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Construction method and application of artificial gene cluster plasmids containing pleocidin and multi-operon

A spinosyn, construction method technology, applied in the field of genetic engineering, can solve problems such as low-cost industrial production, and achieve the effect of high-efficiency heterologous expression

Active Publication Date: 2019-03-19
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, scientists have optimized the spinosyn production of Saccharopolyspora spinosa through traditional methods such as mutation breeding, optimization of fermentation conditions and genetic engineering breeding, but it is far from meeting the requirements of low-cost industrial production

Method used

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  • Construction method and application of artificial gene cluster plasmids containing pleocidin and multi-operon
  • Construction method and application of artificial gene cluster plasmids containing pleocidin and multi-operon
  • Construction method and application of artificial gene cluster plasmids containing pleocidin and multi-operon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 ExoCET technology directly clones the spinosyn gene cluster

[0093] Genomic DNA of Saccharopolyspora spinosa DSM44228 was extracted and digested with XbaI and HpaI to obtain a 71.3kb XbaI-HpaI fragment and a 19.4kb HpaI fragment, wherein the 71.3kb XbaI-HpaI fragment contained 19 spinosads arranged in clusters Biosynthetic genes: spnA, spnB, spnC, spnD, spnE, spnF, spnJ, spnL, spnM, spnO, spnN, spnQ, spnR, spnS, spnG, spnP, spnH, spnI and spnK; 19.4 kb HpaI fragment including spnE gene partial fragment ( figure 1 ). The 71.3kb XbaI-HpaI fragment was cloned into the pBeloBAC11 vector by ExoCET technology to obtain the recombinant plasmid vector pBAC-spn71XH. The pBeloBAC11 vector was prepared by two rounds of PCR: the plasmid pBR322-amp-ccdB-rpsL was used as a template, and the Spn71-1 and Spn71-2 was used as a primer for the first round of PCR; the first round of PCR products were used as templates, and Spn71-3 and Spn71-4 were used as primers for the secon...

Embodiment 2

[0100] Example 2 Cloning of rhamnose biosynthesis gene and construction of spinosyn expression vector

[0101] After PCR amplification, the pBR322-amp vector (primers are BR322-rha-1 and BR322-rha-2), rhamnose synthesis genes gtt, gdh-kre, epi (primers are gtt-1 and gtt-2, gdhkre- 1 and gdhkre-2, epi-1 and epi-2) and kanamycin resistance gene (neo) (primers are neo-1 and neo-2), and introduce 40bp assembly homology arms at both ends of each fragment; The high-fidelity enzyme used in PCR is PrimeSTAR HS DNA Polymerase with GC Buffer (Takara, cat.no.R044A), and the templates and primers used are shown in Table 4.

[0102] Table 4 Primer sequences and templates for PCR amplification

[0103]

[0104] The five PCR products were separated by agarose gel electrophoresis, purified and recovered by the Universal DNA Purification and Recovery Kit (Tiangen Biochemical Technology Co., Ltd.), and then assembled in one step using the Gibson kit (150ng per fragment). The assembled syste...

Embodiment 3

[0107] Example 3 Heterologous expression of Escherichia coli Streptomyces shuttle plasmid pBAC-spn-phiC31-apra in Streptomyces

[0108] The Escherichia coli Streptomyces shuttle plasmid pBAC-spn-phiC31-apra prepared in Example 2 was transformed into three different Streptomyces hosts (Streptomyces coelicolor CH999, Streptomyces lividans Streptomyces K4-114 and white Streptomyces J1074), and integrated into the attB site of the chromosome by phiC31 site-specific recombination. Specific primers for different sites of the gene cluster were selected, and the integrity of the spinosyn gene cluster in the zygote was identified by colony PCR. The specific primers are shown in Table 5.

[0109] Table 5 identifies the primer sequences of Streptomyces engineering bacteria containing spinosyn gene clusters by colony PCR

[0110] name

[0111] The engineered strain and the wild-type strain carrying the spinosyn gene cluster pBAC-spn-phiC31-apra were inoculated into M1 medium an...

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Abstract

The invention relates to a construction method and application of artificial gene cluster plasmids containing pleocidin and multi-operon. According to the construction method, efficient heterologous expression of macrolide bio-pesticide pleocidin can be realized by utilizing the DNA assembly technology and overexpression strategy driven by constructive promoters; 23 genes in the pleocidin synthesizing process are divided into five groups according to functions, and are respectively controlled by different constructive strong promoters to construct an artificial gene cluster of 102.6kb; and the23 genes bio-synthesized by pleocidin are divided into 7 operons, and the pleocidin yield of the artificial gene cluster in a heterologous host streptomyces albus J1074 is increased by 328 times in comparison with that of an original gene cluster. The multi-operon artificial gene cluster strategy provides a new method for synthetic biological researches of secondary metabolites.

Description

technical field [0001] The invention relates to a construction method and application of a spinosyn multi-operon artificial gene cluster plasmid and belongs to the technical field of genetic engineering. Background technique [0002] Spinosyn (a mixture of spinosyn A and D) is a high-efficiency broad-spectrum macrolide biopesticide produced by the soil actinomycete Saccharopolyspora spinosa through secondary metabolism. Spinosyn is composed of a 21-carbon tetracyclic lactone skeleton, a forosamine residue and a rhamnose residue in three parts. Among the spinosad compounds produced by the fermentation of Saccharopolyspora spinosa, spinosyn A and D were the most abundant. Due to its unique insecticidal mechanism, low toxicity to mammals, and rapid degradation in the environment, spinosad won the "Presidential Green Chemistry Challenge Award" in the United States in 1999. [0003] In Saccharopolyspora spinosa, 23 genes are involved in the biosynthesis of spinosyn, 19 of which...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12N15/66C12P19/62C12R1/47C12R1/465
CPCC07K14/36C12N15/76C12P19/62
Inventor 王海龙宋超逸符军张友明
Owner SHANDONG UNIV
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