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A genetically engineered strain of Saccharomyces cerevisiae, a method for constructing the strain, and a method for producing eriodictyol or quercetin

A technology for genetically engineering strains and Saccharomyces cerevisiae, which is applied in the field of efficient production of cyclic polyhydroxy flavonoids, can solve the problems of high price, high cost, pollution of organic reagents and heavy metals, etc., and achieves the effects of high quality and high level of enzyme activity expression.

Inactive Publication Date: 2016-02-10
安徽清水河生态农业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current market quotation of eriodictyol on the Sigma official website is 907 yuan per 1mg (purity is 99%), the price is relatively high, and quercetin is hardly sold in the market now
[0005] At present, the method of extracting quercetin and eriodictyol is mainly from lemon or peanut shells (Yao Li, HPLC determination of 5,7-dihydroxychromone and eriodictyol in peanut shells, Food Science; Du Fangling, Simultaneous detection of 5,7-dihydroxychromanone, eriodictyol and luteolin in peanut shells, etc.) for natural extraction, these methods are costly, easily cause organic reagents and heavy metal pollution, and cannot obtain high purity tea polyphenols, it is difficult to obtain higher purity and ideal yield

Method used

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  • A genetically engineered strain of Saccharomyces cerevisiae, a method for constructing the strain, and a method for producing eriodictyol or quercetin
  • A genetically engineered strain of Saccharomyces cerevisiae, a method for constructing the strain, and a method for producing eriodictyol or quercetin
  • A genetically engineered strain of Saccharomyces cerevisiae, a method for constructing the strain, and a method for producing eriodictyol or quercetin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Above upstream primer VvF3'5'H-pENTR-F

[0040] (sequence: CACCATGGCCATAGATACAAGCCTCTTGC) and downstream primer CZF3'5'H-R

[0041] (Sequence: AGCTAGAGCAACATGTGGCATGTTACCTAGAAGAGGAAGAGCGCCG) is used as amplification primer, and the plasmid of VvF3'5'H gene is used as template for PCR amplification to obtain the DNA of recombinant CZF3'5'H gene, which is common to the DNA of yF3'5'H gene Template, using the upstream primer VvF3'5'H-pENTR-F (sequence: CACCATGGCCATAGATACAAGCCTCTTGC) and the downstream primer yF3'5'H-R (AGCAGCATAAGCATTTGGAGGCAATC) as amplification primers for overlapping PCR amplification to obtain the CZyF3'5'H gene, and It is cloned in pENTR TM / TEV / D-TOPO carrier to obtain the ligation reaction.

[0042] The above ligation reaction was transferred into E.coliDH5α host cells, and the successful transformants were screened with kanamycin, and their plasmids were extracted as entry vectors.

[0043] The entry vector was LR-exchanged with pYES-dest52, and...

Embodiment 2

[0050] The strain was inoculated in 20 ml of Ura-deficient SD medium, cultured overnight at 28° C., 200 rpm, until OD600 = 0.5.

[0051] Ura-deficient SD medium: weigh SD0.7g, YNB3.4g, Glucose10g, Trp0.05g, Ade0.05g, His0.05g, Leu0.05g, add water to make up to 500mL, and sterilize with high-pressure steam at 115℃ for 15min. Solid medium needs to add 1.5%-1.8% agar powder.

[0052] The obtained bacteria were washed with Ura-deficient SD induction medium, and induced in 20 ml of Ura-deficient SD induction medium at 28° C., 200 rpm for 5 hours, and the initial OD600=0.5. Among them, the Ura-deficient SD induction medium consists of: 8g powdery medium (Universal catalog number: YGM0003A) is added to 950ml of water, sterilized by high-pressure steam at 115°C for 15min, and then 50ml of 40% filter-sterilized aseptic galactose is added. .

[0053] The reaction substrate naringenin (Naringenin) was added to the above-mentioned induced medium, and the culture was continued for 10 h.

...

Embodiment 3

[0059] 20mL of the bacterial liquid after the above-mentioned final induction culture was ultrasonically broken for 10min, centrifuged at 12000g for 10min, the supernatant was taken, extracted with 3 times the volume of ethyl acetate, and the obtained supernatant was dried by a rotary evaporator and then used a 100ul chromatographic Dilute to volume with methanol, then filter the obtained extract through a 0.22um pore size organic phase filter, and wait for HPLC detection. At the same time, the induced empty strain WAT11 was used as the control group.

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Abstract

The invention discloses a saccharomyces cerevisiae gene engineering strain, a construction method of the same and a method for producing eriodictyol or quercetin. The strain disclosed by the invention carries a CZyF3'5' gene and can be used for efficiently expressing CZyF3'5'H flavonoid-3',5'-hydroxylase and producing eriodictyol or quercetin.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae genetically engineered strain carrying a recombinant gene capable of expressing flavonoid-3', 5'-hydroxylase and a method for efficiently producing cyclic polyhydroxy flavonoids using the strain, belonging to the technical field of bioengineering . Background technique [0002] 5, 7, 3', 4', 5'-Quercetin, also known as quercetin or onion, has the strongest antioxidant effect among the hundreds of known bioflavonoids, and its antioxidant power is the highest among vitamins 50 times that of E, 20 times that of vitamin C, and its molecular structure is small, good water solubility, easy to be absorbed by the human body. And it can protect the inner wall of blood vessels from damage, and is of great help to the health of the heart and blood vessels. [0003] Eriodictyol (Eriodictyo1), also known as eriodictyol in North America, or eriodictyol, is a flavonoid. Similar to quercetin, it also has strong an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P17/06C12R1/865
Inventor 王云生许玉娇夏涛高丽萍胡晓婧
Owner 安徽清水河生态农业有限公司
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