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Method for detecting eriodictyol with high flux

An eriodictyol, high-throughput technology, applied in the field of synthetic biology, can solve problems such as high time and labor costs, affect product detection, and difficult to achieve rapid detection, and achieve improved screening efficiency, high accuracy, and easy acquisition and production. Effect

Inactive Publication Date: 2019-02-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the HPLC detection method has low throughput and high time and labor costs
During pathway transformation, especially random mutations, thousands to hundreds of millions or more mutants will be generated, and it is difficult to achieve rapid detection only by HPLC and other means
Moreover, when microbial fermentation produces eriodictyol, there is naringenin in the intermediate product, or naringenin is directly used as a precursor, and the existence of these precursors may also affect the detection of the corresponding product

Method used

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  • Method for detecting eriodictyol with high flux
  • Method for detecting eriodictyol with high flux
  • Method for detecting eriodictyol with high flux

Examples

Experimental program
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Effect test

Embodiment 1

[0036] 1 High-throughput color reaction

[0037] Configure naringenin and eriodictyol mother solution, dissolve in 100% ethanol solution, and configure 4g / L standard solution. Then prepare different concentrations of naringenin and eriodictyol mixtures, dissolve and dilute them with water or YNB medium respectively, and make up to 100 μL, and then add 100 μL of 4M sodium hydroxide solution and 4M potassium hydroxide solution respectively. Observe the color reaction after reacting at room temperature for 5 minutes. The color of the reaction solution changes as Figure 1-4 As shown, the higher the eriodictyol concentration, the deeper the purple color and the faster the color development.

[0038] 2 Reaction solution full wavelength scanning

[0039] From the reaction solution treated with 4M potassium hydroxide and 4M sodium hydroxide, select the final concentration of naringenin as 50mg / L, the final concentration of eriodictyol as 50mg / L, and the final concentration of nari...

Embodiment 2

[0044] Saccharomyces cerevisiae C800 (CENPK2-1D, Δgal80::G418) was used for fermentation and plasmid construction. Escherichia coli JM109 was used for plasmid storage and amplification. pY26-P GPD -F3'H-P TEF -CPR is a shuttle plasmid, used as a control plasmid and a starting plasmid, and the screening label in Saccharomyces cerevisiae is uracil. pY26-P GPD -F3'H-P TEF -CPR can be simultaneously amplified and replicated in Saccharomyces cerevisiae and Escherichia coli, wherein the F3'H gene is transcribed by the promoter GPD, and the CPR gene is transcribed by the promoter TEF.

[0045] 1 Plasmid construction and high-throughput detection process

[0046] Select 20 promoters of different strengths from Saccharomyces cerevisiae, and divide them into two groups of AB, 10 promoters in each group. There are three promoters in each group, which can realize the expression of different strengths of corresponding genes, and generate 100 combinations (10×10 ). Among them, group ...

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Abstract

The invention discloses a method for detecting eriodictyol with high flux and belongs to the technical field of synthetic biologics. A developing agent used in the method is sodium hydroxide or potassium hydroxide, after developing treatment, eriodictyol is purple, the higher the concentration is, the darker the purple is, the longer the developing is, the longer the standing time is, the color isalso darkened, the reaction is completed within 5 minutes generally, and high-yield strains can be still selected by using a visual method directly even in the presence of an interfering substance naringenin. The reaction liquid can be further detected by using a microplate reader. In a water solution, NaOH is recommended for treatment, the optimal detection wavelength is 550-580nm, in a colorless synthesis culture medium, KOH (Potassium Hydroxide) is recommended for treatment, and the optimal detection wavelength is 560-570nm. The method is simple, convenient and efficient, good in interference prevention, high in precision, low in time and labor cost and applicable to high-flux screening of high yield strains of eriodictyol.

Description

technical field [0001] The invention relates to a method for high-throughput detection of eriodictyol, which belongs to the technical field of synthetic biology. Background technique [0002] Eriodictyol is synthesized by naringenin at the 3' position and is an important health product. Most of eriodictyol is extracted from plants, the output is low and the price is high. In microbial synthesis, the enzyme that catalyzes the synthesis of eriodictyol from naringenin is the P450 enzyme, which requires flavonoid 3'-hydroxylase (F3'H) and its coenzyme (CPR) to cooperate to transfer electrons to complete the hydroxylation, and the operation is more complicated. When transforming microorganisms to synthesize eriodictyol, high-performance liquid chromatography (HPLC) is often used to detect its content. Before detection, it is necessary to use a 0.22 μm filter head to filter, and then use chromatographically pure methanol-water (45:55, V / V ) solution is used as the mobile phase, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78G01N33/53
CPCG01N21/78G01N33/53
Inventor 周景文陈坚高松徐晓宇曾伟主堵国成吕云斌
Owner JIANGNAN UNIV
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