Genetically engineered bacterium used for biological catalysis of glucuronidation 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 aglycon compounds, achieve the effects of improving bioavailability, reducing toxic and side effects, and improving drug efficacy

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

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of low solubility of natural flavonoid aglycone compo

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0045] 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:

[0046] (1) Take 1-5ml of bacterial culture solution, centrifuge at 10,000rpm for 1min, and remove the supernatant.

[0047] (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.

[0048] (3) Add 20 μl of Proteinase K solution to the tube and mix well.

[0049] (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.

[0050] (5) Add 220 μl of absolute ethanol, shake and mix well for 15 sec.

[0051] (6) Add the solution and flocculent precipitate obtained in the previou...

Embodiment 2

[0057] Embodiment 2: Escherichia coli BL21 (DE3) PGM, the construction of the acquisition of GalU gene and expression plasmid

[0058] Acquisition of Escherichia coli PGM gene:

[0059] Based on the nucleic acid sequence of the gene database GenBank registration number EG12144, PCR reaction primers were designed and synthesized:

[0060] PGM_1:5′-ACGTTGCAGACAAAGGACAAAGCA-3′

[0061] PGM_2:5′-GATATA CCATGG CAATCCACAATCGTGCAG-3' (NcoI site is underlined)

[0062] PGM_3:5′-TGTGTG GCTAGC TTACGCGTTTTTCAGAACTTCGCTAAC-3' (NheI site is underlined)

[0063] PGM_4:5′-GCGTAGCGCATCAGGCAATTCTGT-3′

[0064] 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, 30c...

Embodiment 3

[0085] Embodiment 3: the construction of the acquisition of Escherichia coli BL21 (DE3) UGDH gene and expression plasmid

[0086] Acquisition of Escherichia coli UGDH gene:

[0087] The primers for PCR reaction were designed and synthesized based on the nucleic acid sequence of the American gene database GenBank registration number EG13407:

[0088] UGDH_1:5′-AATAAATATCAGCTATTCTTATAAAGAAAATCTG-3′

[0089] UGDH_2:5′-GGATCCCATATGAAAATCACCATTTCCGG-3′(NdeI site is underlined)

[0090] UGDH_3:

[0091] 5′-AAGCTT GTC GAC GGAGCTCGGATCCTAGTAAATCAATCAAATCAATCTGTTC-3′ (the underline is the SalI site)

[0092] UGDH_4:5′-CATCTTGCCACGCCACAACTGCACT-3′

[0093] Using the genomic DNA of Escherichia coli BL21 (DE3) as a template, the first round of PCR was carried out with primers UGDH_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 use primer UGDH_2 / 3 to carry out Nested-PCR amplificati...

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Abstract

The invention relates to genetically engineered organisms, especially to microbes like Escherichia coli with activity in catalysis of glucuronidation of flavonoids, and provides a genetically engineered bacterium used for biological catalysis of glucuronidation of flavonoids. The genetically engineered bacterium is prepared by coexpression of four genes respectively coding phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase, uridine diphosphate glucose dehydrogenase and uridine diphosphate glucuronyltransferase in a 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 glucuronidation 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 constructing a genetically engineered microbial cell capable of catalyzing the glucuronidation of flavonoid compounds by using synthetic biology technology. Background technique [0002] Flavonoids widely exist in nature and are the active ingredients of various medicinal plants. They mostly exist in the free state or in the form of glycosides combined with sugars in plants; as potential drugs, they have good antioxidant, anti-inflammatory, anti- Biological activities such as virus, anti-tumor and immune regulation. For example, the glucuronidated flavonoid scutellarin is widely used clinically for ischemic cardiovascular and cerebrovascular diseases such as cerebral thrombosis, coronary heart disease and angina pectoris. Products related to Erigeron lanceolata achieved an industrial sales revenue of about 3 billion yuan. Due to the low solubility of most flavonoi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/60C12R1/19
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 AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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