High-throughput screening method for high-organic acid yield strains

A high-yield strain and high-throughput technology, applied in the field of microorganisms, can solve the problems of tediousness and low efficiency, and achieve the effect of simplifying the screening process

Active Publication Date: 2015-02-18
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of organic acids generally uses gas chromatography or HPLC, which has the disadvantages of cumbersome and low efficiency to screen bacterial strains.
There are no related reports on high-throughput screening of α-ketoglutarate-producing strains

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1: ARTP mutagenesis

[0060] Spread the strains of Yarrowia lipolytica CCTCC NO: M207143 stored in a glycerol tube on the slope of an eggplant-shaped bottle and activate it for 24 hours, pick a ring of bacteria and insert it into the seed medium (50mL in a 500mL shaker bottle), 200r / min, Incubate at 28°C for 17-18h, take 1mL of bacterial suspension in a 1.5mL sterile centrifuge tube, centrifuge at 4500r for 10min, wash with normal saline twice, and then dilute with normal saline to make the bacterial concentration at 10 6 ~10 7 Spread the diluted bacterial suspension evenly on the surface of the sterile slide, place the slide on the stage, and use the ARTP mutagenesis breeding system to treat it with an incident power of 100W and a flow rate of helium Under the condition of 10SLM, irradiate for 10s, 20s, 30s, 40s, 50s, 60s, 90s, 120s respectively. The microorganisms attached to the bacteria slide were eluted into the liquid to form a new bacterial suspension. ...

Embodiment 2

[0061] Embodiment 2: the selection of developer

[0062] According to the change characteristics of pH value in the fermentation process of α-ketoglutaric acid, seven kinds of color reagents were selected as bright green, quinaldine red, orange IV, malachite green, thymol blue, Congo red and bromocresol green Pre-select the chromogen, and the pre-experiment in the 48-deep well plate shows that the output of α-ketoglutaric acid and pyruvic acid are both below 2g / L, so the fermentation medium is used as the solution, and α-ketoglutaric acid is added The gradient concentration of the amount of acid 0.6g / L-4.8g / L is dissolved in 200 μ L of fermentation medium, and the chromogen (1 / 400, 1 / 200, 1 / 100, 1 / 50, 1 / 25), react at room temperature for 30 minutes, and measure the absorbance value OD at the maximum visible light absorption value of various chromogens. The result shows that bromocresol green indicator just has obvious discoloration reaction under the low acid concentration, ...

Embodiment 3

[0063] Example 3: Screening of high-production α-ketoglutarate strains

[0064] Dilute the bacterial suspension after ARTP mutagenesis treatment for 30s to 10 -1 、10 -2 For two concentration gradients, draw 100 μL and spread evenly on the pre-screened plate, culture in a 200r / min, 28°C constant temperature incubator for 48 hours, pick the colony with a large colony shape and a large color change circle, and transfer it to the seeds in a 96-deep well plate Medium, 900r / min, 28 ℃ high-throughput deep-well plate constant temperature shaker for 48h, transfer to 48 deep-well plate with 10% inoculum size (load 1mL fermentation medium (volume after inoculation)), 900r / min, 28°C fermentation culture for 96h, centrifuge the 48 deep-well plate at 3500r / min for 10min, take 200μL supernatant in a 96-well plate, add 4μL quinaldine red pH indicator to react for 30min, and measure it with a microplate reader Absorbance value OD under visible light at 520nm 520 . Choose OD 520 Relatively...

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Abstract

The invention discloses a high-throughput screening method for high-organic acid yield strains, and belongs to the technical field of microbiology. According to the method, a pH indicator is combined with high-throughput screening, so that strains with higher organic acid yield can be screened from a great number of strains, the whole screening process is simplified, and the screening efficiency is improved; high-throughput screening combined with the pH indicator is also combined with a conventional breeding method to obtain a strain with high alpha-ketoglutaric acid yield, and compared with that of an original strain, the yield of the strain is improved by 51.8 percent on a shake flask, and is improved by 45.4 percent on a 3L fermentation tank.

Description

technical field [0001] The invention relates to a high-throughput screening method for high-yield strains of organic acids, belonging to the technical field of microorganisms. Background technique [0002] α-Ketoglutarate (α-KG) is a key intermediate metabolite in the tricarboxylic acid cycle and the main prerequisite substance in the metabolism of amino acids and proteins. It is widely used in food, medicine, agriculture and cosmetics and other fields. However, the defect of low production efficiency severely limits the industrial development and application of α-ketoglutarate production by fermentation. Therefore, the screening of high-yielding α-ketoglutarate strains is imminent. [0003] At present, gas chromatography or HPLC is generally used for the detection of organic acids, and there are disadvantages such as tediousness and low efficiency in screening bacterial strains by this method. There is no relevant report on high-throughput screening of α-ketoglutarate high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12R1/645
Inventor 周景文陈坚曾伟主李江华堵国成
Owner JIANGNAN UNIV
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