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Milbemycin synthetic positive-regulation gene kelR, encoding protein and genetically engineered bacterium and preparation method and application thereof

A technology of milbemycin and genetically engineered bacteria, which is applied in genetic engineering, botany equipment and methods, and methods based on microorganisms, can solve the problems of not finding regulatory genes, increase production, reduce production costs, and improve economic efficiency. benefit effect

Active Publication Date: 2018-11-06
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is only one positive regulatory gene milR in the milbemycin biosynthesis gene cluster. It has been confirmed that milR controls the biosynthesis of milbemycin by directly regulating the transcription of milA4 and milF, but directly regulates the regulation of milA1, milA2 and milA3. Gene still not found (Zhang Y et al. Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis. Microbialcell factories. 2016; 15, 152)

Method used

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  • Milbemycin synthetic positive-regulation gene kelR, encoding protein and genetically engineered bacterium and preparation method and application thereof
  • Milbemycin synthetic positive-regulation gene kelR, encoding protein and genetically engineered bacterium and preparation method and application thereof
  • Milbemycin synthetic positive-regulation gene kelR, encoding protein and genetically engineered bacterium and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Research on the function of milbemycin synthesis positively regulating gene kelR.

[0040] 1. Construction of milbemycin synthesis positive regulatory gene kelR blocking mutant strain

[0041] Using primers 6842up-f / 6842up-r from Streptomyces bingchenggensis BCWT (the Streptomyces bingchenggensis BCWT is recorded in Gao Aili, Wang Xiangjing, et al. (2007). "Screening and identification of new species of Streptomyces hygroscopicus." Northeast Agriculture University Journal 38(3):361-364.) PCR amplification on the genome obtained the homologous recombination left arm fragment of the kelR gene, ie the upstream fragment of kelR.

[0042] Using primers 6842down-f / 6842down-r, the right arm fragment of kelR gene homologous recombination, namely the downstream fragment of kelR, was obtained by PCR amplification from Streptomyces bingchenggensis BCWT genome.

[0043] The Streptomyces bingchenggensis BCWT genomic DNA was extracted according to the method described in ...

Embodiment 2

[0073] Example 2. Analysis of milbemycin synthesis of milbemycin synthesis positive regulatory gene kelR blocking mutant strain and anaplerotic mutant strain.

[0074] 1. Fermentation culture adopts the following medium

[0075] Seed medium (1L): sucrose 10g, skim milk powder 1g, peptone 3.5g, yeast extract powder 5g, K 2 HPO 4 ·3H 2 O0.5g, distilled water 1L, pH 7.0. 121°C, 1.01×10 5 Pa sterilized for 20 minutes.

[0076] Fermentation medium (1L): 80g sucrose, 20g soybean cake powder, 1g skimmed milk powder, K 2 HPO 4 ·3H 2 O 1g, FeSO 4 ·7H 2 O 0.1g, CaCO 3 3g, 1L distilled water, pH 7.0-7.2. 121℃, 1.01×10 5 Pa sterilized for 30 minutes.

[0077] 2. Fermentation process

[0078] The pre-prepared spores of Streptomyces bingchenggensis BCWT, the knockout mutant strain ΔkelR and the complementation mutant strain ΔkelR::kelR prepared above were activated by streaking on the MS plate, and scooped out about 2 cm 2 The mycelium block was inoculated into a Erlenmeyer fl...

Embodiment 3

[0089] Example 3. Construction of high-yielding milbemycin genetically engineered bacteria.

[0090] Transform the recombinant vector pSET152::kelR used in Example 1 into E.coli ET12567\pUZ8002 competent cells to obtain E.coli ET12567\pUZ8002 transformants containing pSET152::kelR, and transfer pSET152 :: kelR is transferred into Streptomyces bingchenggensis BC-120-4 bacterial strain (the BC-120-4 bacterial strain is recorded in Wang H et al. Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3 / A4as main components fromStreptomyces bingchenggensis.Applied microbiology and biotechnology.2014;98:9703-9712), the specific process is:

[0091] The competent state of Escherichia coli ET12567\pUZ8002 was prepared, and the preparation method was described in the Streptomyces Genetic Operation Manual. The constructed plasmid was transformed into competent cells of ET12567\pUZ8002; spread on the LB medium plate containing Apr antibiotics, cultiva...

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Abstract

The invention provides a milbemycin synthetic positive-regulation gene kelR, encoding protein and genetically engineered bacterium and a preparation method and application thereof, and belongs to thefield of microorganism pharmacy. According to the milbemycin synthetic positive-regulation gene kelR, the encoding protein and the genetically engineered bacterium and the preparation method and the application thereof, an overexpression vector containing the milbemycin synthetic positive-regulation gene and an upstream promoter sequence is constructed and transformed into escherichia coli, an obtained transformant and streptomyces bingchenggensis are subjected to intergeneric conjugational transferring, zygotes with apramycin resistance are picked, and then the kelR overexpression geneticallyengineered bacteria are obtained. The milbemycin synthetic positive-regulation gene kelR, the encoding protein and the genetically engineered bacterium and the preparation method and the applicationthereof are suitable for milbemycin biosynthesis.

Description

technical field [0001] The invention belongs to the field of microbial pharmacy, and in particular relates to a milbemycin synthesis positive regulation gene kelR, a protein encoded by the gene, a genetically engineered bacterium overexpressing kelR, a preparation method and application thereof. Background technique [0002] Biopharmaceuticals, especially macrolides, are being and are increasingly being used in veterinary medicine and agriculture. Milbemycin is one of the representative commercialized pesticides and veterinary drugs with broad-spectrum inhibition of pests and good eco-friendliness. Milbemycin is a class of sixteen-membered macrolide antibiotics with a chemical structure similar to abamectin. Milbemycin was first discovered in Japan in 1967, and was first registered as an acaricide in Japan for pest control of crops and commercial crops. It was then registered in Australia in 2006 as an acaricide against the flower-concentrating Tetranychus urticae. Up to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N1/21C12N15/76C12N15/66C07K14/36C12P17/18C12R1/465
CPCC07K14/36C12N15/66C12N15/76C12P17/181
Inventor 向文胜王海燕刘雨晴王相晶张继
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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