Doyle, Sean and Cuskelly, Daragh D. and Conlon, Niall and Fitzpatrick, David A. and Gilmartin, Ciara B. and Dix, Sophia H. and Jones, Gary W. (2022) A Single Aspergillus fumigatus Gene Enables Ergothioneine Biosynthesis and Secretion by Saccharomyces cerevisiae. International Journal of Molecular Sciences, 23 (18). p. 10832. ISSN 1422-0067

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Abstract

The naturally occurring sulphur-containing histidine derivative, ergothioneine (EGT), exhibits potent antioxidant properties and has been proposed to confer human health benefits. Although it is only produced by select fungi and prokaryotes, likely to protect against environmental stress, the GRAS organism Saccharomyces cerevisiae does not produce EGT naturally. Herein, it is demonstrated that the recombinant expression of a single gene, Aspergillus fumigatus egtA, in S. cerevisiae results in EgtA protein presence which unexpectedly confers complete EGT biosynthetic capacity. Both High Performance Liquid Chromatography (HPLC) and LC–mass spectrometry (MS) analysis were deployed to detect and confirm EGT production in S. cerevisiae. The localisation and quantification of the resultant EGT revealed a significantly (p < 0.0001) larger quantity of EGT was extracellularly present in culture supernatants than intracellularly accumulated in 96 h yeast cultures. Methionine addition to cultures improved EGT production. The additional expression of two candidate cysteine desulfurases from A. fumigatus was thought to be required to complete EGT biosynthesis, namely AFUA_2G13295 and AFUA_3G14240, termed egt2a and egt2b in this study. However, the co-expression of egtA and egt2a in S. cerevisiae resulted in a significant decrease in the observed EGT levels (p < 0.05). The AlphaFold prediction of A. fumigatus EgtA 3-Dimensional structure illuminates the bidomain structure of the enzyme and the opposing locations of both active sites. Overall, we clearly show that recombinant S. cerevisiae can biosynthesise and secrete EGT in an EgtA-dependent manner which presents a facile means of producing EGT for biotechnological and biomedical use.

Item Type:	Article
Keywords:	antioxidant; AlphaFold; cell factory; redox stress; ROS; ergothioneine;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	17036
Identification Number:	https://doi.org/10.3390/ijms231810832
Depositing User:	Dr. Sean Doyle
Date Deposited:	20 Mar 2023 11:45
Journal or Publication Title:	International Journal of Molecular Sciences
Publisher:	MDPI
Refereed:	Yes
URI:	Publisher
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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