Application of Flammulina velutipes genes fvegt1, fvegt2 and fvegt3 in the synthesis of ergothioneine

Abstract

The invention discloses an application of needle mushroom genes Fvegt1, Fvegt2 and Fvegt3 to synthesis of ergothioneine, and belongs to the field of biosynthesis. By a homologous clone method and a gene synthesis method, 3 genes in an ergothioneine synthesis way are cloned from a needle mushroom mycelium, expression in vitro is performed, and in vitro biosynthesis of the needle mushroom ergothioneine is realized. The FvEgt1 can catalyze substrates of His, Cys, SAM, FeSO4 and the like to form AdoHcy and HerSul; FvEgt2 can be combined with the Cys, so that SH of cysteine is activated to form sulfur anions; the FvEgt3 can catalyze PLP and HerSul to form a ketimine compound; and the sulfur anions activated by the SH of the cysteine perform nucleophillic attack on the ketimine compound to obtain the ergothioneine. The invention firstly clarifies a biosynthesis way of the ergothioneine in basidiomycetes needle mushrooms except bacteria, ascomycetes and the like.

Description

technical field [0001] The invention belongs to the field of biosynthesis, in particular to an application of Flammulina velutipes genes Fvegt1, Fvegt2 and Fvegt3 in synthesizing ergothioneine. Background technique [0002] Ergothioneine (L-Ergothioneine, EGT for short) is a rare natural 2-thioimidazole amino acid, which has two isomer forms of thione and thiol. Recent studies have shown that ergothioneine has strong antioxidant properties, anti-inflammation, anti-oxidation, protection of cells from ultraviolet damage, and cardiovascular protection. It is widely present in animals and plants and is an important active substance in the body. . Animals and plants cannot synthesize EGT by themselves, but can obtain it from the surrounding environment or diet. Plants mainly obtain it through roots and actinomycete symbionts (Eungjun et al., 2010), while the human body can obtain it from various food species such as mushrooms, garlic, wheat and beans through the cation transpor...

AI Technical Summary

Problems solved by technology

However, since EGT is a chiral amino acid, it is very difficult to chemically synthesize L-EGT. The difficulty lies in the difficulty in preparing the raw material 2-mercaptoimidazole, and the acidity of the α-position carbon will make the reaction prone to racemization and fail to achieve the expected yield. Oxis International Corporation has developed a new method to obtain 2-mercaptoimidazole by introducing a sulfhydryl group into the imidazole ring, which solves the problem of difficulty in obtaining imidazole (Xu.et al., 1995)

However, because chemically synthesized EGT is difficult to guarantee safety, and the synthetic raw materials are expensive and costly, the application of EGT is still limited.

[0003] It can also be extracted from natural organisms such as mushrooms, pig blood, animal tissues and ergot, but the yield obtained by this method is still low. Studies have shown that the content of EGT extracted from pig blood is relatively high, which is only 60mg / L. Moreover, many impurities, drug residues, and high extraction costs still limit the industrial development of EGT.

Osawa et al. used the egt gene of Mycobacterium smegmatis to express EGT heterologously in E. coli, and obtained 24 mg / L (104 μM) secreted EGT (Osawa et al., 2017), but because the gene was derived from Mycobacterium smegmatis is not easily accepted by consumers

Obtaining EGT by submerged fermentation of mushroom mycelium is the main means of obtaining EGT in recent years (patent application number 201110460029.4, Jiang Wenxia, ​​Liu Qi, Zhou Tao, Yang Ping), but because the content of ergothioneine obtained from mushroom mycelium submerged fermentation is very low, The production cost is high and the price is very expensive

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