Genetically engineered bacterium used for biological catalysis of glucosidation of flavonoids

A technology of genetically engineered bacteria and glucose coke, which is applied in the field of genetically engineered microbial cells, can solve the problems of low solubility of natural flavonoid aglycone compounds, and achieve the effects of improving bioavailability, increasing drug efficacy, and reducing side effects

Active Publication Date: 2015-11-25
INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Another object of the present invention is to solve the problem of low solubility of natural flavonoid aglycon compounds, and provide a genetically engineered microbial cell that biocatalyzes the glucosidation of flavonoid compounds

Method used

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  • Genetically engineered bacterium used for biological catalysis of glucosidation of flavonoids
  • Genetically engineered bacterium used for biological catalysis of glucosidation of flavonoids
  • Genetically engineered bacterium used for biological catalysis of glucosidation of flavonoids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Escherichia coli genomic DNA extraction: extract the genomic DNA of Escherichia coli BL21 (DE3) with reference to the bacterial genome extraction kit of Tiangen Biochemical (Beijing) Co., Ltd., the specific steps are as follows:

[0051] (1) Take 1-5ml of bacterial culture solution, centrifuge at 10,000rpm for 1min, and remove the supernatant.

[0052] (2) Add 200 μl of buffer GA to the cell pellet, shake until the cells are completely suspended; add 4 μl of RNaseA (100 mg / ml) solution, shake for 15 sec, and place at room temperature for 5 min.

[0053] (3) Add 20 μl of Proteinase K solution to the tube and mix well.

[0054] (4) Add 220 μl buffer GB, shake for 15 sec, place at 70°C for 10 min, the solution becomes clear, and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0055] (5) Add 220 μl of absolute ethanol, shake and mix well for 15 sec.

[0056] (6) Add the solution and flocculent precipitate obtained in the previou...

Embodiment 2

[0062] Example 2 Escherichia coli BL21 (DE3) PGM, the acquisition of GalU gene and the construction of expression plasmid

[0063] Acquisition of Escherichia coli PGM gene:

[0064] Based on the nucleic acid sequence of the gene database GenBank registration number EG12144, PCR reaction primers were designed and synthesized:

[0065] PGM_1:5′-ACGTTGCAGACAAAGGACAAAGCA-3′

[0066] PGM_2:5′-GATATA CCATGG CAATCCACAATCGTGCAG-3' (NcoI site is underlined)

[0067] PGM_3:5′-TGTGTG GCTAGC TTACGCGTTTTTCAGAACTTCGCTAAC-3' (NheI site is underlined)

[0068] PGM_4:5′-GCGTAGCGCATCAGGCAATTCTGT-3′

[0069] Using the genomic DNA of Escherichia coli BL21 (DE3) as a template, the first round of PCR was carried out with primers PGM_1 / 4 (95°C, 5min; 95°C, 50S, 50°C, 1min, 72°C, 1.5min, 30cycles; 72°C, 10 min; 4°C, 10 min). Then use the first-round PCR product as a template, and carry out Nested-PCR amplification with primers PGM_2 / 3 (95°C, 5min; 95°C, 50S, 50°C, 1min, 72°C, 1.5min, 30cycle...

Embodiment 3

[0090] Embodiment 3: the construction of gene cloning and expression vector encoding UGT in the present invention

[0091] Cloning of Scutellaria baicalensis UGT gene:

[0092] Firstly, the Scutellaria baicalensis seeds sterilized by 70% ethanol and 0.1% mercuric chloride were cultivated in hormone-free plant tissue culture medium (MS solid medium) to obtain sterile seedlings; the total RNA of Scutellaria baicalensis was purified by QIANGENRNeasyPlantMiniKit, referring to American Gene Synthetic primers for the nucleic acid sequence of the database registration number AB031274:

[0093] UGT1: 5′-GATTCCATTATAGAATCTTGAAATG-3′

[0094] UGT2: 5′-ATCTTGAC GAATTCATG GGACAACTCCACATAGTCC-3′ (underline is the EcoRI site)

[0095] UGT3: 5′-CAAGAACC CTCGAG GTTTAAGCCCTGTTTCATAGGAGG-3' (XhoI site is underlined) UGT4: 5'-CGTAATTTATATGAGACAAGAACC-3'

[0096] UGT5: 5′-TCATGTGTGGCACCAAATCCACA-3′

[0097] Using UGT5 as the leading primer and using Invitrogen's GeneRacer TM RACEKit perf...

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Abstract

The invention relates to genetically engineered organisms, especially to microbes like Escherichia coli with activity in catalysis of glucosidation of flavonoids, and provides a genetically engineered bacterium used for biological catalysis of glucosidation of flavonoids. The genetically engineered bacterium is prepared by coexpression of three genes respectively coding phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase and uridine diphosphate glucuronosyltransferase in a microbial cell and introduction of the functional enzyme genes into the cell through expression vectors. The genetically engineered bacterium induces expression of functional enzyme protein under the condition of addition of an inductive agent--isopropyl thiogalactoside (IPTG) and is directly used for biological catalysis of glucosidation of flavonoids; and the advantages of good cell growth, a short fermentation period and low cost are obtained.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the construction of a genetically engineered microbial cell capable of catalyzing the glucosidation reaction of flavonoid compounds by using synthetic biology technology. Background technique [0002] Flavonoids widely exist in nature and are active ingredients of various medicinal plants. They mostly exist in the free state or in the form of glycosides combined with sugars in plants; many flavonoids have good antioxidant, anti-inflammatory and anti-viral properties. , anti-tumor and immune regulation and other biological activities have been used clinically, such as rutin (rutin) has the effect of regulating vascular permeability and similar to vitamin P, and has become a legal drug in many countries; baicalin (baicalin) has been used in my country Made into injections and used as antibacterial drugs. Due to the low solubility of most flavonoid aglycones and some fl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/60C12P19/58C12P19/44C12R1/19
Inventor 王伟吴松杨燕童元峰王慧敏林霖唐亮陈成娟刘忞之程克棣孔建强
Owner INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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