Introduction

Ergothioneine (EGT) is a natural amino acid derived from histidine, also known as 2-mercaptohistidine trimethylbetaine, which was first discovered fromClaviceps purpurea in 1909 by Tanret et al. The molecular formula is C9H16N3O2S, and the relative molecular mass is 229.3. EGT exists in the form of thiol-thione tautomer in solution, and thione are the main form at physiological pH [1]. As a natural antioxidant, EGT has many physiological functions, such as anti-oxidation[2], anti-aging [3], anti-inflammatory [4, 5], anti-cancer[6, 7], protecting cells from ultraviolet damage[8, 9], relieving nervous system diseases[10, 11] and so on. It is closely related to human health, and has been paid more and more attention, which makes the market demand of EGT increase.
Although EGT is widely found in organisms, EGT cannot be synthesized by animals, can only be ingested and accumulated through food. EGT is transported into cells through the organic cation transporter OCTN1 [12]. At present, the preparation methods of EGT include chemical synthesis[13], natural biological extraction[14] and biosynthesi [15]. The natural biological extraction method mainly extracts EGT from plants, animal blood, edible and medicinal fungi. However, this method often has some problems such as complicated technology and low extraction efficiency[16]. There are many routes for chemical synthesis of EGT, most of the methods require five to eight steps of reductive amination, sulfhydrylation, methylation and other reactions to obtain EGT, and the yield is low [17]. Some researchers use L-histidine betaine, the precursor of EGT, as the raw material to obtain the product EGT after two steps of reaction, which greatly shortens the synthesis route, but the raw material is extremely expensive [13], and the EGT is difficult to levorotation, and the chemical synthesis of EGT is relatively troublesome, and the safety is not guaranteed. Biosynthesis refers to the fermentation of microorganisms that can synthesize EGT to obtain the target product. It has been reported that many microorganisms have the ability to synthesize EGT, such as Methylobacterium[18],Cyanobacteria[19],Schizosaccharomyces pombe[20],Mycobacterium smegmatis[21], Neurospora crassa[22], etc. The production of EGT by natural microorganisms can reduce costs, but the efficiency of EGT synthesis is low, and the yield cannot be significantly improved by fermentation optimization. Moreover, some microorganisms are opportunistic pathogens and have potential safety hazards. Therefore, exploring the EGT biosynthetic pathway, selecting appropriate and safe gene sources and expression hosts, and using heterologous expression methods to produce EGT on a large scale can not only ensure safety, but also shorten the fermentation cycle.
Synthesis pathways of EGT in a variety of microorganisms have been characterized, mainly divided into the following two pathways: one exists in most prokaryotes, represented by M. smegmatis , discovered and characterized by Seeback [21] in 2010, EGT is synthesized by five gene clusters, involving five enzymatic reaction steps of EgtABCDE . The other exists in most eukaryotes, represented by N. crassa . In 2014, Hu et al.[22] discovered and confirmed that EGT can be synthesized by only two synthetases, Egt1 and Egt2 , by means of bioinformatics and biochemistry. Egt1 is a bi-functional enzyme that catalyzes a continuous reaction, first catalyzes histidine and S-adenosylmethionine (SAM) to form hercynine (HER), and then catalyzes cysteine (Cys) to form S-hercynylcysteine sulfoxide (Cys-HER). Egt2 is a pyridoxal phosphate (PLP) -dependent cysteine desulfurase that cleaves C-S and catalyzes Cys-HER to produce EGT. The fungal pathway is more suitable for EGT production than the bacterial pathway because it requires fewer genes involvemen. With the discovery of synthetic pathway and the development of biotechnology, the heterologous expression of Escherichia coli[23], yeast [24],Aspergillus oryzae[25], etc., has been realized, but most of the genes come from N. crass a and M. smegmatis , and the safety is difficult to guarantee. Edible fungi belong to fungi and are the main source of EGT in diet [26]. Compared with bacteria, edible fungi have more concise EGT synthesis pathway and are also safe and reliable gene sources. However, its growth cycle is long and many conditions need to be controlled during the growth process. Moreover, studies on EGT in edible fungi mainly focus on extraction, purification and antioxidant. At present, the biosynthetic pathway of EGT has been reported only from Flammulina velutipes[27], Grifola frondosa[28], Cordyceps militaris[29], and Pleurotus[30]. Excavation of EGT synthase genes in edible fungi will help us to further understand the mechanism of EGT synthesis in fungi, in order to obtain EGT synthase with higher activity, and then obtain EGT quickly, conveniently and efficiently by gene editing technology.
P. eryngii is one of the rare species in China. It is rich in nutrition, crisp and refreshing in texture, and enjoys the reputation of “mushroom king”. In our previous studies, we have discovered thePeEgt1 gene in P. eryngii , which is an enzyme composed of two functional domains, corresponding to the functions of EgtBand EgtD in M. smegmatis respectively, and its main function is to catalyse the formation of histidine methylation and the formation of cysteine C-S bond [30]. In this study, the EGT synthesis genes in P. eryngii were mined, and the potential synthase genes, single or combined, were integrated into the genome of S. cerevisiae IMX581 to construct engineered strains for heterologous expression, and to explore the synthesis mechanism of potential genes in the EGT synthesis pathway. The results of the study will enrich the types of EGT biosynthetic genes and provide effective genes for the construction of engineered bacteria with efficient expression of EGT, which has scientific significance and broad application prospects.