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Gene engineering bacteria capable of highly producing pleocidin and preparation method thereof

A technology of genetically engineered bacteria and spinosyn, which is applied in the field of genetically engineered bacteria with high yield of spinosyn and its preparation, can solve the problems of no spinosad production, spinosyn PKS impact, and no breakthrough progress in molecular breeding, etc. Achieve the effect of stabilizing the fermentation unit, increasing the fermentation unit and increasing the output

Inactive Publication Date: 2011-09-21
SHANGHAI INST OF PHARMA IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Professor Krishnamurthy of Dow AgroSciences doubled the entire cosmid synthesis gene of this common precursor through a homologous probe. On the basis of the wild strain, the production of spinosad has been significantly improved, but at the same time half of the strain Due to the transfer of large fragments, it has an impact on the spinosyn PKS, resulting in no spinosyn production
However, in China, traditional methods such as natural selection are mainly used to increase its yield, and there are no breakthroughs in molecular breeding.

Method used

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  • Gene engineering bacteria capable of highly producing pleocidin and preparation method thereof
  • Gene engineering bacteria capable of highly producing pleocidin and preparation method thereof
  • Gene engineering bacteria capable of highly producing pleocidin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Cloning of gtt and gdh gene sequences and erythromycin resistance gene promoter (ErmEP)

[0034] Firstly, primers were designed according to the published corresponding sequences of S. spinosad (GenBank accession numbers: AF355467 and AF355468), respectively, the primers for amplifying gtt were: upstream 5'-AAA TCTAGA CGGCAAGAAGAAGG-3'; downstream 5'-AAA TCTAGA CCGACCGCATTCG-3'. The primers for amplifying GDH were: upstream 5'-AAAAAGCTTCCTGCTTCGTAGCTC-3'; downstream 5'-AAAAAGCTTACCAAGCCCTGACC-3'. The total genome of Saccharopolyspora spinosa NRRL 18395 [purchased from the American Agricultural Research Culture Collection (NRRL)] was used as a template, and the primer star high-fidelity polymerase of Bao Bio Company was used to clone the fragments. The PCR conditions are: 98°C, 10s, 68°C, 15s, 72°C, 2min. After PCR product electrophoresis, the target band was recovered. The full lengths of PCR products 1 and 2 are 1131bp and 2259bp, respectively. The rec...

Embodiment 2

[0036] Example 2 Construction and conjugative transfer of single exchange site-directed insertion plasmids

[0037] ErmEP 1 (BamHI / EcoRI) was ligated into plasmid pOJ260 (BamHI / EcoRI) purchased from Takara Company to obtain plasmid pYG-1154. ErmEP2 (XbaI / HindIII) was ligated into pYG-1154 (XbaI / HindIII), resulting in plasmid pYG-1157. gtt (XbaI / XbaI) was positively ligated into pYG-1157 (XbaI / XbaI, dephosphorylated) to obtain plasmid pYG-1158. gdh(HindIII / HindIII) was positively ligated into pYG-1158 ((HindIII / HindIII), dephosphorylated) to obtain plasmid pYG-1159. This recombinant plasmid is the integrated plasmid that has multiplied the gtt and gdh genes. This recombinant plasmid is used to transform large intestine DH5α, and the transformant is obtained and cultured in LB. The plasmid is extracted for enzyme digestion and PCR verification, and finally a single exchange site-directed insertion plasmid pYG is constructed. -1159. The schematic diagram of the above plasmid c...

Embodiment 3

[0039] The screening of embodiment 3 single exchange engineered bacteria

[0040] The zygotes were picked and cultured in TSB containing Abramycin (50 μg / ml), and then the bacterial solution was applied to slant medium (glucose 0.3%, peptone 0.5%, beef cream 0.3%, sodium chloride 0.5%, agar 2.7%, pH 7.0), cultivated at 28°C. Because pOJ260 is a suicide plasmid, only the integrants obtained by homologous single exchange have abramycin resistance and can grow. The zygote can be obtained by two homologous single exchange methods, the schematic diagram is shown in figure 2 . The zygotes were picked and placed in a 4ml TSB small test tube (Am 50 μg / ml, 2-3 glass beads were added to the test tube), cultured with shaking at 30°C, and the total DNA was extracted. Primers were designed on pOJ260 plasmid and ErmEP2 (upstream primer: 5'-AAATCTAGAAGCCCGACCCGAGCA-3'; downstream primer: 5'-AAAAAGCTTTCCGGAGGTCGCACC-3'). This primer does not amplify a band from the genome of the wild str...

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Abstract

The invention discloses gene engineering bacteria capable of highly producing pleocidin and a preparation method thereof. In the gene engineering bacteria, an expression box of NDP-glucose synthesized enzyme gene gtt of NDP-4-ketone-6-deoxidation-Dglucose and an expression box of genes of NDP-glucose reductase gene gdh are integrated in genomes of Saccharopolyspora spinosa wild bacterial strains. According to fermentation validation, the fermenting units of the pleocidin of the gene engineering bacteria is increased by more than three times compared with that of the wild bacterial strains.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a genetically engineered bacterium with high spinosad production and a preparation method thereof. Background technique [0002] Spinosyn is a new type of green broad-spectrum biopesticide, which belongs to microbial source biochemical pesticides. Spinosad has both the safety of biological pesticides and the quick effect of chemically synthesized pesticides. It can be effective on the same day after spraying. The safe harvest period registered by the Chinese and the US Department of Agriculture is only one day, which is most suitable for the production of pollution-free vegetables. Because of its low toxicity, low residue, safety to natural enemies of insects, and fast natural decomposition, it won the "President's Green Chemical Challenge Award" in the United States. Today, with increasing awareness of environmental protection and safety, spinosad, as a high-quality green biologic...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/52C12N15/53C12N15/63C12P19/62C12R1/01
Inventor 李继安邵雷何泞君
Owner SHANGHAI INST OF PHARMA IND
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