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

确保日益增长的世界人口的全球粮食安全，并在气候变化时期满足碳中性能源的需求，目前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，以确保在不进一步造成农业造成的生态系统损失的情况下增加粮食和生物燃料产量。一种有望将作物产量提高高达60%的方法是在作物中设计藻类二氧化碳浓缩机制(CCM)。藻类CCM的中心是叶绿体内液-液分离的细胞器，即类蛋白核。蛋白核含有细胞Rubisco的大部分，并通过主动运输机制不断地向细胞供应二氧化碳。二氧化碳最终通过类囊体膜的特殊区域进入类核，这些区域横跨类核，即所谓的类囊体管。尽管类囊体小管对CO2的供应和代谢产物在类核和叶绿体基质之间的交换是重要的，但我们对类囊体管和类囊体管中含有Rubisco的基质之间的相互作用和类囊体管的生物发生几乎一无所知。在拟议的项目中，我将调查模式生物莱茵衣藻的这些知识空白。通过文献和生物信息学分析，我已经确定了可以作为类囊体小管和蛋白核基质之间的系链蛋白的候选蛋白，以及其他可能参与类囊体管生物发生的候选蛋白。在拟议的项目中，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，我将使用已确定的基本的蛋白核组装基本原理来指导体外合成CCM系统的工程设计。总而言之，拟议项目中获得的数据将指导在作物中设计类蛋白核以提高产量。

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.