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Transposon plasmids for saccharopolyspora spinosa and application of transposon plasmisd

A technology of Saccharopolyspora spinosa and plasmids, which is applied in the field of genetic engineering, can solve the problems of cumbersome work, difficulty in obtaining random mutant strains, and no in vivo transposition mutagenesis system, and achieves the goal of simple method and high transposition efficiency Effect

Active Publication Date: 2020-11-10
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, one of the disadvantages of this method is that the work is tedious and tedious: it is necessary to construct a cosmid library with E. Double-crossover strains can then be screened for mutants with phenotypic changes
Due to the inability to directly transpose within S. rubrum cells, it is difficult to obtain sufficient numbers of random mutants
[0006] At present, there is no transposon that can directly function in vivo (In Vivo) in Saccharopolyspora, and there is no in vivo transposition mutagenesis system available in Saccharopolyspora spinosa

Method used

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  • Transposon plasmids for saccharopolyspora spinosa and application of transposon plasmisd
  • Transposon plasmids for saccharopolyspora spinosa and application of transposon plasmisd
  • Transposon plasmids for saccharopolyspora spinosa and application of transposon plasmisd

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Experimental program
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Effect test

Embodiment 1

[0077] Embodiment 1, the construction of transposable plasmid

[0078] pHL734 is a Tn5-type transposition plasmid, with the inverted repeat sequence ME as the transposition boundary. The mini-Tn5 of this plasmid contains the E. coli replication origin site ori-pUC (SEQ ID NO.5) and apramycin The promoters of the resistance gene aac(3)IV (SEQ ID NO.6) and the transposase gene tnp(5)(SEQ ID NO.13) are the strong promoter PermE*. This plasmid has high transposition efficiency in Streptomyces coelicolor. pHL734 was constructed and preserved by our research group, in the literature "Zhong Xu, Yemin Wang, Keith F. Chater, Hong-Yu Ou, H. Howard Xu, Zixin Deng, Meifeng Tao; Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis. Appl Environ Microbiol. 2017 Mar 15; 83(6):e02889-16." It has been published, and the public can obtain it from the research group of Shanghai Jiao Tong University.

[0079] Gene sy...

Embodiment 2

[0081] Embodiment 2, improve the activity of transposase in the transposable plasmid, its promoter KasOP * Replaced with Saccharopolyspora Medium High promoter for constitutive expression

[0082] 1) In order to ensure the stable and efficient expression of the transposase gene in S. spinosa, the present invention screens out six constitutively expressed protein genes by analyzing the proteome data of S. spinosa, and selects these six genes The promoters of the six genes are used as the promoters controlling the expression of the transposase genes, that is, promoter1-promoter6. The functions of these six genes are shown in Table 2, and their corresponding sequences are shown in SEQ ID NO.17-22 in sequence.

[0083] Table 2. Sources of transposase promoters

[0084] transposase promoter source 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 prot...

Embodiment 3

[0087] Example 3. Transposable plasmids were introduced into Saccharopolyspora rubrum by conjugative transfer to form transposable mutants

[0088] Saccharopolyspora erythraea (S.erythraea) NRRL23338 (the strain in "Oliynyk M, Samborskyy M, Lester J B, et al.Complete genome sequence of the erythromycin-producingbacterium Saccharopolyspora erythraea NRRL23338[J].NATURE BIOTECHNOLOGY,250 (4):447-453.", the public can obtain from the Peter Francis Leadlay laboratory) to carry out the transposition mutagenesis experiment for the recipient bacteria.

[0089] 1) The plasmids pHHB736, pJTn1, pJTn2, pJTn3, pJTn4, pJTn5, pJTn6 were electrotransformed into E.coliDH10B / pUZ8002, screened for resistance to apramycin and ampicillin, and verified by digesting the plasmids extracted from the transformants, Positive transformants 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.col...

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Abstract

The invention discloses transposon plasmids for saccharopolyspora spinosa and an application of the transposon plasmids. The transposon plasmids are Tn5 transposon plasmids pJTn1-pJTn6, the transposonplasmids can be subjected to efficient transposon in saccharopolyspora erythraea, pJTn1 and pJTn5 are successfully subjected to in-vivo transposon in saccharopolyspora spinosa, and 31 transposon mutants with different insertion sites are obtained. The yield of the saccharopolyspora spinosa in fermentation liquor is detected through transposon mutant fermentation, and it is found that transposon insertion has an obvious influence on the yield of saccharopolyspora spinosad. The transposon system has important significance in biosynthesis regulation and control of spinosad in the saccharopolyspora spinosa.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a transposable plasmid for Saccharopolyspora and its application. Background technique [0002] Spinosyn (spinosad) is mainly composed of two organic compounds with complex structures, spinosad A and D. It is a natural product biopesticide, which is effective against a variety of insects and is often used to control a variety of pests, including Thrips, Liriomyza, spider mites, mosquitoes, ants, fruit flies, and more. Since the U.S. Environmental Protection Agency (EPA) registered spinosyn as an insecticide for use as a pesticide in 1997, many countries have used spinosad in agricultural production to control pests. [0003] Spinosyns are produced by fermentation of an actinomycete, Saccharopolyspora spinosa, isolated from soil. The spinosyn fermentation titer of wild-type Saccharopolyspora spinosa is very low. Through traditional mutation breeding, genetic engine...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/74C12N15/66C12N1/21C12P19/62C12R1/01
CPCC12N15/70C12N15/74C12N15/66C12P19/62
Inventor 陶美凤白露露王业民邓子新
Owner SHANGHAI JIAO TONG UNIV
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