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Engineering bacterium of gynostemma pentaphyllum glycosyl transferase, and construction method and application thereof

A technology of glycosyltransferase and construction method, which is applied in the application field of the gynostemma glycosyltransferase in the synthesis of rare ginsenoside CK, can solve the problems of low yield, environmental pollution, complex ginsenoside process, etc., and achieve high yield High efficiency, simple process and low consumption

Active Publication Date: 2017-03-22
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the growth cycle of ginseng is long, and the process of extracting ginsenosides is complicated, the yield is low, and it is easy to cause environmental pollution.

Method used

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  • Engineering bacterium of gynostemma pentaphyllum glycosyl transferase, and construction method and application thereof
  • Engineering bacterium of gynostemma pentaphyllum glycosyl transferase, and construction method and application thereof
  • Engineering bacterium of gynostemma pentaphyllum glycosyl transferase, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Cloning of Glycosyltransferase UGTGp4 from Gynostemma pentaphyllum

[0045] The glycosyltransferase genes that may catalyze the glycosylation of protopanaxadiol were screened with the help of search tools based on compounds and chemical reactions, combined with NCBI and other database resources, and plant PSPG boxes. Using primers SEQ ID NO.1-SEQ ID NO.2, and using a polymerase to amplify the gene from the Gynostemma cDNA library by PCR. The amplified fragment was gel-cut and purified, and double-digested with XhoI and BamHI. The digested fragment was ligated with the plasmid pET-28a (+) that had also been double-digested with XhoI and BamHI. The carrier: the target fragment Mix at a molar ratio of 1:3, add T4 DNALigase, and perform enzyme ligation at 22°C for 5 hours. The ligation product is transformed into E.coliDH5α, and positive clones are screened on a Karna plate and verified by sequencing. The recombinant plasmid pET-28a(+)-comp20426 was obtained.

Embodiment 2

[0047] Establishment of Escherichia coli expression strain and purification of Gynostemma glycosyltransferase UGTGp4

[0048] The recombinant expression plasmid was transformed into the host strain E.coli BL21(DE3) (NEB Company) competent cells to obtain the recombinant expression strain of Gynostemma glycosyltransferase. When the recombinant bacteria were cultured until the OD was 0.6-0.8, 0.1 mM IPTG was added, and the expression was induced at a low temperature of 16° C. for 20 h. The cells were collected by centrifugation at 5500 rpm at 4°C, and the cells were ultrasonically disrupted. It was purified using Ni-NTA His-binding resin.

Embodiment 3

[0050] in vitro enzyme experiment

[0051] According to the preparation of the in vitro reaction system, the experiment of glycosyltransferase was carried out.

[0052]

[0053] After incubating the reaction mixture for 3 h at 30° C., the same volume of n-butanol was added to terminate the reaction.

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Abstract

The invention discloses an engineering bacterium of a gynostemma pentaphyllum glycosyl transferase, and a construction method and an application thereof. The gynostemma pentaphyllum glycosyl transferase can catalyze protopanoxadiol to synthesize rare ginsenoside CK and is named as UGTGp4. The invention also discloses a method for synthesizing the rare ginsenoside CK and an application. A gene of the glycosyl transferase from gynostemma pentaphyllum is heterologously expressed in escherichia coli, and functional identification is performed through in-vitro enzymatic reaction. A product is detected to show relatively higher signals by ESI mass spectrometry and HPLC. The ginsenoside prepared by the method has the advantages of high yield, less by-product, easy industrialized production and the like, and a foundation is laid for heterologous biosynthesis of gypenoside.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and relates to a method for synthesizing rare ginsenosides in vitro by expressing gypenosyltransferase heterologously in Escherichia coli, and also relates to the application of the gypenosyltransferase in synthesizing rare ginsenoside CK. Background technique [0002] Gynostemma Gynostemma is a perennial herbaceous vine of Cucurbitaceae Gynostemma Gynostemma. Gynostemma Gynostemma contains a variety of medicinal ingredients, the most important of which is Gypenoside. The structure of Gypenoside is a tetracyclic triterpene dammarane type, which is the same as that of ginsenoside. Gypenoside is composed of two parts, which is a biomacromolecule formed by the combination of glycoside and glycoside. So far, it has been found that eight kinds of gypenosides are identical in structure to protopanaxadiol-type ginsenosides, and the total amount of these eight kinds of saponins reaches about ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/10C12N15/54C12P33/20C12P33/02C12R1/19
CPCC12N9/1048C12N15/70C12P33/02C12P33/20
Inventor 王磊刘斌许莹莹徐艳丽田鑫黄笛
Owner NANKAI UNIV
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