The discovery of ergothioneine
biosynthesis genes from Pleurotus eryngii and its combinatorial
expression in yeast
Shan
Zhang1,2,Tao Pan1,2,Ying-Hao
Yu1,2,Zhi-Wei Ye1,2*,Tao
Wei1,2,Na
Wang3, Jing-Ru Zhong3,Li-Qiong
Guo1,2*,Jun-Fang
Lin1,2*
1College of Food Science & Institute of Food
Biotechnology, South China Agricultural University, Guangzhou 510640,
Guangdong, China;
2Research Center for Microecological Agents of
Guangdong Province, Guangzhou 510642, China
3Guangzhou Alchemy Biotechnology Co., Guangzhou
510760, Guangdong, China
Corresponding author: Professor Jun-Fang Lin
E-mail address: linjf@scau.edu.cn and 2263253429@qq.com; Address:
Department of Biotechnology, College of Food Science, South China
Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou
510640, China.
Abbreviations
EGT, Ergothionein
SAM, S-adenosylmethionin
HER, hercynine
Cys, cysteine
PLP, phosphate
5-FOA, 5-fluoroorotic acid
Keywords: Ergothioneine;Pleurotus eryngii ; biosynthesis; Saccharomyces cerevisiae ;
heterologous expression
Abstract:
Ergothioneine (EGT) is a natural amino acid derived from histidine,
which has many active functions such as anti-oxidation,
anti-inflammation, anti-cancer and prevention of neurodegenerative
diseases. Pleurotus eryngii is a delicious edible fungus with
high EGT content. The purpose of this study was
to
explore the synthesis mechanism of EGT in P. eryngii and realize
heterologous expressio of EGT in Saccharomyces cerevisiae . The
EGT synthase genes in P.
eryngii were discovered by bioinformatics method, and four synthase
genes were obtained: PeEgt1 , PeEgt2a , PeEgt2b andPeEgt2c , which were heterologously expressed in S.
cerevisiae IMX581 for functional identification. Single-, double- and
triple-gene
heterologous expression strains were constructed, and the results showed
that S. cerevisiae could synthesize EGT only by integratingPeEgt1 ; then integrating PeEgt2a or PeEgt2b on this
basis could significantly increase EGT production, while integratingPeEgt2c did not increase EGT production. The yield of EGT of
MX581-PeEgt1 -2a-2b was not significantly higher than that
of the double-gene engineered
strains, suggesting that the low enzyme activity of PeEgt1 led to
the accumulation of
S-adenosylmethionine or hercynine,
which might be the key rate-limiting step. The EGT synthase genes inP. eryngii were mined and
successfully heterologously expressed in S. cerevisiae , and the
synthesis pathway of EGT in P. eryngii was speculated. The
results of this study enrich the resource pool of EGT synthetic genes,
and provide ideas for the construction of engineered strains with high
EGT expression.