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Glycosyl transferases and method of catalyzing sugar chain extension

A technology of glycosyltransferase and glycosyl transfer, which is applied in the fields of biotechnology and plant biology, and can solve the problems of low activity and inability to fully meet the needs of applications.

Pending Publication Date: 2021-05-25
SYNBIOTECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese scholars have disclosed in patents (PCT / CN2015 / 081111 and PCT / CN2018 / 087678) that glycosyl extension can be carried out at the C20 position of protopanaxadiol type and protopanaxatriol type saponins, and at the C6 position of protopanaxatriol type saponins Glycosyltransferase for glycosyl extension, but its activity is still relatively low, which cannot fully meet the needs of applications

Method used

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  • Glycosyl transferases and method of catalyzing sugar chain extension
  • Glycosyl transferases and method of catalyzing sugar chain extension
  • Glycosyl transferases and method of catalyzing sugar chain extension

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0240] Example 1, separation of notoginseng glycosyltransferase and its coding gene

[0241] The RNA of Panax notoginseng was extracted and reverse-transcribed to obtain the cDNA of Panax notoginseng. Use primer pair 1 (SEQ ID NO:1 and SEQ ID NO:2) or primer pair 2 (SEQ ID NO:19 and SEQ ID NO:20) or primer pair 3 (SEQ ID NO:21 and SEQ ID NO:20) or primer pair 3 (SEQ ID NO:21 and SEQ ID NO:2) with this cDNA as template NO:22) or primer pair 4 (SEQ ID NO:23 and SEQ ID NO:24) for PCR amplification to obtain an amplified product of 1.3-1.4kb. The DNA polymerase was selected from the high-fidelity DNA polymerase PrimeSTAR of Bao Biological Engineering Co., Ltd. The PCR product was detected by agarose gel electrophoresis ( figure 1 ).

[0242] Under UV irradiation, the target DNA band is excised. Then AxyPrep DNA Gel Extraction Kit (AXYGEN Company) was used to recover DNA from the agarose gel, which was the amplified DNA fragment. The DNA fragment was ligated with the commercia...

Embodiment 2

[0245] Embodiment 2, the expression of the glycosyltransferase of Panax notoginseng in Escherichia coli

[0246] The plasmids PNUGT29-17-pMD18T, PNUGT29-pNUGT29-17-pMD18T, PNUGT29- 18-pMD18T, PNUGT29-19-pMD18T, PNUGT29-20-pMD18T, PNUGT29-21-pMD18T, PNUGT29-22-pMD18T, PNUGT29-23-pMD18T, PNUGT29-24-pMD18T were used as templates to amplify the targets shown in Table 3 Genes PNUGT29-17, PNUGT29-18, PNUGT29-19, PNUGT29-20, PNUGT29-21, PNUGT29-22, PNUGT29-23 and PNUGT29-24.

[0247] table 3

[0248]

[0249] After the expression vector pET28a (purchased from Merck) was digested with NcoI / SalI, PNUGT29-17, PNUGT29-18, PNUGT29-19, PNUGT29-20, PNUGT29-21, PNUGT29-22, PNUGT29-23, PNUGT29-24 were cloned into pET28a (one-step cloning kit, purchased from Novizan), respectively constructing Escherichia coli expression vectors PNUGT29-17-pET28a, PNUGT29-18-pET28a, PNUGT29-19-pET28a, PNUGT29-20-pET28a, PNUGT29-21 - pET28a, PNUGT29-22-pET28a, PNUGT29-23-pET28a and PNUGT29-24-pET28a expre...

Embodiment 3

[0250]Example 3, Glycosyltransferases PNUGT29-17, PNUGT29-18, PNUGT29-19 and PNUGT29-20 in vitro transglycosylation activity and product identification

[0251] The supernatant of the cell lysate of recombinant Escherichia coli BL21-PNUGT29-17, BL21-PNUGT29-18, BL21-PNUGT29-19 and BL21-PNUGT29-20 in Example 2 was used as the crude enzyme solution to carry out the transglycosylation reaction. The cell lysate of recombinant Escherichia coli with vector pET28a was used as a control. Ginseng glycosyltransferase gGT29-7 derived from the patent PCT / CN2015 / 081111 was selected as a positive control. According to the reaction system presented in Table 4, in vitro transglycosylation test was carried out at 35°C overnight.

[0252] Reaction result is detected with thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) respectively:

[0253] Table 4. Enzyme activity assay reaction system

[0254]

[0255] like image 3 As shown, protopanaxatriol-type ginseno...

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Abstract

The invention relates to a group of high-activity glycosyl transferases responsible for sugar chain extension and an application of the glycosyl transferases. Specifically, the invention provides glycosyl transferase and a derivative polypeptide thereof, which can efficiently catalyze the reaction of extending sugar chains on the first glycosyl at the C-20 site and the first glycosyl at the C-6 site of a tetracyclic triterpenoid substrate. The ginsenoside products such asginsenoside Rb1, the ginsenoside Rb3, the ginsenoside DMGG, the ginsenoside DMGX, the gynostemma pentaphyllum saponin LXXV, the gynostemma pentaphyllum saponin XVII, the gynostemma pentaphyllum saponin XIII, gynostemma pentaphyllum saponin IX, notoginsenoside U and the notoginsenoside R1, notoginsenoside R2, notoginsenoside R3, 3-O-beta-(D-xylopyranosyl)-beta-(D-glucopyranosyl)-CK, 20-O-glucosyl ginsenoside Rf, Rd-C20-O-Rha, ginsenoside Rg2, and ginsenoside Re and the like can be obtained. The glycosyl transferase disclosed by the invention can also be applied to construction of artificially synthesized ginsenoside and a plurality of new ginsenosides and derivatives thereof.

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): C12N9/10C12N15/54C12P33/00
CPCC12N9/1048C12P33/00
Inventor 周志华李超静杨成帅严兴王平平
Owner SYNBIOTECH (SUZHOU) CO LTD
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