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Glycosyl transferases and applications of glycosyl transferases

A technology of glycosyltransferase and glycosyl transfer, applied in the field of biotechnology and plant biology

Active Publication Date: 2014-12-24
SYNBIOTECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] At present, there is still a lack of an effective method for producing rare ginsenosides CK, F1, Rh1, Rh2 and Rg3 in this field, so it is urgent to develop a variety of specific and efficient glycosyltransferases

Method used

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  • Glycosyl transferases and applications of glycosyl transferases
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  • Glycosyl transferases and applications of glycosyl transferases

Examples

Experimental program
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Effect test

Embodiment 1

[0390] Isolation of Glycosyltransferases and Their Encoding Genes

[0391] More than 100 cDNA sequences of predicted glycosyltransferases were extracted from the published expression profile data of Panax genus plants, from which 60 full-length cDNA sequences were cloned and their expression and transglycosylation reactions were analyzed, among which 11 expressed The product has transglycosylation activity to ginsenogenin and saponin.

[0392] Ginseng RNA was extracted and reverse-transcribed to obtain ginseng cDNA. Carry out PCR amplification with this cDNA as template, use wherein primer pair 1 (SEQ ID NOs.:7,8); Primer pair 2 (SEQ ID NOs.:9,10); Primer pair 3 (SEQ ID NOs.:11 , 12); primer pair 5 (SEQ ID NOs.:34,35); primer pair 7 (SEQ ID NOs.:46,47); primer pair 8 (SEQ ID NOs.:62,63); primer pair 9 ( SEQ ID NOs.: 64, 65) all obtained amplified products. The DNA polymerase was selected from the high-fidelity KOD DNA polymerase of Treasure Bioengineering Co., Ltd. PCR pro...

Embodiment 2

[0421] Construction of Yeast Recombinant Expression Vectors of Glycosyltransferase Genes gGT25, gGT25-1, gGT25-3 and gGT25-5

[0422] Using the plasmids gGT25-pMD18T, gGT25-1-pMD18T, gGT25-3-pMD18T and gGT25-5-pMD18T constructed in Example 1 containing gGT25, gGT25-1, gGT25-3 and gGT25-5 genes as templates to amplify the target gene .

[0423] The forward primers used were:

[0424] 5'-GCCGGAGCTCATGAAGTCAGAATTGATATTC-3'(SEQ ID NO.:13), a SacI recognition site is added to the 5' end: GAGCTC;

[0425] The reverse primers used were:

[0426] 5'-GCCGCTCGAGTTAATGATGATGATGATGATGCATAATTTCCTCAAATAGCTTC-3'(SEQ ID NO.:14), XholI recognition site: CTCGAG was added to the 5' end, and 6×His Tag was introduced into the reverse primer for Western Blot detection, expression and purification.

[0427] The gGT25, gGT25-1, gGT25-3 and gGT25-5 genes were amplified by the PCR method using the above primers and templates. The high-fidelity DNA polymerase kod from Toyobo Company was selected as ...

Embodiment 3

[0429] Expression of Glycosyltransferase Genes gGT25, gGT25-1, gGT25-3 and gGT25-5 in Saccharomyces cerevisiae

[0430] The constructed expression plasmid gt25-pYES2 was transformed into Saccharomyces cerevisiae by electroporation, and spread on the screening plate SC-Ura (0.67% basic nitrogen source without amino acid for yeast, 2% glucose). Yeast recombinants were verified by colony PCR. The yeast recombinant colonies were picked and cultured in 10 mL of SC-Ura (2% glucose) medium at 200 rpm at 30° C. for 20 h. Collect the bacteria by centrifugation at 3500g at 4°C, wash the bacteria twice with sterile deionized water, resuspend the bacteria with induction medium SC-Ura (2% galactose), and inoculate into 50mL induction medium to make the OD 600 At about 0.4, the expression was induced at 30°C and 200rpm. Centrifuge at 3500g at 4°C to collect the cells induced to express for 12 hours, wash the cells twice with sterile deionized water, resuspend them in yeast lysis buffer, a...

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Abstract

The invention relates to a group of glycosyl transferases and applications of the glycosyl transferases. Particularly, the invention provides applications of glycosyl transferases gGT25, gGT13, gGT30, gGT25-1, gGT25-3, gGT25-5, gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-7, 3GT1, 3GT2, 3GT3 and 3GT4, and derived peptides of the glycosyl transferases in glycosylation catalysis and new saponin synthesis of terpenoids. The glycosyl transferases can specifically and efficiently catalyze hydroxyl glycosylation of C-20 bit and / or C-6 bit and / or C-3 of a tetracyclic triterpenoid compound substrate, and / or transfers the glycosyl groups from glycosyl donors to the first glycosyl groups of the C-3 bit and C-6 bit of the tetracyclic triterpenoid compound to extend a carbohydrate chain. The glycosyl transferases disclosed by the invention can also be applied to building synthetic rare ginsenosides and a plurality of novel ginsenosides and derivatives of the ginsenosides.

Description

technical field [0001] The invention relates to the fields of biotechnology and plant biology, in particular, the invention relates to a glycosyltransferase and its application. Background technique [0002] Ginsenoside is a general term for the saponins isolated from ginseng and its congeners (such as Panax notoginseng, American ginseng, etc.), belonging to triterpenoid saponins, and is the main active ingredient in ginseng. At present, at least 60 saponins have been isolated from ginseng, some of which have been proven to have a wide range of physiological functions and medicinal value: including anti-tumor, immune regulation, anti-fatigue, heart protection, liver protection and other functions. [0003] From a structural point of view, ginsenosides are biologically active small molecules formed by glycosylation of saponins. There are only a limited number of saponins in ginsenosides, mainly dammarane-type protopanaxadiol and protopanaxatriol, and oleanolic acid. In rece...

Claims

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Application Information

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IPC IPC(8): C12P33/20C12N9/10C12N15/54C12N15/70C12N5/10A01H5/00
Inventor 周志华严兴王平平魏勇军魏维范云
Owner SYNBIOTECH (SUZHOU) CO LTD
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