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

光合作用的碳同化作用产生的食物，燃料和材料是现代社会的核心。因此，增强光合作用将在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是由称为RuBisCO（核酮糖二磷酸羧化酶/加氧酶）的中心酶限制的。RuBisCO因其缓慢且容易出错的活性而臭名昭着，但改善这种酶的尝试在很大程度上失败了。本项目的目标是通过测试改善其关键限制步骤的可行性，为提高光合碳同化奠定基础。在此过程中，这项研究将通过研讨会吸引其他科学家参与，以发展更广泛的社区和讨论，重点是改善光合作用。此外，从工作中得出的原则将用于开发新的本科课程，使生物化学教学使用定量的原则，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是否被迫进行速率与特异性权衡的基本问题。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，测定这个问题历来是困难的。在这里，研究人员将开发一种新的代谢工程E。大肠杆菌菌株，适合于直接选择的RuBisCO活性在大肠杆菌中。然后将使用该菌株对RuBisCO活性进行深度突变扫描，并鉴定具有扰动动力学特性的酶变体。总的来说，这项工作将为这种关键酶的序列-功能景观带来新的曙光，并可能导致适合工程增强光合作用的改进酶的开发。该奖项反映了NSF的法定使命，并通过使用基金会的智力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.