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Avermectin producing bacteria and preparation method and application thereof

A technology for producing abamectin and bacteria, which is applied in the biological field and can solve problems such as low yield and complicated processing process

Active Publication Date: 2014-06-04
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the production of abamectin and ivermectin needs to be purified from natural fermentation products. The processing process is very complicated, the yield is low, and toxic solvents such as toluene are also used.
In addition, there is no strain whose fermentation product only has B2a components

Method used

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  • Avermectin producing bacteria and preparation method and application thereof
  • Avermectin producing bacteria and preparation method and application thereof
  • Avermectin producing bacteria and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Construction of Abamectin Biosynthesis Gene aveD Inactivation (aveD Point Mutation) Strain

[0140] 1. Construction of recombinant plasmids for aveD point mutation

[0141] The primer sequences of the upstream DNA fragments of the aveD gene in the avermectin biosynthetic gene cluster were cloned by PCR as follows:

[0142] Primer 1: 5'-TAT GAA TTC CCT CGT CGA GGT GGC CGA G-3' (the underline is the EcoRI site) (SEQ ID NO: 4)

[0143] Primer 2: 5'-TTA AAG CTT ACC GCA GCC GAC GTC CAG G-3' (underlined is HindIII) (SEQ ID NO: 5)

[0144] The primer sequences of the downstream DNA fragments of the aveD gene in the avermectin biosynthetic gene cluster cloned by PCR are as follows:

[0145] Primer 3: 5'-TTA AAG CTT AAG CCG GCG GTG CGG CTC G-3' (underlined as HindIII) (SEQ ID NO: 6)

[0146] Primer 4: 5'-TTA TCT AGA TCC TCA CCC TTT CCC CCG GC-3' (XbaI is underlined) (SEQ ID NO: 7)

[0147]Using the extracted total DNA of the avermectin-producing bacteria Streptom...

Embodiment 2

[0158] Construction of aveC aveD double mutant strain of abamectin biosynthesis gene

[0159] 1. Construction of aveC in-frame knockout plasmid

[0160] The primer sequence of the upstream DNA fragment of the aveC gene in the avermectin biosynthetic gene cluster cloned by PCR is as follows:

[0161] Primer 7: 5'-TTA GAA TTC CAT CAC GCT GCT GGA CTT CG-3' (the underline is the EcoRI site) (SEQ ID NO: 10)

[0162] Primer 8: 5'-ACG C CT GCA G GG CCA GAA ACA G-3' (underlined is PstI) (SEQ ID NO: 11)

[0163] The primer sequences of the downstream DNA fragments of the aveC gene in the avemectin biosynthetic gene cluster were cloned by PCR as follows:

[0164] Primer 9: 5'-TAT CTG CAG GTG AAT GCC GTG ATG TTC CTC-3' (underlined is PstI) (SEQ ID NO: 12)

[0165] Primer 10: 5'-TAT TCT AGA TGC TCT CCT CCA CCA CAT CCT C-3' (XbaI is underlined) (SEQ ID NO: 13)

[0166] Using the extracted total DNA of the avermectin-producing bacteria StreptomycesavermectiniusATCC31267 as a ...

Embodiment 3

[0175] Construction of meiC heterologous anaplerotic strain

[0176] 1. Construction of plasmids for heterologous complementation of meiC

[0177] The primer sequences for PCR cloning of meilingmycin biosynthetic gene meiC are as follows:

[0178] Primer 13: 5'-TAT GGA TCC CTG CGC AAT AGG CTC ACC AC-3' (the underline is the BamHI site) (SEQ ID NO: 16)

[0179] Primer 14: 5'-TAT TCT AGA CGG GAG GTG TCT ACA AGT GC-3' (the underline is the XbaI site) (SEQ ID NO: 17)

[0180] Using the extracted total DNA of the meilingmycin-producing bacterium Streptomyces nanchangensis as a template, the meilingmycin biosynthetic gene meiC was amplified by PCR with the above-mentioned pair of specific primers, and a 1.7kb complete meiC gene was obtained Fragmented PCR products. After the PCR product was digested with BamH-XbaI, it was connected to the commercially available BamH-XbaI of pSET152 (an integrated E. coli-Streptomyces shuttle plasmid, see US1,171,583) containing erythromycin...

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Abstract

The invention relates to avermectin producing bacteria and a preparation method and application thereof. Specifically, aveD gene in wild type avermectin producing bacteria is inactivated to obtain a strain getting rid of an A component and only producing a B component; by anaplerosis of a homologous gene of the aveC gene (preferably meiC gene in a Meilingmycin biosynthetic gene cluster) on the basis of simultaneous inactivation the aveD gene and aveC gene, 95wt% above of the obtained mutant fermentation product is avermectin B2a. The product avermectin B2a can be used as a precursor for being directly used for large-scale synthesis of high purity avermectin B1a and ivermectin.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to an abamectin-producing bacterium and a preparation method and application thereof. Background technique [0002] Avermectin is a class of sixteen-membered macrolide antibiotics produced by Gram-positive bacteria Streptomyces avermectinius. Abamectin is a highly efficient, pollution-free biopesticide with broad-spectrum insecticidal and anti-mite activity, and Abamectin has low toxicity and side effects to humans and animals, and low residual in the body. These characteristics make Abamectin parasitic in livestock It is widely used in the treatment of insect infection and the control of agricultural diseases and insect pests. It is the best medicine for the production of green food. [0003] Usually, during the fermentation process of abamectin, A components (A1a and A2a, etc.) and B components (B1a and B2a, etc.) are produced (see figure 1 ), with the B1a com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/62C12N15/76C12R1/465
Inventor 刘文赵群飞
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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