Difunctional C-glycoside glycosyltransferase and application thereof

A technology of glycosyltransferase and carbon glycosides, which is applied in the field of synthetic biology and can solve problems that have not yet been solved

Pending Publication Date: 2021-08-17
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

So far, there are no reports of glycosyltransferases for arabinose carbonyl glycosylation of flavonoids

Method used

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  • Difunctional C-glycoside glycosyltransferase and application thereof
  • Difunctional C-glycoside glycosyltransferase and application thereof
  • Difunctional C-glycoside glycosyltransferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Embodiment 1, novel uridine diphosphate (UDP)-glycosyltransferase

[0122] 1. Cloning of carboside glycosyltransferase gene and construction of expression plasmid

[0123] Take mature leaves or seedlings of gramineous plants (moso bamboo, japonica rice, indica rice, sorghum, millet, Brachypodium distachyon, corn), clean the surface stains with clean water, gently wipe the water stains on the surface of dry leaves with absorbent paper, and weigh them for later use. Genomic DNA was extracted according to the instructions provided by the TIANGEN Plant Genome Extraction Kit.

[0124] The amino acid sequence of the described carboside glycosyltransferase is as follows:

[0125] >PhCGT3 (SEQ ID NO: 1)

[0126] MAPPATLRSGEPDRDTPHVIFIPSAGMGHLYPFFRFIAALSSYGVDISVVTILPTVSAAEADHFAGLFAAFPSIRRIDFHLLPLDTNEFPGADPFFLRWEELRRSAHLLGPLIASATPRVSTIVSDVTLASHVIPIAKELHIPCHVLFISCATMLSLNAYFPVYLDTKAGMDGIGDVDIPGVCRIPKSSLPQALLDLNKLFTKQFIENGREITKADGILVNTFDALEPKALATLRDGTVVTRFPPVFAIGPLTSHSPAAGERVDAGFP...

Embodiment 2

[0168] Embodiment 2, the synthesis of flavonoid carbon glycoside compound schaftoside etc.

[0169] In this example, PhCGT1 and OsiCGT4 were used to synthesize schaftoside and its isomers in Escherichia coli.

[0170] Predicted biosynthetic pathways of schaftosides and their isomers such as Figure 10 shown. Arrows 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 + ).

[0171] 1. Plasmid construction

[0172] (1) Construction of the expression cassette for the synthetic precursor gene of the flavanone naringenin

[0173] The artificially synthesized and codon-optimized precursor synthetic gene sequence was constructed on the pET28a vector to ...

Embodiment 3

[0201] Embodiment 3, the synthesis of flavonoid carbon glycosides apigenin-C-arabinoside etc.

[0202] In this example, OsiCGT6 was used to synthesize apigenin-6-C-arabinoside, apigenin-8-C-arabinoside and apigenin-6,8-C-diarabinoside in Escherichia coli.

[0203] The synthetic pathway of apigenin-6-C-arabinoside, apigenin-8-C-arabinoside and apigenin-6,8-C-bisarabinoside is as follows: Figure 15 shown. Arrows PAL (or TAL), 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 + ).

[0204] 1. Plasmid construction

[0205] (1) The construction of the expression cassette for the synthetic precursor gene of the flavanone naringenin is the same as in Example 2.

...

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Abstract

The invention provides a group of novel uridine diphosphate (UDP)-glycosyltransferases. The group of novel uridine diphosphate (UDP)-glycosyltransferases are composed of a difunctional carbon glycoside arabinosyl transferase and a difunctional carbon glycoside glucosyl transferase. The glycosyltransferases can specifically and efficiently catalyze arabinoglycosylation and glucosylation of C-glycoside of a dihydrochalcone (dihydrochalcone-series) compound or a 2-hydroxyflavanone (hydroxyflavanone-series) compound, so C-glycoside dihydrochalcone-series and C-glycoside-2-hydroxyflavanone-series compounds are generated; and the C-glucoside-2-hydroxyflavanone-series compounds are subjected to further dehydration reaction to form the flavone C-glycoside-series compounds. The invention also relates to application of the novel UDP glycosyl transferases to an artificially constructed recombinant expression system, and production of C-glycoside dihydrochalcone and flavone C-glycoside 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 carboside glucosyl and arabinosyltransferase and application thereof. Background technique [0002] Flavonoids are C 6 -C 3 -C 6 A class of polyphenol hydroxyl (class) 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 (classes) 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 (like) compounds in which the sugar group is directly connected to the aglycone core by C-C bonds are called flavone carbon glycosides. [0003] Flavone carbon glycosides (classes) compounds have a variety of pharmacological activities, according to reports, flavone carbon glycosi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/18C12P19/02C12P19/12C12N9/10C12N15/54C12N15/70C12N1/21C12R1/19
CPCC12N9/1051C12N15/70C12P19/18C12P19/02C12P19/12C12N9/1048C12Y204/02043C12N2800/101C12P17/06
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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