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Genetically engineered bacterium producing 5-keto-22,23-dihydro doramectin and preparation method and application of bacterium

A technology of dihydrodoramectin and genetically engineered bacteria is applied to the genetically engineered bacteria producing 5-keto-22,23-dihydrodoramectin and the fields of preparation and application thereof, and can solve the problems of loss and the like, Achieve the effect of improving production efficiency, reducing production costs and reducing negative impacts

Active Publication Date: 2016-07-13
NORTHEAST AGRICULTURAL UNIVERSITY
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  • 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

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  • Genetically engineered bacterium producing 5-keto-22,23-dihydro doramectin and preparation method and application of bacterium
  • Genetically engineered bacterium producing 5-keto-22,23-dihydro doramectin and preparation method and application of bacterium
  • Genetically engineered bacterium producing 5-keto-22,23-dihydro doramectin and preparation method and application of bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the construction of genetically engineered bacteria StreptomycesavermitilisMil

[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 Streptomycesavermitilis NEAU1069 genome to obtain the homologous recombination left arm fragment encoding the DH2-KR2 gene in the abamectin biosynthesis gene cluster.

[0050] The total system of the PCR reaction was 25 μL, including 2.5 μL of 10×pfuBuffer, 2 μL of dNTP, 1 μL of Streptomycesavermitilis NEAU1069 genomic DNA, 2 μL of each primer a1 and primer a2, 2 μL of pfuDNA polymerase (5U / μL), 2.5 μL of DMSO, and 6 μL of water. 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 e...

Embodiment 2

[0061] Embodiment 2, the construction of genetically engineered bacteria StreptomycesavermitilisMil-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 Streptomycesavermitilis NEAU1069 genome to obtain the homologous recombination left arm fragment of the aveF gene in the abamectin biosynthesis gene cluster.

[0064] The total PCR reaction system was 25 μL, including 10×pfuBuffer 2.5 μL, dNTP 2 μL, Streptomycesavermitilis NEAU1069 genomic DNA 1 μL, primer aveF-F1 and primer aveF-R1 2 μL each, pfuDNA 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, the target band ...

Embodiment 3

[0073] Example 3, Fermentation of genetically engineered bacteria StreptomycesavermitilisMil and StreptomycesavermitilisMil-AveF and MS identification of target compounds

[0074] Pick the original strain Streptomycesavermitilis NEAU1069, the genetically engineered strains StreptomycesavermitilisMil and StreptomycesavermitilisMil-AveF, respectively, and culture them on the Gaoshi No. CoCl 2 ·6H 2 O0.01 (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 O0.1%, 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 volume of methanol and soaked fo...

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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

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Application Information

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Patent Type & Authority Applications(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
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