Method for increasing yield of avermectin by using regulatory protein gene

A technology of abamectin and regulatory protein, which is applied in the field of genetic engineering and microbiology, and can solve problems such as decreased viability

Inactive Publication Date: 2010-06-09
SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since abamectin was used in actual production as a broad-spectrum antibiotic, the research work on improving the fermentation yield of strains has never stopped, and the most frequently used method is conventional mutagenesis breeding, but this method has considerable influence. Randomness, and the viability of the strains will slowly decline after long-term treatment with mutagen

Method used

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  • Method for increasing yield of avermectin by using regulatory protein gene
  • Method for increasing yield of avermectin by using regulatory protein gene
  • Method for increasing yield of avermectin by using regulatory protein gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment one: the impact of knocking out the avaL2 gene on the production of avermectin (avermectin)

[0093] 1: Obtaining homology arms by left and right exchange

[0094] Using the genome of S. avermitilis as a template, using primers 5'-ATT AAG CTT CGA CCGGAT CAT CGC GGT GAA C-3' and primers TTA CTC GAG GGC GGG GAC CGA AGC ATA AAAG to clone a PCR product of about 2kb in the left arm, using Ligate the restriction site into pGEM-7zf, then use primer 5'-AAT TCT AGA GGC CGG CCA CGG CCC GGT GAG-3' and primer 5'-AAT GAT ATCACG GTC ACC CGC TCC CGC CTG-3' to get the right arm The PCR product of about 2kb was ligated into pSP72 using restriction sites.

[0095] 2: Acquisition of resistance gene fragments

[0096] The erythromycin resistance gene is obtained by digesting the plasmid pAGE-1 with restriction sites XhoI and XbaI, and is about 1.8kb in length.

[0097] 3: Obtaining the recombinant plasmid pWJb360

[0098] The left arm was cut out from the vector with endonuc...

Embodiment 2

[0110] Embodiment 2: the impact of knocking out avaR1 on the production of avermectins (avermectins) B1a

[0111] 1: Obtaining homology arms by left and right exchange

[0112] Using the genome of S. avermitilis as a template, using primers 5'-ATT AAG CTT ACC CCTTGG CGA CCG CCG TC-3' and primers 5'-TTA TCT AGA CTC GAG TCG CTC CTG CCGCGC CAC AC-3' to clone the left arm about The 2kb PCR product was ligated into pGEM-3zf using restriction sites, and then primers 5'-AAT TCT AGA GGC ACG GAC GTT GGA GTG AC-3' and primers 5'-TTA GAA TTC GTG CGG ATC GCG CGT TCC TG-3' got a PCR product of about 2kb in the right arm, which was ligated into pGEM-3zf using restriction sites.

[0113] 2: Acquisition of resistance gene fragments

[0114] The erythromycin resistance gene is obtained by digesting the plasmid pAGE-1 with restriction sites XhoI and XbaI, and is about 1.8kb in length.

[0115] 3: Obtaining the recombinant plasmid pWJb309

[0116] The left arm was cut out from the vector wit...

Embodiment 3

[0121] Embodiment 3: the impact of knocking out avaR2 on the production of avermectins (avermectins)

[0122] 1: Obtaining homology arms by left and right exchange

[0123] Using the genome of S. avermitilis as a template, using primers 5'-GAA AAG CTT GAC GCCCCC TTC ATA G-3' and primers 5'-CAC CTC GAG CTT GCT CTC GAA GTG-3' to clone a PCR product of about 2 kb in the left arm , using the enzyme cutting site to connect into pGEM-7zf, and then use primer 5'-AATTCT AGA GCC GTA CGG CAG CCG CTC-3' and primer 5'-TAA GAA TTC TGC CCC TACGCC CTG GAC-3' to get the right arm about The 2kb PCR product was ligated into pGEM-3zf using restriction sites.

[0124] 2: Acquisition of resistance gene fragments

[0125] The erythromycin resistance gene is obtained by digesting the plasmid pAGE-1 with restriction sites XhoI and XbaI, and is about 1.8kb in length.

[0126] 3: Obtaining the recombinant plasmid pWJb374

[0127]The left arm was cut out from the vector with endonucleases HindIII an...

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Abstract

The invention discloses use of a regulatory protein gene in streptomyces avermitilis. The inactivation of the gene can obviously change the ability of the streptomyces avermitilis to generate an avermectin. The regulatory protein gene comprises an avaL2 gene, an avaR1 gene or an avaR2 gene, wherein the gene replaced mutation strains of the avaL2 gene and the avaR1 gene can obviously improve avermectin generating ability and the gene replaced mutation strain of the avaR2 gene can obviously reduce the avermectin generating ability. The invention also discloses a method for generating the streptomyces avermitilis mutation strain by using the genes, and the application of the streptomyces avermitilis mutation strain, wherein the streptomyces avermitilis mutation strain is increased in the yield. Compared with the yield of the avermectin obtained from an unmodified strain, the yield of the avermectin obtained by fermenting the corresponding mutation strain is greatly increased.

Description

Technical field: [0001] The invention relates to the fields of genetic engineering and microbiology, in particular to a class of genes related to the production of abamectin and a method for increasing the production of abamectin by operating the gene. technical background: [0002] Avermectins (AVM) are a class of widely used agricultural antibiotics, which are produced by Streptomyces avermitilis and have a chemical structure of a sixteen-membered macrolide. -23, The difference in structure at the C26 position can be divided into eight components: A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b. Among them, B1a is the most active component, which is widely used in the treatment of livestock parasite infection and the control of agricultural pests, and has become a very important biological pesticide. [0003] Since abamectin was used in actual production as a broad-spectrum antibiotic, the research work on improving the fermentation yield of strains has never stopped, and the ...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12P19/62C12R1/465
Inventor 唐功利王健博
Owner SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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