Supercharge Your Innovation With Domain-Expert AI Agents!

A genetically engineered bacterium producing 5-keto-22,23-dihydrodoramectin and its preparation method and application

A technology of dihydrodoramectin and genetically engineered bacteria, applied in the field of genetically engineered bacteria producing 5-keto-22,23-dihydrodoramectin and its preparation and application, can solve problems such as loss, Achieve the effects of improving production efficiency, reducing production costs and reducing negative impacts

Active Publication Date: 2019-07-02
NORTHEAST AGRICULTURAL UNIVERSITY
View PDF7 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this mutant strain, due to the mutation inactivation of the bkd gene, the strain lost the ability to form the abamectin initiation unit 2-methylbutyryl-CoA and isobutyryl-CoA

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A genetically engineered bacterium producing 5-keto-22,23-dihydrodoramectin and its preparation method and application
  • A genetically engineered bacterium producing 5-keto-22,23-dihydrodoramectin and its preparation method and application
  • A genetically engineered bacterium producing 5-keto-22,23-dihydrodoramectin and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the construction of genetically engineered bacteria Streptomyces avermitilis Mil

[0047] method:

[0048] 1. Construction of the replacement plasmid pER-4

[0049] Using primer a1 (SEQ ID NO.1, 5'-CC AAGCTT CCGCATTCATCTGCTCCG-3', the underline is the HindIII site) and a2 (SEQ ID NO.2, 5'-GC TCTAGA CCGGCTGCTGACACGTTGC-3', the underline is the XbaI site) PCR amplified from the Streptomyces avermitilis NEAU1069 genome to obtain the homologous recombination left arm fragment of the DH2-KR2 gene in the avermectin biosynthesis gene cluster.

[0050] The total system of the PCR reaction is 25 μL, including 10×pfu Buffer 2.5 μL, dNTP 2 μL, Streptomycesavermitilis NEAU1069 genomic DNA 1 μL, primer a1 and primer a2 2 μL each, pfu DNA polymerase (5U / μL) 2 μL, DMSO 2.5 μL, water 6 μL . The reaction conditions are: 98°C for 1min; 98°C for 10s, 61.3°C for 30s, 72°C for 1min, 30cycles; 72°C for 10min. After the PCR product was loaded for electrophoresis, the targ...

Embodiment 2

[0061] Embodiment 2, the construction of genetically engineered bacteria Streptomyces avermitilis Mil-AveF

[0062] 1. Construction of knockout plasmid pER-aveF

[0063] Using primer aveF-F1 (SEQ ID NO.7, 5'-CCC AAGCTT GGGCCGAACTCTCGCTGTGCCGTG-3', the underline is the HindIII site) and aveF-R1 (SEQ ID NO.8, 5'-GC TCTAGA CGACATGAACCGCGCGATGCGCGA-3', the underline is the XbaI site) PCR amplified from the Streptomyces avermitilis NEAU1069 genome to obtain the homologous recombination left arm fragment of the aveF gene in the avermectin biosynthesis gene cluster.

[0064] The total system of the PCR reaction is 25 μL, including 10×pfu Buffer 2.5 μL, dNTP 2 μL, Streptomycesavermitilis NEAU1069 genomic DNA 1 μL, primer aveF-F1 and primer aveF-R1 2 μL each, pfu DNA polymerase (5U / μL) 2 μL, DMSO 2.5 μL, water 6 μL. The reaction conditions are: 98°C for 1min; 98°C for 30s, 56°C for 30s, 72°C for 1min, 30cycles; 72°C for 10min. After the PCR product was loaded for electrophoresis,...

Embodiment 3

[0073] Example 3, Fermentation of genetically engineered bacteria Streptomyces avermitilis Mil and Streptomycesavermitilis Mil-AveF and MS identification of target compounds

[0074] Pick the original strain Streptomyces avermitilis NEAU1069, the genetically engineered strains Streptomyces avermitilis Mil and Streptomyces avermitilis Mil-AveF, respectively, and culture them on Gaoshi No. 5.0, soy peptone 2.0, CoCl 2 ·6H 2 O 0.01 (g / 1000mL), pH7.0), and then cultured at 28°C and 250r / min for 42h. Get 2.0mL of seed culture solution and transfer to 25mL of fermentation medium (glucose 0.5%, cornstarch 10%, peptone 1%, cottonseed meal 1%, NaCl 0.1%, K 2 HPO 4 0.2%, MgSO 4 ·7H 2 O 0.1%, CaCO 3 0.7%, pH7.2), 28°C, 250r / min for 10 days. At 24h and 168h of fermentation, cyclohexanecarboxylic acid with a final concentration of 100mg / L and 60mg / L was added to the fermentation medium, respectively.

[0075] After the fermentation, the fermented liquid was mixed with an equal vo...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a genetically engineered bacterium producing 5-keto-22,23-dihydro doramectin and a preparation method and application of the bacterium.The genetically engineered bacterium takes Streptomyces avermitilis NEAU1069 producing the doramectin as an original strain, an encoding gene aveDH2-KR2 of an AveDH2-KR2 structural domain in avermectin polyketide synthase is replaced with an encoding gene milDH2-ER2-KR2 of an MilDH2-ER2-KR2 structural domain in Streptomyces bingchengsis milbemycin polyketide synthase, and the genetically engineered bacterium is obtained after an encoding gene aveF of AveF is broken off.The genetically engineered bacterium can be directly used for fermentation production of the 5-keto-22,23-dihydro doramectin.

Description

technical field [0001] The present invention relates to a genetically engineered bacterium producing 5-keto-22,23-dihydrodoramectin and its preparation method and application Background technique [0002] Avermectin (avermectin) is a sixteen-membered ring macrolide polyketide compound produced by the fermentation of Streptomyces avermitilis, which has good acaricidal and nematicidal activities. Taking Abamectin as the parent compound for structural modification, has formed including Ivermectin (Ivermectin), Doramectin (Doramectin), Selamectin (Selamectin) and Eprinomectin (Eprinomectin), etc. A series of avermectins have been further improved and improved in terms of insecticidal activity and pharmacokinetic properties. Abamectin drugs can effectively control agricultural pests and various harmful mites, especially for pests and harmful mites that are resistant to commonly used pesticides. They are relatively safe for crops and do not harm natural enemies. They have been wi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P19/62C12R1/465
CPCC12N9/0006C12N9/1029C12P19/62C12Y101/01184
Inventor 向文胜张继王相晶刘重喜
Owner NORTHEAST AGRICULTURAL UNIVERSITY
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com