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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
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 it is only based on Neurospora crassa, and there are no more Fermentation-based applications and related research based on edible fungi

Method used

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  • 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

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Experimental program
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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...

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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

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Application Information

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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
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