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Novel C-glycoside glycosyl transferase and application thereof

A technology of glycosyltransferase and carbon glycosides, applied in the field of synthetic biology

Pending Publication Date: 2021-07-20
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And, at present basically can only be by separating flavone carbon glycoside (class) compound from nature, the production technology of aspects such as large-scale low-cost biosynthesis or artificial synthesis all needs further research and development

Method used

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  • Novel C-glycoside glycosyl transferase and application thereof
  • Novel C-glycoside glycosyl transferase and application thereof
  • Novel C-glycoside glycosyl transferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1, novel uridine diphosphate (UDP)-glycosyltransferase

[0105] 1. Cloning of carboside glycosyltransferase gene and construction of expression plasmid

[0106] Take mature leaves or seedlings of gramineous plants (japonica rice, corn, wheat, sorghum, millet, Brachypodium distachybes, moso bamboo, light bamboo, sargassum), clean the surface stains with clean water, and gently wipe the water on the surface of dry leaves with absorbent paper. stains, weighed for later use. Genomic DNA was extracted according to the instructions provided by the TIANGEN Plant Genome Extraction Kit.

[0107] The amino acid sequence of the described carboside glycosyltransferase is as follows:

[0108] >PhCGT1 (SEQ ID NO: 1)

[0109] MPTSGGRTGDRPHVILLPSAGMGHLVPFSRLAVALSSGHGCDVSVVAVLPTVSAAESRHLQGLLDASPAVRRLDLHLAPFDESEFPGADPFFLRFEAMRRSAPLLGPLLADTGASALVTDIALASVVIPVAKEIHLPCYVLFTASAAMLSFCAYFPTYLDTNAGRVVGDVDIPGVYRIPKASIPQALHDPNHLFTRQFVANGQDLAKADGVLVNTFDALELEAVTALRQGSVAAGFPPVFSVGPLVTVN...

Embodiment 2

[0163] Example 2. Using the glycosyltransferase of the present invention to synthesize vitexin, isovitexin, orientin and isoorientin In this example, PhCGT1 was used to synthesize vitexin and isovitexin in Escherichia coli Isovitexin, orientin and isoorientin

[0164] The predicted biosynthetic pathway of flavone-C-monoglucoside as Figure 11 . Arrows TAL (or PAL), 4CL, CHS, CHI represent the main enzymes responsible for naringenin biosynthesis. Arrows F2H, F3'H are flavanone 2-hydroxylase (F2H) and flavanone 3'-hydroxylase (F3'H), which constitute the backbone of hydroxylated flavanones. C-Glycosyltransferase (CGT) is indicated by the arrow CGT. After the formation of a C-glycosylated intermediate, the dehydration reaction occurs spontaneously in an acidic solvent (arrow H + ) or catalyzed by a dehydratase (arrow DH).

[0165] 1. Plasmid construction

[0166] (1) Construction of the expression cassette for the synthetic precursor gene of the flavanone naringenin

[016...

Embodiment 3

[0203] Embodiment 3, use the glycosyltransferase of the present invention to synthesize Wei Cai Ning-2

[0204] In this example, TaCGT1-D was used to synthesize vicenin-2 in Escherichia coli. The predicted biosynthetic pathway of Wei Cai Ning-2 (pigenin-6,8-diglucoside) is as follows: Figure 12 . TAL (or PAL), 4CL, CHS, CHI represent the main enzymes responsible for naringenin biosynthesis. Arrow F2H is flavanone 2-hydroxylase (F2H), which constitutes the backbone of 2-hydroxyflavanone. C-Glycosyltransferase (CGT) is indicated by the arrow CGT. After the formation of a C-glycosylated intermediate, the dehydration reaction occurs spontaneously in an acidic solvent (arrow H + ).

[0205] 1. Plasmid construction

[0206] (1) The construction of the expression cassette for the synthetic precursor gene of the flavanone naringenin is the same as in Example 2.

[0207] (2) The construction of the flavanone-2-hydroxylase / CPR expression cassette is the same as in Example 2.

...

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Abstract

The invention provides a group of novel uridine diphosphate (UDP)-glycosyl transferase, which is a C-glycoside glycosyl transferase. The glycosyl transferase can be used for specifically and efficiently catalyzing the carbon glycoside glucosylation of a dihydrochalcone (class) compound or a 2-hydroxyflavanone (class) compound, so that a class of carbon glycoside dihydrochalcone (class) compounds or a class of carbon glucoside-2-hydroxyflavanone (class) compounds are generated; and the carbon glucoside-2-hydroxyflavanone (class) compound is subjected to further dehydration reaction to form the flavone C-glycoside (class) compound. The invention also relates to application of the novel UDP glycosyl transferase to an artificially constructed recombinant expression system, and production of C-glycoside dihydrochalcone (class) compounds or flavone C-glycoside (class) compounds through fermentation engineering.

Description

technical field [0001] The invention belongs to the field of synthetic biology, and more specifically, the invention relates to a new type of carbonoside glycosyltransferase and its application. Background technique [0002] Flavonoids are C 6 -C 3 -C 6 A class of polyphenol hydroxyl compounds that are structural cores can be divided into flavones, flavonols, flavanones, anthocyanins, and chalcones according to the structure of the core and the substitution of functional groups. Flavonoids often exist in the form of glycosides in plants, and such glycosylation modification is mainly catalyzed by uridine diphosphate (UDP)-glycosyltransferase (UGT). The flavonoid glycosides in which the sugar group is directly connected to the aglycone core by a C-C bond are called flavone carbon glycosides. [0003] Flavonoid carbon glycoside (class) compounds have a variety of pharmacological activities, according to reports, flavonoid carbon glycoside (class) compounds have a significan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12P7/26C12P17/06C12P19/44C12N9/02C12N15/70C12N1/21
CPCC12N9/1048C12Y204/00C12P7/26C12P17/06C12P19/44C12Y114/11009C12N9/0071C12N15/70C12N15/52C12N9/1051C12P19/18C12P19/32C12P13/22C12Y114/14C12Y203/01074C12Y505/01006C12Y403/01023C12Y403/01005C12Y602/01012C07H15/203C12N9/10C12N15/74
Inventor 王勇孙雨伟陈卓张前张英刘海利李建华
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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