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.