Application of genes Gfegt1 and Gfegt2 of ergothioneine of grifola frondosa to synthesis of ergothioneine

A technology of ergothioneine and frondosa, applied in application, genetic engineering, plant genetic improvement and other directions

Active Publication Date: 2019-12-24
SOUTH CHINA AGRI UNIV
View PDF6 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on egt1 and 2 of fungi is mainly based on protein structure research (Hu et al., 2014, Irani et al., 2018), and i

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of genes Gfegt1 and Gfegt2 of ergothioneine of grifola frondosa to synthesis of ergothioneine
  • Application of genes Gfegt1 and Gfegt2 of ergothioneine of grifola frondosa to synthesis of ergothioneine
  • Application of genes Gfegt1 and Gfegt2 of ergothioneine of grifola frondosa to synthesis of ergothioneine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1.1 Materials and methods

[0044] 1.1.1 Strains and plasmids

[0045] Grifola frondosa strains were purchased from Beijing Jijiyuan Technology Co., Ltd.; pMD18T plasmid was purchased from Baori Biotech (Beijing) Co., Ltd. (Takara China), and Escherichia coli DH5α was purchased from Shanghai Weidi Biotechnology Co., Ltd. for gene Cloning; Saccharomyces cerevisiae EC1118 and corresponding pYES2 plasmid: Saccharomyces cerevisiae EC1118 was purchased from Lallemand, pYES2 plasmid was purchased from Invitrogen; pRS42k was gifted by Professor Christof Taxis and Professor Michael Knop, and it is also in the literature "System of centromeric, episomal, and integrative vectors based on drugresistance markers for Saccharomyces cerevisiae[J].Biotechniques 2006,40(1):73-78." published for gene expression.

[0046] 1.1.2 Medium and reagent preparation

[0047] PDB medium: used for the cultivation of Grifola frondosa hypha. Potatoes (peeled) 200g, glucose 20g, MgSO 4 ·7H 2 O1.5g, KH 2 PO ...

Embodiment 2

[0140] 2.1 Materials and methods (refer to 1.1 Materials and methods)

[0141] 2.2 Study on the function of ergothioine biosynthetic genes

[0142] 2.2.1 Obtaining the thioneine synthase gene, refer to 1.2.1 Obtaining the thioneine synthase gene in Example 1.

[0143] 2.2.2 The connection between the cloning vector and the target gene, refer to the connection between the 1.2.2 cloning vector and the target gene in Example 1.

[0144] 2.2.3 Construction and transformation of expression plasmid

[0145] 2.2.3.1 Add homology arms for gene fragments

[0146] The method of constructing the vector this time is the homologous recombination method. The multi-segment plasmid is connected to the expression vector pRS42k by using homologous recombinase, and the DNA polymerase PrimeStar Max is used to add the homology arm using primers (1) Sal1-TEF1p-F&TEF1p -Gfegt1-R / (2)Gfegt1-F&Gfegt1-CYC1t-R / (3)CYC1t-F&CYC1t-TEF1p-R / (4)TEF1p-F&TEF1p-Gfegt2-R / (5)Gfegt2-F&Gfegt2-CYC1t-R / ( 6) CYC1t-F&CYC1t-EcoRI-R...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses application of genes Gfegt1 and Gfegt2 of ergothioneine of grifola frondosa to synthesis of the ergothioneine, and belongs to the field of biosynthetic biology. The possible ergothioneine synthetase genes Gfegt1 and Gfegt2 in the grifola frondosa are found out through a homologous comparison method, and RNA of the grifola frondosa is extracted and then subjected to reversetranscription to generate cDNA for cloning obtaining. Carriers for expression, started by utilizing yeast promoters, of the two genes Gfegt1 and Gfegt2 are constructed, and converted into saccharomyces cerevisiae EC1118, then the saccharomyces cerevisiae is absorbed by adding a substrate, reaction and catalysis are conducted in vivo for synthesizing the ergothioneine, after extracting, generationof ergothioneine products is detected through high performance liquid chromatography (HPLC), in-vivo biosynthesis of the ergothioneine is achieved. A brand new biosynthetic pathway is provided for production of the ergothioneine.

Description

Technical field [0001] The present invention belongs to the field of biosynthetic biology, and particularly relates to the application of thioneine genes Gfegt1 and Gfegt2 of Grifola frondosa in the synthesis of thioneine. Background technique [0002] Ergothioneine (EGT) is an antioxidant substance, which plays important roles in maintaining redox homeostasis, signal transduction, and cell metabolism in bacteria and fungi. Tanret (1909) first discovered the substance in Clavicepspurpurea and named it. Ergothioine is a derivative of histidine trimethyl betaine. A sulfhydryl group is attached to the second C atom on the imidazole ring. It exists in the form of thioketone, and its standard reduction potential is higher than that in the form of thiol. The reduction potential is low, so ergothioneine is more stable than other thiol compounds (such as glutathione) (Cheah et al., 2012). [0003] Ergothioine has a variety of antioxidant and cell protection functions in vitro and in a sm...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N9/02C12N9/88C12N15/53C12N15/60C12N15/81C12N1/19C12P17/10C12R1/865
CPCC12N9/0083C12N9/88C12N15/81C12P17/10C12Y404/01
Inventor 林俊芳余颖豪郭丽琼廖寒露李浩莹
Owner SOUTH CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap