Liquid-liquid phase separation around a membrane: Investigating the assembly of the pyrenoid around the thylakoid membrane in Chlamydomonas reinhardtii

目前，确保全球粮食安全以满足不断增长的世界人口，并在气候变化时期满足对碳中和能源的需求，是人类面临的一项重大挑战。应对这一挑战的关键是提高作物产量，以确保实现粮食和生物燃料产量的增加，同时避免农业导致的生态系统进一步丧失。一种有希望的方法是将藻类二氧化碳浓缩机制（CCM）工程引入作物植物中，预计可将作物产量提高60%。藻类CCM的核心是叶绿体内液-液相分离的细胞器，即类核蛋白。类芘含有大部分细胞的Rubisco，并通过主动运输机制不断提供二氧化碳。二氧化碳最终通过类囊体膜的特殊区域，即所谓的类囊体小管，进入类囊体。尽管类囊体小管在类pyrenox和叶绿体基质之间的CO2供应和代谢物交换中起着重要作用，但我们对类pyrenox和类囊体小管的含Rubisco基质与类囊体小管之间的相互作用以及类囊体小管的生物发生基本一无所知。在这个项目中，我将研究模式生物莱茵衣藻（Chlamydomonas reinhardtii）的知识缺口。通过文献和生物信息学分析，我已经确定了候选蛋白质，这些蛋白质可以作为类囊体小管和类pyrenox基质之间的系链蛋白，以及其他可能参与类囊体小管生物发生的蛋白质。在这个项目中，我将对这些蛋白质在体内和体外的功能进行生化分析，以阐明它们的功能。此外，我将使用已确定的类芘组装的基本原理来指导体外合成CCM系统的工程设计。总之，在拟议项目中获得的数据将指导作物中的类芘工程以提高产量。

It is currently a major challenge for humankind to ensure global food security for a growing world population and to fulfil the demands of carbon-neutral energy sources in times of climate change. Critical to addressing this challenge is an increase in crop yields to ensure that increases in food and biofuel production are achieved without further agriculture driven ecosystem loss. One promising approach that is predicted to increase crop yields by up to 60% is the engineering of an algal CO2 concentrating mechanism (CCM) into crop plants. The heart of the algal CCM is a liquid-liquid phase separated organelle within the chloroplast, the pyrenoid. The pyrenoid contains most of the cells Rubisco and is constantly supplied with CO2 by active transport mechanisms. CO2 enters the pyrenoid ultimately through specialised regions of the thylakoid membrane that transverse the pyrenoid, the so-called thylakoid tubules. Even though the thylakoid tubules are important for the CO2 supply and the exchange of metabolites between the pyrenoid and the chloroplast stroma, we basically know nothing about the interaction between the Rubisco containing matrix of the pyrenoid and thylakoid tubules and the biogenesis of the thylakoid tubules. In the proposed project, I will investigate these knowledge gaps in the model organism Chlamydomonas reinhardtii. Through literature and bioinformatic analysis I have identified candidate proteins that could act as tether proteins between the thylakoid tubules and the pyrenoid matrix, and others that are potentially involved the biogenesis of the thylakoid tubules. In the proposed project, I will biochemically analyse the function of those proteins both in vivo and in vitro to elucidate their function. Furthermore, I will use the identified underlying fundamental principles of pyrenoid assembly to guide the engineering of a synthetic CCM system in vitro. Together, the data obtained in the proposed project will guide the engineering of a pyrenoid in crops to increase yields.