Saccharomyces cerevisiae is a GRAS (generally regarded as safe) microorganism. It has a clear genetic background, and is commonly applied in simple genetic operations. It has been widely employed as the chassis organism for the biosynthesis of natural products in food, medicine, and other fields. In this work, we tried to construct the ergothioneine metabolic pathway in Saccharomyces cerevisiae CEN.PK113-5D. Two genes, NcEgt1 and CpEgt2, were integrated into genome by homologous recombination mediated by CRISPR-Cas9 technology. The strain we used in the project for cloning unmethylated DNA is E. coli JM110. It is a commonly used strain, whose safety has been verified, and only requires laboratory safety level P1. There is no risk to our community and the environment.

DNA sequences of NcEgt1 and CpEgt2 were derived from Neurospora crassa OR74A and Claviceps purpurea 20.1, and were codon-optimized for S. cerevisiae. They are also environmentally friendly and there is no safety concern.

Our main goal is to achieve higher production of ergothioneine, which is a natural antioxidant. As an important active substance in the body, it is distributed in some tissues and organs of mammals. It can be safely used in new food supplements in the diet of infants, young children, and women who are pregnant or lactating.

The laboratory environment is in line with safety regulations, and there are corresponding emergency equipment and emergency protocols. Professor Dai and Professor Yao are responsible for safety in the lab.

They are professors of Hubei University of Technology. They have provided support in the experiment design to maintain project safety. The link below contains the biosafety guideline for our laboratory: https://zcc.hbut.edu.cn/info/1112/1544.htm

Everyone involved in this project has already received security training. Training includes: the operating principles of instruments and equipment, the handling of chemical substances, laboratory emergency protocols, the maintenance of experiment equipment, and waste disposal measures. During all experimental operations, each member wore proper attire to protect personal safety.