin resistance gene, SEQ ID NO.6), lox66 site (SEQ ID NO.7), lox71 site (SEQ ID NO.4), specific DNA recombination site [ attB φ C31(SEQ ID NO.2), attB φ BT1(SEQ ID NO.3), attB φ Rv1(SEQ ID NO.8), attB φ Joe (SEQ ID NO.9), attB φ Tp901-1(SEQ ID NO.10) ], transposon end sequences ME (SEQ ID NO.1, 11). The promoter of the transposase gene tnp (5) is derived from a promoter capable of efficiently initiating transcription in Saccharopolyspora spinosa, and is used for enhancing the expression level of the transposase gene tnp (5).

The construction process of the transposition mutagenesis system of saccharopolyspora in the invention is as follows:

(1) gene synthesis DNA fragment 1: contains transposon terminal sequence ME, specific DNA recombination sites attB phi C31, attB phi BT1, lox71 sites, cuts the fragment by restriction enzymes EcoRI and PciI, and then connects to EcoRI and PciI cutting sites of plasmid pHL734 to obtain plasmid pHHB 735;

(2) gene synthesis DNA fragment 2: contains the sequence of the 3 'end part of the apramycin resistance gene, lox66 site, specific DNA recombination site attB phi Rv1, attB phi Joe, attB phi Tp901-1, transposon end sequence ME, promoter KasOP, the sequence of the 5' end part of transposase gene, the fragment is cut by restriction enzymes BstEII and AsiSI, and then the fragment is connected to the corresponding site (BstEII and AsiSI cutting site) of plasmid pHHB735 to obtain plasmid pHHB 736;

(3) six constitutive high-efficiency expressed protein genes are screened from the proteome data of saccharopolyspora spinosa by analyzing the proteome data, and promoters of the six corresponding genes in the saccharopolyspora spinosa genome are selected as promoters of transposase genes, namely Promoter 1-Promoter 6. Synthesizing DNA fragments SEG-pro 1-SEG-pro 6 (the two ends of the sequence of Promoter 1-Promoter 6 are respectively added with NheI 'GCTAGC'/BamHI 'GGATCC', namely the sequence of SEG-pro 1-SEG-pro 61) containing the sequences of Promoter 1-Promoter 6 by using restriction endonucleases NheI and BamHI to respectively cut DNA fragments SEG-pro 1-SEG-pro 6, then respectively connecting to corresponding sites (NheI and BamHI cutting sites) at the upstream of tnp (5) gene in pHHB736, respectively replacing the Promoter of transposase gene tnpp (5) with Promoter 1-Promoter 6, and obtaining plasmids named pJTn1, pJTn2, pJTn 48, pJTn4, pJTn5 and pJTn 6;

(4) in the process of constructing plasmids pHHB736, pJTn1, pJTn2, pJTn3, pJTn4, pJTn5 and pJTn6, the enzyme connecting products are respectively electrically transformed into E.coli ET12567 to obtain positive transformants E.coli ET12567/pHHB736, ET12567/pJTn1 (strain preservation number CGMCC NO.17351), ET12567/pJTn2 (strain preservation number CGMCC NO.17352), ET12567/pJTn3 (strain preservation number CGMCC NO.17353), ET12567/pJTn4 (strain preservation number CGMCC NO.17421), ET12567/pJTn5 (strain preservation number CGMCC NO.17422) and ET12567/pJTn6 (strain preservation number CGMCC NO.17423), which are used as host for providing plasmids.

(5) The seven transposon plasmids pHHB736, pJTn1, pJTn2, pJTn3, pJTn4, pJTn5 and pJTn6 were transferred into E.coli DH10B/pUZ8002 by an electrotransformation method to obtain E.coli DH10B (pUZ8002, pHHB736), E.coli DH10B (pUZ8002, pJTn1), E.coli DH10B (pUZ8002, pJTn2), E.coli DH10B (pUZ8002, pJTn3), E.coli 10B (pUZ8002, pJTn4), E.coli DH10B (pUZ8002, pJTn5) and E.coli DH10B (pUZ8002, pJTn 6). These seven E.coli strains were used as donor bacteria and Saccharopolyspora erythraea was used as recipient bacteria, and conjugation transfer was performed. Based on the obtained number of the zygotes, the level of transposition efficiency is determined. Among them, many of the four transposon plasmids pJTn1, pJTn2, pJTn5 and pJTn6 can obtain the spliceosome.

(6) pJTn1, pJTn2, pJTn5, and pJTn6 were transferred to E.coli DH10B/pUZ8002 by an electrotransformation method to obtain E.coli DH10B (pUZ8002, pJTn1), E.coli DH10B (pUZ8002, pJTn2), E.coli DH10B (pUZ8002, pJTn5), and E.coli DH10B (pUZ8002, pJTn 6). These four E.coli strains were used as donor bacteria and Saccharopolyspora spinosa as recipient bacteria, and conjugation transfer was performed. Based on the obtained number of the zygotes, the level of transposition efficiency is determined. Among them, the two transposon plasmids pJTn1 and pJTn5 have many adapters.

(7) PCR primers were designed using mini-Tn5DNA sequences (SEQ ID NO.16, Zhong Xu, Yemin Wang, Keith F. Chater, Hong-Yu Ou, H. Howard Xu, Zixin Deng, Meifeng Tao; Large-Scale translocation Mutagenesis of microorganisms peptides of Genes infection identification of Genes, apple Environ Microbiol.2017Mar 15; 83(6): e02889-16.) as templates to determine the transposon insertion site of each mutated Saccharopolyspora spinosa strain by transposon rescue method and to confirm the mutated gene;

(8) fermenting the wild strain and the transposition mutant strain by using a proper fermentation method to produce the spinosad, and detecting the yield of the spinosad of the mutant strain and the wild strain by using a High Performance Liquid Chromatography (HPLC) method;

(9) the influence of gene mutation caused by transposition on spinosyn production by saccharopolyspora spinosa was confirmed by analyzing spinosyn production of mutant strains and wild-type strains.

The following are specific medium formulations and culture conditions according to the examples of the present invention

(1) Glycopolyspora saccharopolyspora seed activation and sporulation BM media is used, the formulation of which is disclosed in the literature "Dhakal D, Pokhrel A R, Jha A K, et al.

Inoculating spore or strain preservation solution to a solid culture medium plate, and culturing at 30 ℃ for 10-12 days.

(2) Saccharopolyspora liquid culture is carried out by using Tryptone Soy Broth (TSBY) culture medium:

30g/L of tryptone, 103g/L of sucrose, 10g/L of yeast extract and 7.0-7.2 of pH.

Inoculating the single colony or the strain preservation solution into a 250mL shaking bottle filled with 50mL of TSBY culture medium, and carrying out shaking culture at 30 ℃ and 220rpm/min for 24-48 hours;

(3) the fermentation of saccharopolyspora spinosa for producing spinosad adopts CTF4 culture medium, and the formula of the culture medium is disclosed in "Nonpeng," research on the breeding and fermentation process of spinosad producing strains, university of agriculture in Huazhong, 2004 ".

1mL of the Saccharopolyspora spinosa seed solution was transferred to a 250mL shake flask containing 50mL of CTF4 medium, and cultured at 30 ℃ and 220rpm/min for 10 days.

(4) coli-Saccharopolyspora conjugal transfer R6 solid medium was used, the medium formulation is described in "Illing G T, Normansel I D, Peberdy J F. protoplast Isolation and Regeneration in Streptomyces clavuligerus [ J ]. Microbiology,1989,135(8): 2289-"

During conjugation and transfer, the mixed solution of the escherichia coli and the saccharopolyspora spinosa mycelia is coated on an R6 solid plate and cultured for 7-14 days at 30 ℃.

The strains, plasmids and primers used in the present invention are shown in table 1:

TABLE 1 strains, plasmids and primers used in the invention

Example 1 construction of transposable plasmid

pHL734 is Tn5 type transposition plasmid, and uses inverted repetitive sequence ME as transposition boundary, the mini-Tn5 of the plasmid contains Escherichia coli replication initiation site ori-pUC (SEQ ID NO.5) and apramycin resistance gene aac (3) IV (SEQ ID NO.6), and the promoter of transposase gene tnp (5) (SEQ ID NO.13) is strong promoter PermE. The plasmid has high transposition efficiency in streptomyces coelicolor. pHL734 for the subject group construction and preservation, in the literature "Zhong Xu, Yemin Wang, Keith F.Chater, Hong-Yu Ou, H.Howard Xu, Zixin Deng, Meifong Tao; Large-Scale transformation Mutagenesis of Streptomyces coelicolor identities, Hundreds of Genes underfluoring biological biosynthesis, apple Environ Microbiol, 2017Mar 15; 83(6) e02889-16 ", publicly available from the school subjects group of Shanghai university of transportation.

The gene synthesis comprises ME1(SEQ ID NO.1), lox71 site (SEQ ID NO.4) and specific DNA recombination site

(SEQ ID NO.2)、

(SEQ ID NO.3) DNA fragment 1(SEQ ID NO.14) (both ends have EcoRI and PciI cleavage sites, respectively); cutting the DNA fragment 1 and the plasmid pHL734 by using restriction endonuclease EcoRI-PciI, separating the required DNA fragment by agarose gel electrophoresis and recovering the DNA fragment; the fragment and the vector were ligated, the ligation product was transformed into E.coli DH10B, and transformants were selected with the antibiotics apramycin and ampicillin. Designing a primer pair pHHB736-seg1-F/R (SEQ ID NO.23 and SEQ ID NO.24), carrying out PCR verification on the improved particles of the transformant, detecting a PCR product (337bp) by agarose gel electrophoresis, and obtaining a plasmid pHHB735 if the size is consistent with the expected size.

The gene synthesis comprises the 3' end part sequence (SEQ ID NO.30) of the apramycin resistance gene, lox66 site (SEQ ID NO.7) and specific DNA recombination site [ attB^Φrv1(SEQ ID NO.8)、attB^ΦJoe(SEQ ID NO.9)、 attB^ΦTp901-1(SEQ ID NO.10)]ME2(SEQ ID NO.11), promoter KasOP^*(SEQ ID NO.12) and a 5' -terminal sequence (SEQ ID NO.31) of a transposase gene (both ends of which have BstEII and AsiSI cleavage sites, respectively, SEQ ID NO. 15); the restriction enzyme BstEII-AsiSI is used for cutting the DNA fragment 2 and the plasmid pHHB735, and the required DNA fragment is separated and recovered by agarose gel electrophoresis; the fragment and the vector were ligated, the ligation product was transformed into E.coli DH10B, and transformants were selected with the antibiotics apramycin and ampicillin. Because the DNA sequence of the target clone is only partially different from the starting plasmid pHHB735 in basic group, the PCR method is not suitable for verification, so that the plasmid is verified by double enzyme digestion by using the restriction endonuclease SpeI-EcoRI, the positive clone can be cut into 1969bp and 2825bp DNA fragments, and the enzyme digestion product is detected to be consistent with the expected size by agarose gel electrophoresis, thus obtaining the plasmid pHHB 736. FIG. 1 is a schematic representation of the procedure for constructing plasmid pHHB 736.

^*Example 2 increasing transposase Activity in transposable plasmids, promoter KasOP is replaced by Saccharopolyspora sinensis Promoters for efficient constitutive expression

1) In order to ensure that the transposase gene is stably and efficiently expressed in saccharopolyspora spinosa, six constitutive expression protein genes are screened from saccharopolyspora spinosa proteome data by analyzing the data, promoters of the six genes are selected as promoters for controlling transposase gene expression, namely promoter 1-promoter 6, the functions of the six genes are detailed in table 2, and the corresponding sequences are sequentially shown in SEQ ID NO. 17-22.

TABLE 2 sources of transposase promoters

Transposase promoter	Origin of origin
		promoter1	superoxide dismutase[Fe-Zn]1(WP_010308738.1)
promoter 2	superoxide dismutase[Fe-Zn]2(WP_101376528.1)
		promoter 3	cold-shock DNA-binding protein(WP_010308657.1)
promoter 4	chaperonin GroEL 1(WP_010316107.1)
		promoter 5	chaperonin GroEL 2(WP_010308482.1)
promoter 6	elongation factor Tu(WP_010315430.1)

2) Gene synthesis of SEG-pro 1-SEG-pro 6 fragments containing six Promoter Promoter 1-Promoter 6DNA sequences (Promoter 1-Promoter 6 with NheI (GCTAGC)/BamHI (GGATCC) added at both ends of the sequences to obtain SEG-pro 1-SEG-pro 6 sequences), restriction enzymes NheI and BamHI are used to cut DNA fragments SEG-pro 1-SEG-pro 6 respectively, then the DNA fragments are respectively connected to pHHB736 corresponding sites (NheI and BamHI cutting sites), the promoters of transposase gene tnp (5) are respectively replaced by Promoter 1-Promoter 6, and the obtained plasmids are named pJTn1, pJTn2, pJTn3, pJTn4, pJTn5 and pJTn6, and the plasmid map is shown in figure 2.

3) The primers Prom-F/R (SEQ ID NO.25, SEQ ID NO.26) are used for carrying out PCR verification on plasmids pJTn1, pJTn2, pJTn3, pJTn4, pJTn5 and pJTn6, the PCR amplification band of the positive clone is 470bp, the size of the PCR product detected by agarose gel electrophoresis is consistent with the expectation, and the correct plasmid is obtained, and the agarose gel electrophoresis pattern of the PCR product is shown in figure 1.

Example 3 introduction of transposable plasmid into Saccharopolyspora erythraea by conjugative transfer to form a transposable mutant

Transposable mutagenesis experiments were performed with Saccharopolyspora erythraea (S.erythraea) NRRL23338 (a strain disclosed in "Oliynyk M, Samborsky M, Lester J B, et al. complete genome sequence of the erythromycins-producing bacterium Saccharopolyspora erythraea NRRL23338[ J ]. NATURE BIOTHNECOLOGY, 2007, 25(4): 447. sup. 453." publicly available from the Peter Francis Leadlay laboratory) as the recipient strain.

1) Plasmids pHHB736, pJTn1, pJTn2, pJTn3, pJTn4, pJTn5 and pJTn6 are electrically transformed into E.coli DH10B/pUZ8002, and positive E.coli DH10B (pUZ8002, pHHB736), E.coli DH10B (pUZ8002, pJTn1), E.coli DH10B (pUZ8002, pJTn2), E.coli DH10B (pUZ8002, pJTn3), E.coli DH10B (pUZ8002, pJTn4), E.coli DH10 DH B (pUZ 2, pJTn5), E.coli DH10 (pJTn 80027 ), and seven strains of co-bacillus.

2) The seven strains were used as donor bacteria, and spores of Saccharopolyspora erythraea were used as recipient bacteria to perform conjugation transfer experiments. Because the transposition plasmid does not contain a replicon capable of working in saccharopolyspora, only under the action of transposase, mini-Tn5 is inserted into chromatin of a recipient bacterium, and thus, the new host can be endowed with apramycin resistance. The resulting conjugal transferor was screened for transposition mutators with apramycin.

3) The mutagenesis efficiency of seven transposable plasmids was compared based on the number of zygotes that grew on the conjugative transfer plates. Under the same bacterial concentration and operation conditions, the plasmid pHHB736 does not obtain a conjugant, pJTn3 and pJTn4 can obtain hundreds of positive conjugants, the number of the positive conjugants of pJTn1, pJTn2, pJTn5 and pJTn6 is more than 1000, and the effect of the conjugal transfer experiment is shown in figure 3. Therefore, it is considered that pJTn1, pJTn2, pJTn5, and pJTn6 have high mutagenesis efficiency on Saccharopolyspora erythraea among seven transposable plasmids, and these four plasmids were selected for transposable mutagenesis on Saccharopolyspora erythraea.

Example 4 transposable plasmid mutagenesis of Saccharopolyspora spinosa

Transposable mutagenesis experiments were performed using mycelia of Saccharopolyspora spinosa (S.spinosa) NRRL18395(PAN Y, YANG X, LI J, et al genome Sequence of the spinosyn-Producing Bacterium Saccharomyces spinosa NRRL18395 [ J ]. Journal of Bacteriology,2011,193(12):3150 and 3151.) as recipient bacteria, using transposable plasmids pJTn1, pJTn2, pJTn5 and pJTn 6.

1) The plasmids pJTn1, pJTn2, pJTn5 and pJTn6 are electrically transformed into E.coli DH10B/pUZ8002, and positive transformants E.coli DH10B (pUZ8002, pJTn1), E.coli DH10B (pUZ8002, pJTn2), E.coli DH10B (pUZ8002, pJTn5) and E.coli DH10B (pUZ8002, pJTn6) are obtained by screening apramycin and ampicillin resistance and carrying out enzyme digestion verification on the extracted plasmid of the transformant.

2) Preparation of donor bacteria: donor bacterial transformants E.coli DH10B (pUZ8002, pJTn1) and E.coli DH10B (pUZ8002, p) were selectedJTn2), E.coli DH10B (pUZ8002, pJTn5), E.coli DH10B (pUZ8002, pJTn6) into LB liquid medium (tryptone 10g, yeast extract 5g, NaCl 5g, deionized water to 1L, pH natural), to which apramycin, ampicillin and kanamycin were added to final concentrations of 50. mu.g/mL, 100. mu.g/mL and 50. mu.g/mL, respectively; shaking culture at 37 deg.C to OD₆₀₀0.5 to 0.6.

3) And (3) recipient bacterium culture: inoculating 3mL of Saccharopolyspora spinosa seed solution into 27mL of Tryptone Soy Broth (TSBY), and culturing overnight at 30 ℃ and 220 rpm/min; the cells were transferred once, inoculated with 5mL of a culture solution into 15mL of Tryptone Soy Broth (TSBY), and cultured under the same conditions for 4-7 hours.

4) Collecting recipient bacteria: taking 3mL of bacterial liquid into a centrifugal tube, centrifuging at 5000rpm/min for 1min, and removing supernatant; then, 1mL of TSBY medium was added, the mixture was shaken well, centrifuged at 5000rpm/min for 2min to collect the cells, and the cells were resuspended in 500. mu.L of TSBY medium.

5) Bonding and transferring: mixing donor bacteria and acceptor bacteria, fully and uniformly mixing the donor bacteria and the acceptor bacteria, coating the mixture on an R6 solid culture medium flat plate, and performing inverted culture at 30 ℃ for 16-20 hours; the resulting conjugal transferor was screened for transposition mutators with apramycin.

Example 5 mutant Gene mapping of transposable mutants

In transposition mutagenesis with e.coli DH10B (pUZ8002, pJTn1) and e.coli DH10B (pUZ8002, pJTn5) as donor bacteria, 14 and 17 mutants were obtained, respectively. Streaking and inoculating 31 strains of polyspora spinosa transposable mutate, namely BLL01-BLL31, to a BM culture medium (yeast extract 1g, beef extract 1g, N-Z-Amine A2 g, glucose 10g, agar 20g and pH 7.2-7.4) plate containing apramycin (working concentration is 50 mug/mL), wherein resistance verification is correct; transferring the transposable mutants to a non-resistant BM culture medium to produce spores when the transposable mutants grow well; after spores are mature, the spores are inoculated into a TSBY liquid culture medium, and the culture is carried out for 48 hours at the temperature of 30 ℃ and at the speed of 220 rpm/min; centrifuging at 5000rpm/min for 10min to collect thallus and extracting mutant genome DNA. Because the occurrence frequency of the restriction enzyme ApaI in the actinomycete genome is high, the extracted genome DNA is cut by using ApaI, and the fragments are self-connected by using T4DNA ligase; the ligation product was transformed into E.coli DH10B and transformants were selected with apramycin. Selecting three transformants from the rescue plate obtained from each mutant, extracting plasmids, and designing PCR primers Tn-F and Tn-R (SEQ ID NO.27 and SEQ ID NO.28) for sequencing by taking a mini-Tn5DNA sequence as a template; according to the sequencing result, the mutant gene locus is determined by comparing the sequence with the saccharopolyspora spinosa NRRL18395 genome sequence.

Example 6 comparison of fermentation with transposable mutant strains with spinosyn production

1) Inoculating saccharopolyspora spinosa transposable mutant strain (BLL01-BLL31) and wild strain NRRL18395 on a BM culture medium for spore production, scraping spores into 50mL of TSBY culture medium after the spores are mature, and culturing at 30 ℃ and 220rpm/min for 48 hours to obtain fermented seed liquid.

2) Transferring 1mL of seed liquid to 50mL of CTF4 fermentation medium (glucose 48g, starch 20g, soybean cake powder 20g, corn steep liquor 15g, cotton seed powder 2.5g, yeast extract 1.2g, soybean oil 3g, CaCO)₃2.5g, adding water to 1L, pH 7.2-7.4), and carrying out shaking fermentation at 30 ℃ and 220rpm/min for 10 days.

3) After the fermentation is finished, 700 mu L of fermentation liquor is added with methanol with one time of volume, and is subjected to ultrasonic treatment for 30 minutes and then is subjected to standing extraction for 12 hours.

4) And after extraction is finished, centrifuging at 12000rpm/min for 15min, taking supernate, filtering by using a 0.22 mu m filter membrane, and detecting the yield of the spinosad by using an agilent 1200 series High Performance Liquid Chromatography (HPLC) for filtrate. The chromatographic column used is ZORBAX SB-C18 column, 4.6X 250mm, 5 μm, sample volume 10 μ L; the mobile phase is methanol/acetonitrile/0.05% sodium acetate solution (volume ratio is 47.5/47.5/5), the flow rate is 1mL/min, the column temperature is room temperature, and the detection wavelength is 250 nm. And analyzing by analysis software carried by an HPLC system to obtain the response peak areas of the spinosad A and the spinosad D, and calculating the yield of the spinosad according to a standard curve. The HPLC profiles of spinosyn components A and D are shown in figure 4.

5) Analysis shows that spinosad cannot be detected by the transposition mutant strain BLL08 on HPLC, the yield of BLL01 and BLL29 is 43.98mg/L and 21.26mg/L respectively, the yield is reduced by 66 percent and 83 percent respectively compared with the yield of 128.57mg/L of a wild strain, and the yield of the rest strains is not obviously changed, which is shown in figure 5. Transposition of the mutant strain BLL08 occurs at the cytochrome biosynthetic protein gene resB, which is associated with post-translational modification of proteins, protein turnover, and molecular chaperones, and transposition insertion may affect synthesis of related enzymes during spinosyn biosynthesis, resulting in loss of the ability of BLL088 to synthesize spinosyns; the mutant strain BLL01 transposes on the transcriptional regulator protein gene of the differentiation regulator, and the generation of the pleocidin is influenced; transposition of mutant BLL09 occurred in a M67 family peptidase gene, whose function and effect on spinosyn synthesis were further investigated.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Sequence listing

<110> Shanghai university of transportation

<120> transposable plasmid for saccharopolyspora and use thereof

<130> DAG38279

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> transposon terminal sequence (ME1)

<400> 1

ctgtctctta tacacatctt 20

<210> 2

<211> 35

<212> DNA

<213> specific DNA recombination site (attB φ C31)

<400> 2

ggtgccaggg cgtgcccttg ggctccccgg gcgcg 35

<210> 3

<211> 36

<212> DNA

<213> specific DNA recombination site (attB φ BT1)

<400> 3

gctggatcat ctggatcact ttcgtcaaaa acctgg 36

<210> 4

<211> 34

<212> DNA

<213> lox71 site (lox71)

<400> 4

taccgttcgt atagcataca ttatacgaag ttat 34

<210> 5

<211> 589

<212> DNA

<213> replicon (ori-pUC)

<400> 5

tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60

gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120

ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180

cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240

caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300

ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360

taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420

taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 480

cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 540

tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaa 589

<210> 6

<211> 804

<212> DNA

<213> apramycin resistance Gene (aac (3) IV)

<400> 6

atgtcatcag cggtggagtg caatgtcgtg caatacgaat ggcgaaaagc cgagctcatc 60

ggtcagcttc tcaaccttgg ggttaccccc ggcggtgtgc tgctggtcca cagctccttc 120

cgtagcgtcc ggcccctcga agatgggcca cttggactga tcgaggccct gcgtgctgcg 180

ctgggtccgg gagggacgct cgtcatgccc tcgtggtcag gtctggacga cgagccgttc 240

gatcctgcca cgtcgcccgt tacaccggac cttggagttg tctctgacac attctggcgc 300

ctgccaaatg taaagcgcag cgcccatcca tttgcctttg cggcagcggg gccacaggca 360

gagcagatca tctctgatcc attgcccctg ccacctcact cgcctgcaag cccggtcgcc 420

cgtgtccatg aactcgatgg gcaggtactt ctcctcggcg tgggacacga tgccaacacg 480

acgctgcatc ttgccgagtt gatggcaaag gttccctatg gggtgccgag acactgcacc 540

attcttcagg atggcaagtt ggtacgcgtc gattatctcg agaatgacca ctgctgtgag 600

cgctttgcct tggcggacag gtggctcaag gagaagtcgc ttcagaagga aggtccagtc 660

ggtcatgcct ttgctcggtt gatccgctcc cgcgacattg tggcgacagc cctgggtcaa 720

ctgggccgag atccgttgat cttcctgcat ccgccagagg cgggatgcga agaatgcgat 780

gccgctcgcc agtcgattgg ctga 804

<210> 7

<211> 34

<212> DNA

<213> lox66 site (lox66)

<400> 7

ataacttcgt atagcataca ttatacgaac ggta 34

<210> 8

<211> 40

<212> DNA

<213> specific DNA recombination site (attB φ Rv1)

<400> 8

gaaggtgttg gtgcggggtt ggccgtggtc gaggtggggt 40

<210> 9

<211> 50

<212> DNA

<213> specific DNA recombination site (attB φ Joe)

<400> 9

atctggatgt gggtgtccat ctgcgggcag acgccgcagt cgaagcacgg 50

<210> 10

<211> 53

<212> DNA

<213> specific DNA recombination site (attB φ Tp901)

<400> 10

atgccaacac aattaacatc tcaatcaagg taaatgcttt ttgctttttt tgc 53

<210> 11

<211> 20

<212> DNA

<213> transposon terminal sequence (ME2)

<400> 11

aagatgtgta taagagacag 20

<210> 12

<211> 97

<212> DNA

<213> transposase promoter (KasOp)

<400> 12

tgttcacatt cgaacggtct ctgctttgac aacatgctgt gcggtgttgt aaagtcgtgg 60

ccaggagaat acgacagcgt gcaggactgg gggatcc 97

<210> 13

<211> 1434

<212> DNA

<213> transposase Gene (Tnp (5))

<400> 13

atgatcacct cggccctcca ccgcgccgcg gactgggcca agtccgtgtt cagctcggcc 60

gccctgggcg acccccggcg gacggcccgg ctggtcaacg tcgcggccca gctcgcgaag 120

tactccggca agtccatcac catctcgtcc gagggcagca aggccgccca ggagggcgcg 180

taccggttca tccgcaaccc gaacgtctcc gccgaggcca tccgcaaggc cggggccatg 240

cagacggtca agctggcgca ggagttcccc gagctcctgg ccatcgagga cacgacgagc 300

ctctcgtacc ggcaccaggt cgcggaggag ctgggcaagc tggggagcat ccaggacaag 360

tcgcgcggct ggtgggtgca cagcgtgctg ctcctggagg ccaccacgtt ccggacggtg 420

gggctgctcc accaggagtg gtggatgcgg ccggacgacc ccgcggacgc cgacgagaag 480

gagagcggca agtggctcgc ggcggcggcc acgagccgcc tccgcatggg ctccatgatg 540

agcaacgtca tcgccgtgtg cgaccgggag gccgacatcc acgcctacct ccaggacaag 600

ctcgcccaca acgagcggtt cgtggtccgg tccaagcacc cgcgcaagga cgtcgagtcg 660

ggcctgtacc tgtacgacca cctcaagaac cagcccgagc tgggcgggta ccagatcagc 720

atcgcccaga agggcgtggt cgataagcgg gggaagcgca agaaccgccc ggcgcgcaag 780

gcgagcctga gcctgcggag cgggcggatc acgctgaagc aggggaacat caccctgaac 840

gcggtgctcg ccgaggagat caaccccccg aagggcgaga cgccgctcaa gtggctcctc 900

ctgacgagcg agccggtcga gtcgctcgcc caggccctcc gggtcatcga catctacacg 960

caccgctggc ggatcgagga gttccacaag gcgtggaaga cgggcgcggg cgcggagcgc 1020

cagcgcatgg agaagccgga caacctcgag cggatggtgt cgatcctcag cttcgtcgcg 1080

gtccggctcc tgcagctgcg cgagagcttc acgccgcccc aggcgctgcg ggcgcagggc 1140

ctcctcaagg aggcggagca cgtggagagc cagagcgccg agaccgtgct gacccccgac 1200

gagtgccagc tgctcgggta cctggacaag ggcaagcgga agcgcaagga gaaggccggg 1260

tcgctccagt gggcctacat ggcgatcgcc cgcctcgggg gcttcatgga ctcgaagcgg 1320

accgggatcg cctcgtgggg ggccctctgg gaggggtggg aggccctgca gtcgaagctc 1380

gacgggttcc tggcggcgaa ggacctgatg gcgcagggca tcaagatcgg gtag 1434

<210> 14

<211> 137

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gaattcctgt ctcttataca catcttggtg ccagggcgtg cccttgggct ccccgggcgc 60

ggctggatca tctggatcac tttcgtcaaa aacctggtac cgttcgtata gcatacatta 120

tacgaagtta tacatgt 137

<210> 15

<211> 2319

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggttaccccc ggcggtgtgc tgctggtcca cagctccttc cgtagcgtcc ggcccctcga 60

agatgggcca cttggactga tcgaggccct gcgtgctgcg ctgggtccgg gagggacgct 120

cgtcatgccc tcgtggtcag gtctggacga cgagccgttc gatcctgcca cgtcgcccgt 180

tacaccggac cttggagttg tctctgacac attctggcgc ctgccaaatg taaagcgcag 240

cgcccatcca tttgcctttg cggcagcggg gccacaggca gagcagatca tctctgatcc 300

attgcccctg ccacctcact cgcctgcaag cccggtcgcc cgtgtccatg aactcgatgg 360

gcaggtactt ctcctcggcg tgggacacga tgccaacacg acgctgcatc ttgccgagtt 420

gatggcaaag gttccctatg gggtgccgag acactgcacc attcttcagg atggcaagtt 480

ggtacgcgtc gattatctcg agaatgacca ctgctgtgag cgctttgcct tggcggacag 540

gtggctcaag gagaagtcgc ttcagaagga aggtccagtc ggtcatgcct ttgctcggtt 600

gatccgctcc cgcgacattg tggcgacagc cctgggtcaa ctgggccgag atccgttgat 660

cttcctgcat ccgccagagg cgggatgcga agaatgcgat gccgctcgcc agtcgattgg 720

ctgaataact tcgtatagca tacattatac gaacggtaga aggtgttggt gcggggttgg 780

ccgtggtcga ggtggggtat ctggatgtgg gtgtccatct gcgggcagac gccgcagtcg 840

aagcacggat gccaacacaa ttaacatctc aatcaaggta aatgcttttt gctttttttg 900

cactagtaag atgtgtataa gagacaggct agctgttcac attcgaacgg tctctgcttt 960

gacaacatgc tgtgcggtgt tgtaaagtcg tggccaggag aatacgacag cgtgcaggac 1020

tgggggatcc atgatcacct cggccctcca ccgcgccgcg gactgggcca agtccgtgtt 1080

cagctcggcc gccctgggcg acccccggcg gacggcccgg ctggtcaacg tcgcggccca 1140

gctcgcgaag tactccggca agtccatcac catctcgtcc gagggcagca aggccgccca 1200

ggagggcgcg taccggttca tccgcaaccc gaacgtctcc gccgaggcca tccgcaaggc 1260

cggggccatg cagacggtca agctggcgca ggagttcccc gagctcctgg ccatcgagga 1320

cacgacgagc ctctcgtacc ggcaccaggt cgcggaggag ctgggcaagc tggggagcat 1380

ccaggacaag tcgcgcggct ggtgggtgca cagcgtgctg ctcctggagg ccaccacgtt 1440

ccggacggtg gggctgctcc accaggagtg gtggatgcgg ccggacgacc ccgcggacgc 1500

cgacgagaag gagagcggca agtggctcgc ggcggcggcc acgagccgcc tccgcatggg 1560

ctccatgatg agcaacgtca tcgccgtgtg cgaccgggag gccgacatcc acgcctacct 1620

ccaggacaag ctcgcccaca acgagcggtt cgtggtccgg tccaagcacc cgcgcaagga 1680

cgtcgagtcg ggcctgtacc tgtacgacca cctcaagaac cagcccgagc tgggcgggta 1740

ccagatcagc atcgcccaga agggcgtggt cgataagcgg gggaagcgca agaaccgccc 1800

ggcgcgcaag gcgagcctga gcctgcggag cgggcggatc acgctgaagc aggggaacat 1860

caccctgaac gcggtgctcg ccgaggagat caaccccccg aagggcgaga cgccgctcaa 1920

gtggctcctc ctgacgagcg agccggtcga gtcgctcgcc caggccctcc gggtcatcga 1980

catctacacg caccgctggc ggatcgagga gttccacaag gcgtggaaga cgggcgcggg 2040

cgcggagcgc cagcgcatgg agaagccgga caacctcgag cggatggtgt cgatcctcag 2100

cttcgtcgcg gtccggctcc tgcagctgcg cgagagcttc acgccgcccc aggcgctgcg 2160

ggcgcagggc ctcctcaagg aggcggagca cgtggagagc cagagcgccg agaccgtgct 2220

gacccccgac gagtgccagc tgctcgggta cctggacaag ggcaagcgga agcgcaagga 2280

gaaggccggg tcgctccagt gggcctacat ggcgatcgc 2319

<210> 16

<211> 1985

<212> DNA

<213> transposon (mini-Tn5)

<400> 16

ctgtctctta tacacatctt ggtgccaggg cgtgcccttg ggctccccgg gcgcggctgg 60

atcatctgga tcactttcgt caaaaacctg gtaccgttcg tatagcatac attatacgaa 120

gttatacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 180

ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 240

gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 300

cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 360

gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 420

tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 480

cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 540

cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 600

gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 660

agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 720

cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 780

tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 840

tttggtcatg agattatcaa aaaggatctt cacctagatc cttttggttc atgtgcagct 900

ccatcagcaa aaggggatga taagtttatc accaccgact atttgcaaca gtgccgttga 960

tcgtgctatg atcgactgat gtcatcagcg gtggagtgca atgtcgtgca atacgaatgg 1020

cgaaaagccg agctcatcgg tcagcttctc aaccttgggg ttacccccgg cggtgtgctg 1080

ctggtccaca gctccttccg tagcgtccgg cccctcgaag atgggccact tggactgatc 1140

gaggccctgc gtgctgcgct gggtccggga gggacgctcg tcatgccctc gtggtcaggt 1200

ctggacgacg agccgttcga tcctgccacg tcgcccgtta caccggacct tggagttgtc 1260

tctgacacat tctggcgcct gccaaatgta aagcgcagcg cccatccatt tgcctttgcg 1320

gcagcggggc cacaggcaga gcagatcatc tctgatccat tgcccctgcc acctcactcg 1380

cctgcaagcc cggtcgcccg tgtccatgaa ctcgatgggc aggtacttct cctcggcgtg 1440

ggacacgatg ccaacacgac gctgcatctt gccgagttga tggcaaaggt tccctatggg 1500

gtgccgagac actgcaccat tcttcaggat ggcaagttgg tacgcgtcga ttatctcgag 1560

aatgaccact gctgtgagcg ctttgccttg gcggacaggt ggctcaagga gaagtcgctt 1620

cagaaggaag gtccagtcgg tcatgccttt gctcggttga tccgctcccg cgacattgtg 1680

gcgacagccc tgggtcaact gggccgagat ccgttgatct tcctgcatcc gccagaggcg 1740

ggatgcgaag aatgcgatgc cgctcgccag tcgattggct gaataacttc gtatagcata 1800

cattatacga acggtagaag gtgttggtgc ggggttggcc gtggtcgagg tggggtatct 1860

ggatgtgggt gtccatctgc gggcagacgc cgcagtcgaa gcacggatgc caacacaatt 1920

aacatctcaa tcaaggtaaa tgctttttgc tttttttgca ctagtaagat gtgtataaga 1980

gacag 1985

<210> 17

<211> 294

<212> DNA

<213> transposase promoter (promoter1)

<400> 17

cgccgctccc cgtggcgtgt ccaatctgcg cgacgagcaa tcggcgcatc gtgccttcca 60

ggggcgagcc ggcctgcggg aaggccggga ccgcacggga agccgcgagt tccagacccc 120

gaccgcagac cacggcggtc gctggagtca tccaaaatgc tcccgcaccg caacgttttc 180

gcagatcaca ggggtttctg gcgcaactcg cccggcagtt gagatcaaca cccccatcca 240

agactaattg ccataagatg gcagtagcta gactctgtcg acagcgaagg gaga 294

<210> 18

<211> 294

<212> DNA

<213> transposase promoter (promoter2)

<400> 18

caggagccgc cgggccagtt cgcggtctga agcgaagttg cagctaccgc ccaggaagta 60

gtcgtagacc ctggcgccgc tcgggcggtc cggggcgagg tcctgcggga gctgggcgtg 120

gtccggcatg gtgcctccgg gtaaccgtgc aggaagcgca gagtagtcgc ccgaacgccg 180

gtcgaacagg atgaaacgtg attgagaaca ctccggacgt gtcattgacc cattgacctt 240

tagcgcgctt gaggttcgag actttcggcg aacgcaagat cagtgaaggg gaag 294

<210> 19

<211> 294

<212> DNA

<213> transposase promoter (promoter3)

<400> 19

aacccgaaca ggaccatcga taccaagagc actgcggacc tggaccttgc actagccatg 60

ctgagcagcg tattcacgac tgaaccgggt gccgcagcgg gccttggtca ataaggtcgg 120

gtgttatcgg ctggttacgc ggtacgcttg gcgtgacgcg tcccggcgcg cccaactgcg 180

tggcgtattc ggggcgcttt cgtcgtgccg gggtcgttcg gtcccactcg gcggagcaga 240

caacgacgac ggaggccccg gagaccgggg cggcgcattc gacagggaac ggtg 294

<210> 20

<211> 294

<212> DNA

<213> transposase promoter (promoter4)

<400> 20

ccgggtcgcg tcgacgacaa gggcaaccgc atcccggtgg atgtcaagga aggcgacgtc 60

gtcatctact cgaagtacgg cggcaccgag gtcaagtaca acggcgagga gtacctgatc 120

ctctccgccc gcgacgtgct ggctgtcgtc aactgacgca gccgcaccaa gcgagcaatg 180

ccgccccggc ggtcccgacc gcgggacccc ggggcggtcg cacgtccggg gcagcgggac 240

ttgtcgatgg aacaggtacg gcctcaatag atcaggtacc gatgaagggc tgtt 294

<210> 21

<211> 294

<212> DNA

<213> transposase promoter (promoter5)

<400> 21

gaaacctccc cgtacaacaa caaccacctg ccgacttcgg tcccgactcg ctgacggtcg 60

cgctccgccg catcggttac ggcgcttgca ctcgactggc gagagtgcta aacacggtat 120

tggcactcag caaggttgag tgccaggtcg ggacggtgag gccgtctccg gcggtgccac 180

cagacggcgc cgccgcacgg tcgtccgtcg cgggcaccga gcctggccga gcacgagtcc 240

tgccgtgggg tgcgcaaacc caccaccgcg gcgtccagac aggtggagga ccac 294

<210> 22

<211> 294

<212> DNA

<213> transposase promoter (promoter6)

<400> 22

actcgatggt gttcgactcc tacgccgagg ttccggcgaa cgtggctaag gagatcatcg 60

ccaaggcaac gggcgagtga cccgctgctc ggtcccggcc agccgggacc gagcgggagg 120

caacgcctcg cgaaggcgac cggggagcaa tcccctccag ttcggcggcg gacgggccgc 180

caccccgcaa ggacagtgtt cttccgggat cggcggcccg ctcgtcacct acccgacagg 240

actccgcctg gcacaacaag tcgtacggcg gaaagttaac aagtccagga ggac 294

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

agcggataca tatttgaatg 20

<210> 24

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gatttttgtg atgctcgtca 20

<210> 25

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

atgtgtataa gagacaggct ag 22

<210> 26

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cgagatggtg atggacttg 19

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

aagcacggat gccaacacaa 20

<210> 28

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cggtaccagg tttttgacg 19

<210> 29

<211> 110

<212> DNA

<213> conjugation transfer initiation site (oriT)

<400> 29

cccgccagcc tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag 60

tgaagaagga acacccgctc gcgggtgggc ctacttcacc tatcctgccc 110

<210> 30

<211> 724

<212> DNA

<213> apramycin resistance gene (aac (3) IV)

<400> 30

ggttaccccc ggcggtgtgc tgctggtcca cagctccttc cgtagcgtcc ggcccctcga 60

agatgggcca cttggactga tcgaggccct gcgtgctgcg ctgggtccgg gagggacgct 120

cgtcatgccc tcgtggtcag gtctggacga cgagccgttc gatcctgcca cgtcgcccgt 180

tacaccggac cttggagttg tctctgacac attctggcgc ctgccaaatg taaagcgcag 240

cgcccatcca tttgcctttg cggcagcggg gccacaggca gagcagatca tctctgatcc 300

attgcccctg ccacctcact cgcctgcaag cccggtcgcc cgtgtccatg aactcgatgg 360

gcaggtactt ctcctcggcg tgggacacga tgccaacacg acgctgcatc ttgccgagtt 420

gatggcaaag gttccctatg gggtgccgag acactgcacc attcttcagg atggcaagtt 480

ggtacgcgtc gattatctcg agaatgacca ctgctgtgag cgctttgcct tggcggacag 540

gtggctcaag gagaagtcgc ttcagaagga aggtccagtc ggtcatgcct ttgctcggtt 600

gatccgctcc cgcgacattg tggcgacagc cctgggtcaa ctgggccgag atccgttgat 660

cttcctgcat ccgccagagg cgggatgcga agaatgcgat gccgctcgcc agtcgattgg 720

ctga 724

<210> 31

<211> 1289

<212> DNA

<213> apramycin resistance gene (aac (3) IV)

<400> 31

atgatcacct cggccctcca ccgcgccgcg gactgggcca agtccgtgtt cagctcggcc 60

gccctgggcg acccccggcg gacggcccgg ctggtcaacg tcgcggccca gctcgcgaag 120

tactccggca agtccatcac catctcgtcc gagggcagca aggccgccca ggagggcgcg 180

taccggttca tccgcaaccc gaacgtctcc gccgaggcca tccgcaaggc cggggccatg 240

cagacggtca agctggcgca ggagttcccc gagctcctgg ccatcgagga cacgacgagc 300

ctctcgtacc ggcaccaggt cgcggaggag ctgggcaagc tggggagcat ccaggacaag 360

tcgcgcggct ggtgggtgca cagcgtgctg ctcctggagg ccaccacgtt ccggacggtg 420

gggctgctcc accaggagtg gtggatgcgg ccggacgacc ccgcggacgc cgacgagaag 480

gagagcggca agtggctcgc ggcggcggcc acgagccgcc tccgcatggg ctccatgatg 540

agcaacgtca tcgccgtgtg cgaccgggag gccgacatcc acgcctacct ccaggacaag 600

ctcgcccaca acgagcggtt cgtggtccgg tccaagcacc cgcgcaagga cgtcgagtcg 660

ggcctgtacc tgtacgacca cctcaagaac cagcccgagc tgggcgggta ccagatcagc 720

atcgcccaga agggcgtggt cgataagcgg gggaagcgca agaaccgccc ggcgcgcaag 780

gcgagcctga gcctgcggag cgggcggatc acgctgaagc aggggaacat caccctgaac 840

gcggtgctcg ccgaggagat caaccccccg aagggcgaga cgccgctcaa gtggctcctc 900

ctgacgagcg agccggtcga gtcgctcgcc caggccctcc gggtcatcga catctacacg 960

caccgctggc ggatcgagga gttccacaag gcgtggaaga cgggcgcggg cgcggagcgc 1020

cagcgcatgg agaagccgga caacctcgag cggatggtgt cgatcctcag cttcgtcgcg 1080

gtccggctcc tgcagctgcg cgagagcttc acgccgcccc aggcgctgcg ggcgcagggc 1140

ctcctcaagg aggcggagca cgtggagagc cagagcgccg agaccgtgct gacccccgac 1200

gagtgccagc tgctcgggta cctggacaag ggcaagcgga agcgcaagga gaaggccggg 1260

tcgctccagt gggcctacat ggcgatcgc 1289

Claims (10)

1. A transposable plasmid for saccharopolyspora comprising the following elements in order: mini-Tn5, transposase gene tnp (5), ampicillin resistance gene bla, conjugation transfer initiation site oriT; the mini-Tn5 sequentially comprises a transposon terminal sequence ME and a specific DNA recombination site

C31, specific DNA recombination site

BT1, lox71 locus, replicon ori-pUC, apramycin resistance gene aac (3) IV, lox66 locus, specific DNA recombination locus attB phi Rv1, specific DNA recombination locus attB phi Joe, specific DNA recombination locus attB phi Tp901-1 and transposon terminal sequence ME.

2. Use of a transposable plasmid for saccharopolyspora as defined in claim 1 for transposing mutagenized saccharopolyspora in vivo.

3. Use according to claim 2, wherein the saccharopolyspora species comprises saccharopolyspora spinosa, saccharopolyspora erythraea.

4. A method of constructing a transposable plasmid for saccharopolyspora as claimed in claim 1, comprising the steps of:

s1, synthesizing gene, sequentially including transposon terminal sequence ME and specific DNA recombination site

C31、

BT1, lox71 site DNA fragment 1, after cutting enzyme, connected to plasmid pHL734, get plasmid pHHB 735;

s2, synthesizing a DNA fragment 2 sequentially comprising a 3 'end part sequence of an apramycin resistance gene, a lox66 site, a specific DNA recombination site attB phi Rv1, attB phi Joe, attB phi Tp901-1, a transposon terminal sequence ME, a promoter KasOP and a transposase gene 5' end part sequence, and connecting to a plasmid pHHB735 after enzyme digestion to obtain a plasmid pHHB 736;

s3, synthesizing SEG-pro1, SEG-pro2, SEG-pro3, SEG-pro4, SEG-pro5 or SEG-pro6 containing Promoter Promoter1, Promoter2, Promoter3, Promoter4, Promoter5 or Promoter6DNA sequence respectively, and connecting the obtained fragments to plasmid pHHB736 after enzyme digestion to obtain plasmids pJTn1, pJTn2, pJTn3, pJTn4, pJTn5 or pJTn 6;

the plasmid pHHB736, the plasmid pJTn1, the plasmid pJTn2, the plasmid pJTn3, the plasmid pJTn4, the plasmid pJTn5 and the plasmid pJTn6 are the transposable plasmids.

5. A host strain providing the transposable plasmid for saccharopolyspora as claimed in claim 1, which is a positive transformant obtained by transferring the transposable plasmid into e.coli ET12567 by an electrotransformation method.

6. A host strain Escherichia coli providing the transposable plasmid for Saccharopolyspora as defined in claim 1, having a accession number of CGMCC NO.17351, CGMCC NO.17352, CGMCC NO.17353, CGMCC NO.17421, CGMCC NO.17422 or CGMCC NO. 17423.

7. Use of a transposable mutant strain obtained by transposon mutagenesis using the transposable plasmid for saccharopolyspora as described in claim 1 for the production of spinosad.

8. A method for identifying a mutation site in the genome of saccharopolyspora spinosa, which is associated with spinosyn biosynthesis, using the transposable plasmid for saccharopolyspora spinosa of claim 1, the method comprising the steps of:

b1, transferring the transposition plasmid into E.coli DH10B/pUZ8002 by an electrotransformation method to obtain a donor bacterium, performing conjugal transfer by taking saccharopolyspora erythraea as a recipient bacterium, and determining the transposition plasmid with high transposition efficiency according to the number of obtained conjugars;

b2, transferring the transposition plasmid with high transposition efficiency obtained in the step B1 into E.coli DH10B/pUZ8002 by an electrotransformation method to obtain a donor bacterium, performing conjugation transfer by using saccharopolyspora spinosa as a recipient bacterium, and obtaining a saccharopolyspora spinosa transposition mutant strain with high transposition efficiency according to the number of obtained zygotes;

b3, designing PCR primers by taking a mini-Tn5DNA sequence as a template, determining a transposon insertion site of each saccharopolyspora spinosa transposition mutant strain by a transposon rescue method, and determining a mutated gene;

b4, fermenting the wild type saccharopolyspora spinosa strain and the saccharopolyspora spinosa transposition mutant strain to produce the spinosad, detecting the yield of the spinosad by an HPLC method, analyzing the association of gene mutation caused by transposition and spinosad biosynthesis, and determining the mutation site of the high-yield spinosad.

9. A Saccharopolyspora spinosa genetically engineered strain obtained by recombining the high-yield spinosad mutation site determined by the method of claim 8 into a Saccharopolyspora spinosa strain.

10. Use of the saccharopolyspora spinosa genetically engineered strain of claim 9 for the production of spinosad.

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