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Efficient screening method for abamectin high-producing strain

A technology of abamectin and high-yield strains, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of long culture period, cumbersome detection methods, and long detection time, etc., to achieve the improvement of spore production Effects of growth rate, prevention of poor extraction results, and simplified screening process

Inactive Publication Date: 2016-12-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The screening of existing avermectin bacterial strains generally adopts shake flask fermentation to detect with HPLC method, and there are defects such as long cultivation period, long detection time, and complicated detection methods

Method used

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  • Efficient screening method for abamectin high-producing strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: optimal solid plate culture medium selection

[0044] Five different formulations of solid plate medium were prepared, and the diluted sterile Streptomyces avermitilis suspension was coated on the solid plate medium. Observe the colony morphology and growth rate. It is found that Streptomyces avermitilis grows fast and has plump spores on No. 1 solid plate medium, which can effectively improve the efficiency of subsequent screening. However, Streptomyces avermitilis grows slowly on No. 2 solid plate medium, grows slowly on No. 3 slant medium, and does not grow on No. 4 and No. 5 slant surfaces.

Embodiment 2

[0045] Example 2: ARTP mutagenesis

[0046]The slant strain of the starting strain was cultured at 28°C for 5 days until the spores matured. Scrape an appropriate amount of spores into a test tube filled with 2 mL of sterile normal saline, shake well to make a bacterial suspension, and filter with sterile filter paper. Draw 10 μL of the bacterial suspension and evenly spread it on the surface of the sterile slide, place the slide on the platform of the ARTP mutagenesis instrument, and use the ARTP mutagenesis instrument for mutagenesis. Conditions: The incident power is 100W, the helium gas flow rate is 10SLM, irradiate for 0s, 10s, 20s, 30s, 40s, 50s, 60s, and 70s respectively. In EP tubes, after full shaking, serially dilute. Pipette 100 μL of the diluted solution and apply it on a solid plate (3 parallel groups for each group). The spores matured after being cultured in a constant temperature incubator at 28°C for 5 days. Count the number of colony-forming units on each...

Embodiment 3

[0047] Embodiment 3: Microplate reader detection

[0048] According to the fact that abamectin has the maximum light absorption at 240nm, but the blank culture medium has no absorption value at 240nm, the detection of abamectin by microplate reader was selected as the primary screening method. Configure a standard product with a certain concentration gradient (the concentration range of the standard product is 0-210 μg / mL), detect it with a microplate reader, and obtain the standard curve equation y=0.01781x+0.06292, R 2 =0.99913 (where y is OD 240nm , x is the concentration of avermectin standard substance, unit μg / mL). The pre-fermentation experiment in the 48-well plate showed that the titer of avermectin was around 1000 μg / mL. Draw 50 μL of the culture solution in the 48 deep-well plate, dilute the abamectin to the linear range of the standard curve with 95% ethanol, ultrasonically extract at 30KHZ, power 40W for 1min, centrifuge at 4000r / min for 15min, and take 200μL of...

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Abstract

The invention discloses an efficient screening method for an abamectin high-producing strain and belongs to the technical field of metabolic engineering. The method includes the steps that firstly, several different abamectin inclined mediums are compared, a proper inclined medium is selected for abamectin, and goodness of an original strain is guaranteed. ARTP mutation and other similar mutation treatment means are carried out on the original strain abamectin, a porous plate culture method and a microplate reader high-throughput test method are combined for use, the whole screening process is simplified, and screening efficiency is improved. A microplate reader preliminary screening method is verified, the pore plate OD value measured by a microplate reader and a pore plate tier obtained after efficient liquid detection are compared, it is found that good relevance is achieved, and therefore the preliminary screening feasibility and accuracy of the microplate reader are proved. The high-producing mutant strain of abamectin is screened out, and compared with original bacteria, the yield of abamectin of the mutant strain is increased by 10% or above on a shake flask and increased by 10% or above on a 30L fermentation tank.

Description

technical field [0001] The invention relates to a high-efficiency screening method for abamectin high-yield bacterial strains, belonging to the technical field of microbial production. Background technique [0002] Avermectins are lactone antibiotics produced by Streptomyces avermitilis and consist of 8 components A1a, A2a, A1b, A2b, B1a, B2a, B1b and B2b, of which B1a component has the effect of most. Abamectin is a highly lipophilic compound with high solubility in organic solvents, such as acetone, ethanol, methanol, and chloroform. As a broad-spectrum antibiotic, abamectin has the characteristics of low toxicity and high efficiency, and is widely used in agriculture, animal husbandry, and medicine. [0003] Abamectin, as a secondary metabolite of Streptomyces avermitilis, has a complex and huge network in its metabolism. Although the whole genome sequencing of abamectin has been completed, its biosynthesis process is complex, regulated at different levels, and its met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N13/00C12N15/01G01N21/31C12R1/465
CPCC12N1/20C12N13/00C12N15/01G01N21/31
Inventor 周景文陈坚曹晓梅堵国成曾伟主
Owner JIANGNAN UNIV
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