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


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.<br/><br/>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.<br/><br/>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.

Winickoff, David
University of California - Berkeley
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