To meet the growing market demand of pharmaceutical, cosmetics and food industries, and make up for the food waste caused by the natural extraction method of mushroom and the environmental pollution caused by the biosynthesis method, the ergothioneine synthesis pathway was successfully constructed in Saccharomyces cerevisiae in this project. The strain with the ability of ergothioneine synthesis was obtained, which provided an economic and easy to scale up approach for the synthesis of ergothioneine.

In order to achieve ergothioneine biosynthesis in Saccharomyces cerevisiae, we first designed the expression module of ergothioneine synthesis gene egt1 from Crassa Neurospora. CRISPR/Cas9 gene editing technology was applied to construct a recombinant yeast strain with egt1 gene expression framework. The fermentation metabolites were detected by high performance liquid chromatography. The results showed that the genetically engineered strain Saccharomyces cerevisiae CEN.PK113-5D E01 had the ability to produce ergothioneine, but the yield was low. To improve yield, we found that another gene, egt2, also plays a key role in the biosynthetic pathway. Then, we designed the ergothioprotein synthesis gene egt2 module of Claviviella violaceus, and constructed recombinant strain Saccharomyces cerevisiae CEN.PK113-5D E02. It was found that the engineered yeast with egt1 and egt2 showed better production capacity. These results indicate that Saccharomyces cerevisiae lacks egt1, which is the key gene for ergothioneine synthesis. The addition of exogenous gene egt2 helps to improve the synthesis of ergothioneine. Next, we will focus on the increase in egt1 gene expression to improve ergothioneine production.

This project popularizes the functions of synthetic biology and natural cytoprotective agents to the society, helps to correctly understand synthetic biology and iGEM events. Cell protective agent refers to the material that has obvious protective effect on cells.