ABSTRACT Ergothioneine is one of the most abundant thiols in many parts of the human body and several diseases have been linked to ergothioneine insufficiency, including rheumatoid arthritis, Crohn's disease, neurodegenerative diseases, cardiovascular disorders and diabetes. Animals do not synthesize ergothioneine and, instead, obtain it from their diet through an ergothioneine-specific transporter (OCTN1) with accumulation up to mM concentrations in several organs and tissues. Due to their differences in reduction potential (E0' = - 0.06 V for ergothioneine and E0' = - 0.24 V for glutathione), a combination of ergothioneine and glutathione provide cellular protection over a wide range of conditions such as against reactive oxygen species (ROS) and reactive nitrogen species (RNS). Despite ergothioneine's demonstrated role in maintaining human health, the current method used to produce it has proved to be a bottleneck in its widespread use. Specifically, the chemical synthesis method suffers from racemization of a stereo-center with the byproduct isomer being toxic. As a result, production at a large scale is both challenging and costly. In our preliminary studies, we developed an alternative, fermentation-based biosynthetic production method capable of producing 10 grams/liter on a 1 L batch-scale. In addition, we have identified the signaling pathway(s) related to ergothioneine function. These breakthroughs in supply and mechanistic understanding provide the scientific and manufacturing support necessary to realize ergothioneine's full commercial potential, which includes: further growth of its share in the cosmetic market, expansion into new markets (e.g., as a component in nutraceuticals), or for drug discovery and development. In this proposal we will address two key go/no-go questions that will guide the further development of this product. Specifically, we will scale-up the synthesis to pilot scale production using our patented ergothioneine production technology and also pinpoint ergothioneine's biological targets. To this end, the aims are: Aim 1: Achieve pilot-scale (100's g – 1 kg) production of ergothioneine with purity >99%. Aim 2: Verify the ergothioneine-related signaling pathways. Outcomes. Upon successful completion of these studies, we will apply for a Phase II grant with aims of large- scale production under good manufacturing practices to support additional market penetration and expansion. At the same time, we will re-examine ergothioneine's impact on ageing and age-related disease in vivo. We will also reach out to other scientists in the anti-ageing field to seek collaboration, including collaboration with an existing NIH anti-ageing program (https://www.nia.nih.gov/research/dab/interventions-testing-program-itp).