Ergothioneine (EGT) is a naturally occurring sulphur-containing modified amino acid which has been shown to exhibit anti-oxidant properties. EGT is produced by selected fungi and prokaryotes, however Saccharomyces cerevisiae does not naturally produce EGT. Up to 5 genes are involved in bacterial EGT biosynthesis while in fungi, two genes have been identified to be involved in EGT biosynthesis, and one such gene is egtA from Aspergillus fumigatus. Here, it is shown that expression of EgtA alone in the GRAS organism Saccharomyces cerevisiae facilities the production of EGT. The novel production of EGT in S. cerevisiae was confirmed by both HPLC and LC-MS analysis, and quantification of resultant EGT revealed a significantly (P < 0.0001) larger quantity of EGT present extracellularly in culture supernatants and accumulated therein until the time point of 96 h was reached. To complete the EGT biosynthetic process, a cysteine desulfurase is required, and two candidates were identified in A. fumigatus, namely AFUA_2G13295 and AFUA_3G14240, termed egt2a and egt2b in this study. Co-expression of egtA and egt2a in S. cerevisiae using high expression 2μ vectors was expected to increase EGT production, however EGT levels were seen to be significantly decreased (P < 0.05). As expression of egtA alone in S. cerevisiae been shown to effect EGT biosynthesis this raised the question of the ability of S. cerevisiae to complete the EGT biosynthetic process. A putative endogenous cysteine desulfurase Nfs1 was identified in S. cerevisiae. Interestingly decreasing the expression of Nfs1 was shown to increase levels of EGT. Moreover, manipulation of the conserved residue Thr213 in EgtA indicates indiscriminate phosphorylation by threonine kinases could be negatively regulating EGT production in S. cerevisiae. This study demonstrates quite remarkably that S. cerevisiae can produce EGT with the expression of just one additional protein. Hence, production of EGT using a synthetic biology approach now opens the possibility to economically viable means of producing EGT.