EAGER: Leveraging Chaperones to Escape the Plant RuBisCO Catalytic Catch-22

光合生物利用大气中的二氧化碳产生糖和生物量，支持所有其他生命。RuBisCO酶负责二氧化碳的化学捕获。不幸的是，这个过程进行得非常缓慢，以至于二氧化碳的捕获常常限制了植物的生长。此外，RuBisCO经常错误地与氧气而不是二氧化碳反应，浪费能量并进一步限制植物生长。改良后的Rubisco如果应用于农业，将显著提高作物产量，改善粮食安全，增强气候适应能力，而改善Rubisco功能一直被视为农业技术的圣杯。考虑到这种酶的潜在价值，研究人员询问是否有可能大幅改善RuBisCO功能的这些区域，因为迄今为止自然进化未能做到这一点。一种被广泛吹捧的可能性是RuBisCO陷入了进化陷阱，无法得到改进。另一种观点是，将这种酶置于新的自然选择压力和环境中，将使进化创新成为可能，实际上可以释放出更好的RuBisCO。该项目的目标是通过将基本的生物物理概念与最先进的实验室进化技术相结合，来测试后一种想法。这项拟议中的研究还将通过每年夏天让高中实习生参与这项工作，帮助培养下一代科学家和工程师。分子伴侣与细胞中的其他蛋白质（客户）相互作用并提供帮助，在这样做的过程中，可以深刻地影响客户的进化轨迹。在植物中，RuBisCO需要近12种这样的分子伴侣来协助其生产、调节和维持。这些伴侣蛋白的序列和活性如何影响植物RuBisCO的进化，是否有方法优化伴侣蛋白为RuBisCO开辟新的序列空间区域？通过将实验室进化应用于微生物宿主中的RuBisCO及其伴侣，该项目将寻求回答这些问题。下一代诱变技术和新型筛选系统将用于实现两个关键目标：(1)证明植物RuBisCO及其伴侣的高通量体内定向进化；(2)验证RuBisCO及其伴侣的共同进化相对于单独进化RuBisCO可以导致更好的进化结果和增强的酶特性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Photosynthetic organisms use carbon dioxide from the atmosphere to produce sugar and biomass that support all other life. The enzyme RuBisCO is responsible for this chemical capture of carbon dioxide. Unfortunately, it carries out this process only very slowly – to the extent that carbon dioxide capture often limits plant growth. Moreover, RuBisCO regularly and mistakenly reacts with oxygen instead of carbon dioxide, wasting energy and further limiting plant growth. An improved Rubisco, if translated into agriculture, would lead to a significant increase in crop yields, improved food security, and better climate resilience, and improving Rubisco function has long been viewed as an agricultural technology holy grail. With the potential value of such an enzyme in mind, researchers have asked whether it is possible to substantially improve these areas of RuBisCO function, since natural evolution has failed to do so thus far. One widely touted possibility is that RuBisCO is caught in an evolutionary trap and cannot be improved. An alternative view is that subjecting the enzyme to new-to-nature types of selection pressures and environments would enable evolutionary innovations that actually can unlock a better RuBisCO. The goal of this project is to test the latter idea, by integrating fundamental biophysical concepts with state-of-the-art techniques for laboratory evolution. The proposed research will also help to train the next generation of scientists and engineers by involving high school interns each summer in the work. Molecular chaperones interact with and assist other proteins (clients) in the cell and, in doing so, can profoundly influence the evolutionary trajectories accessible to their clients. In plants, RuBisCO requires nearly a dozen of these molecular chaperones to assist its production, regulation, and maintenance. How do the sequences and activities of these chaperones impact the evolution of plant RuBisCO, and are there ways to optimize the chaperones to open new regions of sequence space for RuBisCO? By applying laboratory evolution to RuBisCO and its chaperones in a microbial host, this project will seek to answer these questions. Next-generation mutagenesis technologies and novel screening systems will be used to achieve two key goals: (1) Demonstrating high-throughput in vivo directed evolution of plant RuBisCO and its chaperones and (2) Testing the idea that co-evolution of RuBisCO and its chaperones can lead to better evolutionary outcomes and enhanced enzymatic properties relative to evolving just RuBisCO alone.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.