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Method for improving yield of avermectins and producing strain thereof

An abamectin and a technology for producing abamectin, applied in the field of genetic engineering, can solve the problems of high production cost and low fermentation unit, and achieve the effects of increasing yield, increasing fermentation unit and reducing production cost

Inactive Publication Date: 2011-08-24
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production strains of abamectin in my country still have low fermentation units and high production costs.

Method used

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  • Method for improving yield of avermectins and producing strain thereof
  • Method for improving yield of avermectins and producing strain thereof
  • Method for improving yield of avermectins and producing strain thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Construction of Example 1 sig6 gene deletion vector

[0023] 1. Cloning of Streptomyces avermitilis sig6 gene

[0024] According to the sequence of the sig6 gene [NC_003155.4 (820435..821349)] in the Streptomyces avermitilis wild-type strain ATCC 31267 published by Genebank and its upstream and downstream sequences, PCR primers were designed:

[0025] Primer sig6_1: 5′-CCC AAGCTT ACCGAGGCAAGTACGAGG-3′

[0026] Primer sig6_2: 5′-GGAGTTCGAGCCCAGGAGTCACCGAACCGA

[0027] GCAACC-3′

[0028] Primer sig6_3: 5′-GGTTGCTCGGTTCGGTGACTCCTGGGCTC

[0029] GAACTCC-3′

[0030] Primer sig6_4: 5′-CG GAATTC GTCGATGCCGGTGACAAC-3′

[0031] The two ends of primers sig6_1 and sig6_4 introduced HindIII and EcoRI restriction sites (underlined part) respectively. Using Streptomyces avermitilis ATCC 31267 genomic DNA as a template, sig6_1 and sig6_2, sig6_3 and sig6_4 were used for PCR amplification. PCR reaction conditions: 95°C, 5min; (95°C, 1min; 60°C, 1min; 72°C, 0.5min) x 25 cycle...

Embodiment 2

[0034] Transformation of embodiment 2 recombinant plasmids

[0035] The deletion plasmid vector pLB11 of the sig6 gene was constructed according to Example 1, and the original plasmid used as a control was pKC1139.

[0036] Due to the strong restriction modification in Streptomyces avermitilis, the transformation efficiency of Streptomyces avermitilis is very low when the plasmid extracted from E.coliDH5α is used to directly transform Streptomyces avermitilis, sometimes even no transformant can be obtained. However, the transformation efficiency of the plasmid from E.coliET12567, a recipient strain without restriction modification, was significantly improved. Therefore, the constructed recombinant plasmid was transformed into E.coliET12567 (Kieser T, Bibb M J, Buttner M J, etc. Practical Handbook of Streptomyces Genetics, 2000, Norwich: John Innes Foundation) to obtain unmethylated avermitilis protoplasts were transformed with unmethylated plasmid DNA.

[0037]In this exampl...

Embodiment 3

[0039] Embodiment 3 Obtaining of sig6 gene deletion mutant strain

[0040] Collect the above verified correct spores containing the pLB11 plasmid and the control plasmid pKC1139 transformants respectively, apply about 100 spores per culture dish on the YMS plate containing apramycin, and culture at 28°C for 48-72h. The diameter of the colony is about 2mm. When the aerial hyphae are about to form, the plate is transferred to 39°C for 7 days, and some colonies containing pLB11 appear fan-shaped growth, while the colonies containing pKC1139 do not grow after moving to 39°C. This is because pKC1139 is a temperature-sensitive E. coli-Streptomyces shuttle plasmid, which cannot replicate itself when the temperature is higher than 34°C, so it cannot grow on YMS medium containing apramycin; only when the recombinant plasmid pLB11 carries The upper and lower sequences of the sig6 gene undergo homologous recombination with the homologous segment on the chromosome of Streptomyces avermiti...

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Abstract

The invention provides a method for improving the yield of avermectins and a producing strain thereof. A deletion vector of a sig6 gene, which is used for encoding an extracytoplasmic function (ECF) subfamily ribonucleic acid (RNA) polymerase sigma factor, in Streptomyces avermitilis is introduced into the Streptomyces avermitilis to obtain a recombinant strain in which the sig6 gene is lacked toensure that the recombinant strain cannot synthesize sig6 any more, so that the yield of the avermectins is improved. The genetic engineering strain can be directly used for fermentation production of the avermectins, improves the fermentation unit of the avermectins, and reduces production cost.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetic engineering method for increasing the yield of abamectin. Background technique [0002] Avermectins (avermectins) is a group of high-efficiency insecticidal sixteen-membered ring macrolide antibiotics produced by the fermentation of Streptomyces avermitilis. Its natural products have eight components (A1a, A1b) , A2a, A2b, B1a, B1b, B2a and B2b), of which the B1 component has the strongest insecticidal activity, resisting almost all nematodes and arthropods related to agriculture, and is widely used in agricultural production. Ivermectin (ivermectin) is made from avermectin B1 by hydrogenation reduction at C22-C23 position. It has the same insecticidal activity as avermectin B1, but its toxicity is 2- It is more suitable for the prevention and treatment of internal and external parasites in animals. It is a new type of broad-spectrum and effective anti-pa...

Claims

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Application Information

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IPC IPC(8): C12P19/62C12N1/20C12N15/76C12R1/465
Inventor 陈芝姜利滨文莹宋渊李季伦
Owner CHINA AGRI UNIV
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