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

目前，人类面临的一项重大挑战是，为不断增长的世界人口确保全球粮食安全，并在气候变化时期满足对碳中性能源的需求。应对这一挑战的关键是提高作物产量，以确保粮食和生物燃料产量的增加，而不会进一步造成农业驱动的生态系统损失。一种有前景的方法，预计将增加作物产量高达60%，是藻类CO2浓缩机制（CCM）到作物植物的工程。藻类CCM的核心是叶绿体内的液-液相分离的细胞器，即蛋白核。蛋白核含有大部分细胞Rubisco，并通过主动运输机制不断提供CO2。CO2最终通过横穿蛋白核的类囊体膜的专门区域进入蛋白核，即所谓的类囊体小管。尽管类囊体小管对于CO2的供应以及蛋白核和叶绿体基质之间的代谢物交换是重要的，但我们基本上不知道含Rubisco的蛋白核和类囊体小管基质与类囊体小管的生物发生之间的相互作用。在建议的项目中，我将调查这些知识差距的模式生物莱茵衣藻。通过文献和生物信息学分析，我已经确定了候选蛋白质，可以作为类囊体小管和蛋白核基质之间的系链蛋白，以及其他可能涉及类囊体小管的生物发生。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物化学分析，以阐明它们的功能。此外，我将使用蛋白核组装的基本原则来指导体外合成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蛋白核的工程设计以提高产量。

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.