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UDP-glycosyl transferases capable of catalyzing carbohydrate chain extension, and applications thereof

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

Inactive Publication Date: 2018-12-07
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, except for the glycosyltransferase plant that extends a glucosyl group at the C3 position, other glycosyltransferases that can catalyze sugar chain extension in ginseng have not been reported.

Method used

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  • UDP-glycosyl transferases capable of catalyzing carbohydrate chain extension, and applications thereof
  • UDP-glycosyl transferases capable of catalyzing carbohydrate chain extension, and applications thereof
  • UDP-glycosyl transferases capable of catalyzing carbohydrate chain extension, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0214] Embodiment 1 Separation of glycosyltransferase and its coding gene

[0215] Ginseng RNA was extracted and reverse-transcribed to obtain ginseng cDNA. Using the cDNA as a template, use primer pair 1 (SEQ ID NO.:1 and SEQ ID NO.:2) or primer pair 2 (SEQ ID NO.:8 and SEQ ID NO.:9) to carry out PCR amplification to obtain 1.4- 1.5 kb amplification product. The DNA polymerase was selected from the high-fidelity KOD DNA polymerase of Treasure Bioengineering Co., Ltd. PCR products were detected by agarose gel electrophoresis.

[0216] Under UV irradiation, the target DNA band is excised. Then Axygen Gel Extraction Kit (AEYGEN Company) was used to recover DNA from the agarose gel, which was the amplified DNA fragment. The DNA fragment was ligated with the commercially available cloning vector pMD18-T Vector with the rTaq DNA polymerase of Treasure Bioengineering Co., Ltd. at the end, and the ligated product was transformed into commercially available Escherichia coli EPI300...

Embodiment 2

[0230] Example 2 Expression of Glycosyltransferase Gene GT29-32, GT29-33 and GT29-34 in Escherichia coli

[0231] Using the plasmids GT29-32-pMD18T, GT29-33-pMD18T and GT29-34-pMD18T constructed in Example 1 containing the GT29-32, GT29-33 and GT29-34 genes as templates, amplified with the primers shown in Table 1 Target genes GT29-32, GT29-33 and GT29-34.

[0232] After the expression vector pET28a (purchased from Merck) was digested with NcoI / SalI, GT29-32, GT29-33 and GT29-34 were cloned into pET28a (one-step cloning kit, purchased from Novizan), and Escherichia coli was constructed. Expression vectors GT29-32-pET28a, GT29-33-pET28a and GT29-34-pET28a. Using the 6×His tag sequence on pET28a, the C-terminals of the recombinant proteins GT29-32, GT29-33 and GT29-34 were tagged with 6×His tag. The plasmids were respectively transformed into commercially available E.coli BL21 to construct recombinant strains BL21-GT29-32, BL21-GT29-33 and BL21-GT29-34. Inoculate one recombin...

Embodiment 3

[0236] Embodiment 3GT29-32, the in vitro transglycosylation activity and product identification of GT29-33 and GT29-34

[0237]The supernatant of the cell lysate of recombinant Escherichia coli BL21-GT29-32, BL21-GT29-33 and BL21-GT29-34 in Example 2 was used as the crude enzyme solution to carry out the transglycosylation reaction, and the empty vector pET28a recombinant Escherichia coli Cell lysate served as a control.

[0238] Such as figure 2 Shown: Protopanaxadiol-type ginsenoside CK is used as the glycosyl acceptor, UDP-glucose is used as the glycosyl donor, and GT29-32 and GT29-34 can catalyze it to generate a new product;

[0239] Such as image 3 Shown: With ginsenoside Rd as the glycosyl acceptor and UDP-glucose as the glycosyl donor, GT29-32, GT29-33 and GT29-34 can catalyze the formation of Rb1. The results of HPLC were consistent with those of TLC.

[0240] Therefore, GT29-32 and GT29-34 can catalyze C20-O-Glc of CK to extend a molecule of glucose to generate...

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Abstract

The invention relates to a set of UDP-glycosyl transferases capable of catalyzing carbohydrate chain extension, and applications thereof, and more specifically provides a catalytic reaction used for obtaining ginsenoside products including ginsenoside Rb1, ginsenoside Rb3, gypenoside LXXV, gypenoside XVII, notoginsenoside U and, notoginsenoside R1, and notoginsenoside R2 through catalyzing of C-20site 1th glycosyl and C-6 site 1th glycosyl of tetracyclic triterpene substances with glycosyl transferases GT29-32, GT29-33, GT29-34, GT29-4, GT29-5, GT29-7, GT29-9, GT29-11, GT29-13, GT29-17, GT29-18, GT29-24, and GT29-25, and derived peptides thereof through carbohydrate chain extension. The glycosyl transferases can be used for construction of artificially synthesized ginsenosides and a plurality of novel ginsenosides and derivatives of ginsenosides.

Description

technical field [0001] The invention relates to the fields of biotechnology and plant biology, in particular, the invention relates to a group of glycosyltransferases and applications thereof. Background technique [0002] Ginsenoside is a general term for saponins isolated from plants of the genus Panax (such as ginseng, Panax notoginseng and American ginseng, etc.) and Gynostemma pentaphyllum, and is a class of triterpenoids. Ginsenosides are also known as ginsenosides, notoginseng saponins and gypenosides depending on their isolated source. Ginsenosides are the main bioactive components in these medicinal plants. Currently, about 150 saponins have been isolated. From the structural point of view, ginsenosides are mainly biologically active small molecules formed by glycosylation of saponins. There are only a limited number of saponins in ginsenosides, mainly protopanaxadiol and protopanaxatriol of the dammarane-type tetracyclic triterpenes, and oleananoic acid. After ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12P19/18C12P19/56C12N15/54C12N15/82
CPCC12N9/1051C12N15/8245C12P19/18C12P19/56C12N15/8243C12P33/00C12N9/1048C12N9/10C12N15/82
Inventor 周志华魏维严兴杨成帅魏勇军王平平
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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