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

植物和藻类需要抗氧化剂来保护它们免受光合作用产生的活性氧的影响。我们最近发现，许多植物和藻类积累了大量的代谢物麦角硫氨酸(EGT)，这是一种潜在的新型抗氧化剂，但它在这些生物体中的作用尚不清楚。EGT(组氨酸甜菜碱在咪唑环上带有互变的硫酮/硫醇基团)及其前体Hercyine(组氨酸甜菜碱)以前只在真菌和一些细菌群中被发现。饮食中的EGT通过一种特定的转运蛋白在人类体内积累到高浓度。这一观察结果导致了人们对其作为抗氧化剂/细胞保护剂的好处的猜测(有一些实验证据)，并为本项目提供了更广泛的背景。学生将使用以下方法研究光合作用有机体中EGT生物合成的进化和功能1。通过质谱学研究植物和原生生物树中二氢叶青素和EGT的存在情况，并利用现有的基因组序列，确定候选的EGT生物合成基因。通过在大肠杆菌中表达来测试候选生物合成基因的功能(它本身不会产生EGT)。这种方法已经使我们能够在蓝藻中鉴定EGT的生物合成途径。在选定的模式生物中敲除或敲除(CRISPR-Cas，amiRNA)候选EGT生物合成基因：藻类(苔藓)、衣藻(绿藻)和褐指藻(光合原生生物)。这些菌株将被用来通过研究它们对高光、极端温度和金属的反应来研究EGT的功能。将通过基于质谱学的代谢物图谱来评估对新陈代谢的更广泛影响。EGT已被预测为活性氧物种的清除剂和金属离子(如铜、镍、钴)的有效螯合剂。更具推测的是，与其他甜菜碱一样，它可以稳定/保护大分子结构。学生将在重组大肠杆菌中合成EGT(昂贵的)和Hercyine(不可商业获得)，然后测试它们稳定纯化蛋白质、膜(使用叶绿体类囊体膜)和DNA的能力，以抵御氧化处理、高温和有毒金属。

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.