Unravelling the function of the unusual antioxidant ergothioneine in plants and photosynthetic protists

Plants and algae need antioxidants to protect them from photosynthesis-derived reactive oxygen. We have recently discovered that many plants and algae accumulate significant quantities of the metabolite ergothioneine (EGT), a potential novel antioxidant, but its role in these organisms is not understood. EGT (histidine betaine with a tautomeric thione/thiol group on the imidazole ring) and its precursor hercynine (histidine betaine) were previously known only in fungi and some groups of bacteria. Dietary EGT is accumulated to high concentration in humans using a specific transporter. This observation has resulted in speculations (with some experimental evidence) about its benefits as an antioxidant/cytoprotectant and provides a wider context for this project.

The student will investigate the evolution of EGT biosynthesis amongst photosynthetic organisms and functions using the following approaches.
1. Survey the occurrence of hercynine and EGT across the plant and protist tree of life by mass spectrometry and, using existing genome sequences, identify candidate EGT biosynthesis genes.
2. Test the function of candidate biosynthesis genes by expression in E. coli (which does not make EGT itself). This method has already allowed us to identify the EGT biosynthetic pathway in cyanobacteria.
3. Knock out or knock down (CRISPR-Cas, amiRNA) candidate EGT biosynthesis genes in selected model organisms: Physcomitrella (moss); Chlamydomonas (green alga) and Phaeodactylum (photosynthetic protist). These strains will be used to investigate the function of EGT by investigating their response to high light, temperature extremes and metals. Wider effects on metabolism will be assessed by mass spectrometry-based metabolite profiling.
4. EGT has predicted functions as a scavenger of reactive oxygen species and as an effective chelator of metal ions (e.g. Cu, Ni, Co). More speculatively, like other betaines, it could stabilise/protect macromolecular structure. The student will synthesise EGT (expensive) and hercynine (not commercially available) in recombinant E. coli and then test their ability to stabilise purified proteins, membranes (using chloroplast thylakoid membranes) and DNA against oxidative treatments, high temperature and toxic metals.