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Application of arabidopsis thaliana UGT74F2 in catalyzing phenyllactic acid to synthesize phenyllactoyl glucose

A technology for catalyzing phenyllactic acid and Arabidopsis, applied in biochemical equipment and methods, transferases, enzymes, etc., can solve the problems of limited accumulation and weak catalytic activity, and achieve the effect of increasing alkaloid content and expanding application value

Active Publication Date: 2021-08-27
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have found that the catalytic activity of the reported belladonna AbUGT1 is very weak, so the synthesis of phenylalactoylglucose is an extremely critical rate-limiting step in both TA drug source plants and TA de novo synthesis yeasts. Insufficient supply severely limits the accumulation of hyoscyamine and scopolamine downstream (Prashanth Srinivasan1 & Christina D. Smolke. Biosynthesis of medicinal tropane alkaloids in yeast. Nature. 2020)

Method used

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  • Application of arabidopsis thaliana UGT74F2 in catalyzing phenyllactic acid to synthesize phenyllactoyl glucose
  • Application of arabidopsis thaliana UGT74F2 in catalyzing phenyllactic acid to synthesize phenyllactoyl glucose
  • Application of arabidopsis thaliana UGT74F2 in catalyzing phenyllactic acid to synthesize phenyllactoyl glucose

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Embodiment 1

[0023] Example 1. Molecular docking screening for potential glucosylphenyllactate transferases

[0024] 909 glucosyltransferase protein crystal structures were downloaded from the Protein Data Bank PDB. Homology modeling of belladonna AbUGT1 was performed using Swissmodel to obtain its protein structure model. Download the three-dimensional structures of D-(+)-Phenyllactic acid (PLA) and Uridine Diphosphate Glucose (UDPG) from the PubChem database. Using the Autodock Vina software, the glycosyltransferase and AbUGT1 protein models from the PDB database were used to dock PLA and UDPG respectively. Each docking calculation was repeated 10 times, and the binding free energy was calculated. Finally, pymol was used to generate the complex model of glycosyltransferase, PLA and UDPG, and the substrate binding pocket was analyzed.

[0025] Molecular docking calculation results show that the binding free energy of AbUGT1 of belladonna to PLA is -5.5kcal.mol -1 , the binding free ene...

Embodiment 2

[0026] Embodiment 2, enzyme activity analysis

[0027] (1) Arabidopsis total RNA extraction

[0028] Take an appropriate amount of Arabidopsis plant tissue, put it in liquid nitrogen for quick freezing, grind it into a fine powder at low temperature, add it to a 1.5mL Eppendorf (EP) centrifuge tube containing 1mL of lysate, shake and mix well, and follow the TIANGEN kit Total RNA was extracted according to the instruction manual. The integrity of the total RNA was detected by agarose gel electrophoresis, and the concentration of the extracted RNA was measured and analyzed on an ultra-micro ultraviolet spectrophotometer.

[0029] (2) Gene cloning and prokaryotic expression vector construction

[0030] Using the extracted total RNA as a template, cDNA was synthesized according to the instructions of the Tiangen FastKing cDNA First Strand Synthesis Kit; the UGT74F2 sequence was obtained from the NCBI public database, and the recombinant primers were designed in combination with...

Embodiment 3

[0048] Embodiment 3, the effect of UGT74F2 in tropane alkaloid plant metabolism engineering

[0049] (1) Construction of plant overexpression vector

[0050] Enzyme activity experiments in vitro have shown that UGT74F2 has higher catalytic efficiency than AbUGT1, suggesting that UGT74F2 may have a better role in promoting the biosynthesis of conchine downstream. Therefore, in order to understand the promotion effect of overexpression of UGT74F2 and AbUGT1 on the synthesis of downstream alkaloids in vivo, this study constructed overexpression vectors and compared plant metabolic engineering with belladonna hairy roots to evaluate the role of the above two genes in tropicane biogenesis. Significance in base biosynthesis.

[0051] Based on the UGT74F2 coding region sequence obtained above, recombinant primers were designed in conjunction with the overexpression vector pBI121. Primers were designed as follows:

[0052] 121-UGT74F2-F: 5'-cgggggactctagaggatccatggagcataagagaggaca-...

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Abstract

The invention discloses an application of arabidopsis thaliana UGT74F2 in catalyzing phenyllactic acid to be subjected to glycosyl transfer reaction to synthesize phenyllactoyl glucose. Research finds that UGT74F2 from arabidopsis thaliana can catalyze phenyllactic acid to synthesize phenyllactoyl glucose, and the catalytic activity of the UGT74F2 is higher than that of AbUGT1 reported at present and used for tropine alkaloid synthesis. The UGT74F2 can be used for tropane alkaloid metabolic engineering to improve the alkaloid content, the application value of UGT74F2 is widened, and the UGT74F2 has very important significance on medicinal TA metabolic engineering and synthetic biology.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of Arabidopsis thaliana UGT74F2 in catalyzing the transglycosylation reaction of phenyllactate to synthesize phenyllactylglucose. Background technique [0002] Tropane alkaloid (Tropane Alkaloid, TA) is a class of anticholinergic drugs acting on the parasympathetic nerve. Its representative drugs hyoscyamine, scopolamine and their derivatives have been widely used in anesthetics, treatment of Parkinson's, various organ colic and asthma caused by smooth muscle spasm, and the market demand is huge. At present, tropane alkaloids still completely rely on the extraction from a few medicinal plants of Solanaceae, including belladonna (Atropa belladonna), datura (Datura stramonium) and hyoscyamus niger. Belladonna is a commercially cultivated drug source plant for the production of hyoscyamine and scopolamine included and stipulated in the Chinese Pharmacopoeia. The mass f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12P17/18C12P19/02C12P19/18
CPCC12N9/1051C12P19/02C12P19/18C12P17/182
Inventor 曾俊岚邱飞赵腾飞张巧卓刘雪超廖志华
Owner SOUTHWEST UNIV
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