Mapping sequence-function landscapes to isolate improved variants of the dominant carbon-fixing enzyme

光合作用的碳同化产生了现代社会的核心食物、燃料和材料。因此，加强光合作用将对这些关键应用领域产生重大的积极影响。光合作用的碳同化被认为受到中心酶RuBisCO（二磷酸核酮糖羧化酶/加氧酶）的限制。RuBisCO因其缓慢和易出错的活性而臭名昭著，然而改进这种酶的尝试基本上失败了。该项目的目标是通过测试改善其关键限制步骤的可行性，为增强光合作用碳同化奠定基础。在此过程中，这项研究将通过专题讨论会吸引其他科学家，以发展更广泛的社区和讨论，重点关注改善光合作用。此外，从这项工作中得出的原理将用于开发新的本科课程，使生物化学教学使用定量原理，而不是传统的基于记忆的方法。这些课程将在加州大学伯克利分校的本科生物化学课程中使用和测试，以评估其有效性。研究人员将应用蛋白质工程和DNA测序的新进展来回答RuBisCO是否被迫在速率和特异性之间做出权衡的基本问题。由于系统地构建必要的文库和测定体内RuBisCO的羧化酶活性的挑战，分析这个问题在历史上一直很困难。在这里，研究人员将开发一种新的代谢工程大肠杆菌菌株，适合直接选择RuBisCO活性。然后，该菌株将用于执行RuBisCO活性的深度突变扫描，并识别具有扰动动力学性质的酶变体。总的来说，这项工作将为这种关键酶的序列功能景观提供新的思路，并可能导致适合工程增强光合作用的改良酶的发展。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 carbon assimilation produces the foods, fuels, and materials that are central to modern society. Enhancing photosynthesis would thus have significant positive impact in these critical application areas. Photosynthetic carbon assimilation is thought to be limited by the central enzyme called RuBisCO (Ribulose Bisphosphate Carboxylase/Oxygenase). RuBisCO is notorious for its slow and mistake-prone activity, yet attempts to improve this enzyme have largely failed. The goal of this project is to lay the groundwork for enhancing photosynthetic carbon assimilation by testing the feasibility of improving its key, limiting step. In the process, this research will engage other scientists, via symposia, to develop a broader community and discussion focused on improving photosynthesis. In addition, principles derived from the work will be used to develop novel undergraduate curricula that enables teaching biochemistry using quantitative principles, rather than traditional memorization-based methods. These curricula will be employed and tested in an undergraduate biochemistry course at UC Berkeley to evaluate their effectiveness.The investigators will apply new advances in protein engineering and DNA sequencing to answer the fundamental question of whether RuBisCO is forced to make rate versus specificity trade-offs. Assaying this question has historically been difficult due to the challenge of systematically constructing the necessary libraries and assaying for the carboxylase activity of RuBisCO in vivo. Here, the investigators will develop a novel metabolically engineered E. coli strain suitable for the direct selection of RuBisCO activities en masse. This strain will then be used to perform a deep mutational scan of RuBisCO activity and identify enzyme variants with perturbed kinetic properties. In total, this work will shed new light into the sequence-function landscape of this critical enzyme and could lead to the development of improved enzymes suitable for engineering enhanced photosynthesis.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.