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Genetically engineered bacterium for catalyzing glucose glycosylation of flavonoids compound and application thereof

A technology of flavonoids and genetically engineered bacteria, applied in the field of genetic engineering, can solve problems such as accumulation of unfavorable flavonoid glucoside products and increase in yield, and achieve the effects of improving bioavailability, improving water solubility, and improving drug efficacy

Active Publication Date: 2017-09-15
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

[0011] One object of the present invention is to solve the problem of β-glucoside hydrolase hydrolyzing flavonoid glucoside in Saccharomyces cerevisiae cells, and to provide a Saccharomyces cerevisiae project that reduces or eliminates the hydrolysis of flavone-β-glucoside by β-glucoside hydrolase in host cells cells, because the host cells have high endogenous β-glucoside hydrolase activity, which is not conducive to the accumulation and yield increase of flavone glucoside products synthesized by biocatalysis

Method used

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  • Genetically engineered bacterium for catalyzing glucose glycosylation of flavonoids compound and application thereof
  • Genetically engineered bacterium for catalyzing glucose glycosylation of flavonoids compound and application thereof
  • Genetically engineered bacterium for catalyzing glucose glycosylation of flavonoids compound and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: Preparation of Saccharomyces cerevisiae engineering bacteria

[0065] Step 1: Genomic DNA extraction of Saccharomyces cerevisiae: Use the Yeast Genomic DNA Extraction Kit of Beijing Kangwei Century Biotechnology Co., Ltd. to extract genomic DNA. The specific operation steps are as follows:

[0066] (1) Pick a single clone and inoculate it in 10mL liquid YPD (10g / L yeast extract, 20g / L tryptone, 20g / Lglucose) medium, culture at 30°C, 250rpm for 15-20h (logarithmic phase).

[0067] (2) 12000rpm, 1min multiple centrifugation to collect the bacterial cells in a 1.5mL EP tube (4-5 times), and discard the supernatant by centrifugation.

[0068] (3) Removal of yeast cell wall: add 600 μL Lyticase Working Buffer (before use, add β-mercaptoethanol to make the final concentration 0.1%), and add 5 μL Lyticase (10 U / μL), mix well, 30 Treat at ℃ for 30 min, centrifuge at 4000 rpm for 10 min, discard the supernatant, and collect the precipitate.

[0069] (4) Add 200 μ...

Embodiment 2

[0145] Example 2: Analysis of the activity of scutellarein catalyzed by glycosyltransferase SbGT34 in engineering bacteria knocked out of glycoside hydrolase

[0146] Engineering bacteria W303-1b / ES / SbGT34 was the experimental group. Based on the original bacteria W303-1b, the engineered bacteria W303-1b / SbGT34 containing only the glycosyltransferase SbGT34 was obtained by LiAc transformation; The gene SbGT34 was used to obtain the engineering strain W303-1b / ES / SbGT34. Using scutellarein as a substrate to identify the changes in the glycosylation activity of flavonoid substrates before and after the completion of knockout of glycoside hydrolase, the specific method is as follows:

[0147] (1) The single clone generated by transformation was inoculated in YPD liquid medium, 30°C, 220rpm, cultured to OD 600 to around 3.0.

[0148] (2) Transfer to SC synthetic medium according to 1% inoculum amount, and cultivate for about 10 h at 30° C. and 220 rpm.

[0149] (3) Regulate the...

Embodiment 3

[0153] Example 3: Effects of Saccharomyces cerevisiae sugar metabolism pathway PGM2 and UGP1 genes on conversion rate of flavone substrates catalyzed by engineering bacteria

[0154] The engineering bacteria W303-1b / ES / PU / SbGT34 was used as the experimental group, and the engineering bacteria W303-1b / ES / SbGT34 was used as the control group to identify the effects of PGM2 and UGP1 genes on the conversion rate of flavonoid substrates catalyzed by engineering bacteria. Using scutellarein as a substrate to identify the changes in the glycosylation activity of flavonoid substrates before and after the completion of knockout of glycoside hydrolase, the specific method is as follows:

[0155] (1) The single clone generated by transformation was inoculated in YPD liquid medium, 30°C, 220rpm, cultured to OD 600 to around 3.0.

[0156] (2) Transfer to SC synthetic medium according to 1% inoculum amount, and cultivate for about 10 h at 30° C. and 220 rpm.

[0157] (3) Regulate the biom...

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Abstract

The invention discloses a genetically engineered bacterium for catalyzing glucose glycosylation of a flavonoids compound and an application thereof. Specifically, a Beta-glycoside hydrolases gene of a chassis biological cell is removed according to the genetically engineered bacterium, and endogenous degradation of a glycosylation product is reduced; the high-expression regulation is performed to two key enzymes of glucophosphomutase (PGM) and uridine diphosphate glucose pyrophosphorylase (UGP1) of the chassis biological cell for participating in the synthesis of glycosylated-donor uridine diphosphate glucose (UDP-Glu), and a UDP-glucuronosyltransferase gene for catalyzing the flavonoid aglycones glucose glycosylation is combined so as to obtain a genetically engineered strain for effectively catalyzing the glucose glycosylation of the flavonoids compound, and finally scutellarein is used as a catalytic substrate for fermenting for 54 h in 10 L of a fermentation tank, wherein the yield of scutellarein-7-O-glucose of the glycosylation product is up to 1200 mg / L. The used strategy provides a technology reference for biologically-catalyzing chemical micromolecular glycosylation by using a microbiological method.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the construction of a genetically engineered bacterium capable of catalyzing the glucosidation of flavonoid compounds by using synthetic biology technology and its application. technical background [0002] Flavonoids (flavonoids) are polyphenolic compounds that widely exist in nature. They first referred to a class of compounds derived from 2-phenylchromone (flavone) as the mother nucleus, and now generally refer to two benzene rings (A ring A series of compounds with C6-C3-C6 as the basic skeleton formed by connecting with each other through the middle three-carbon ring (C ring). Flavonoids are the active ingredients of various medicinal plants. For example, baicalein is the main medicinal active ingredient of the plant Scutellaria baicalensis. It exists in the free state or in the form of glycosides combined with sugar in plants, and has various physiological and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P19/60C12R1/865
CPCC12N9/1051C12N9/2402C12P19/60C12Y204/01
Inventor 王伟王惠敏杨燕林霖周文龙刘忞之田雷瑜
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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