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

光合作用的有机体利用大气中的二氧化碳来生产糖和生物质，以维持所有其他生命。Rubisco酶负责这种二氧化碳的化学捕获。不幸的是，它进行这一过程的速度非常慢--以至于二氧化碳捕获往往会限制植物的生长。此外，Rubisco经常错误地与氧气而不是二氧化碳发生反应，浪费能源，进一步限制植物的生长。一个改进的Rubisco，如果转化为农业，将导致作物产量的显著增加，粮食安全的改善，以及更好的气候适应能力，而改善Rubisco的功能长期以来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功能的这些领域，因为自然进化到目前为止还没有做到这一点。一种被广泛吹捧的可能性是，Rubisco陷入了进化陷阱，无法改进。另一种观点认为，将这种酶置于新的自然类型的选择压力和环境中，将使进化创新成为可能，实际上可以释放出更好的Rubisco。这个项目的目标是通过将基本的生物物理学概念与实验室进化的最先进技术相结合来测试后一种想法。这项拟议的研究还将通过每年夏天让高中实习生参与这项工作，帮助培训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所能接触到的进化轨迹。在植物中，Rubisco需要近12个这样的分子伴侣来协助其生产、调节和维护。这些伴侣的序列和活性如何影响植物Rubisco的进化，有没有方法优化这些伴侣来为Rubisco开辟新的序列空间区域？通过将实验室进化应用于Rubisco及其在微生物宿主中的伴侣，该项目将寻求回答这些问题。下一代突变技术和新颖的筛选系统将被用来实现两个关键目标：(1)展示植物Rubisco及其伴侣的高通量体内定向进化，以及(2)测试与单独进化相比，Rubisco及其伴侣的共同进化可以带来更好的进化结果和增强的酶特性的想法。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.