Exploring RuBisCO function using synthetic biology

Strategies to study or improve the biochemical properties of the ubiquitous photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) often link CO2 fixation to bacterial growth. However, such viability-driven phenotypes may not directly reflect RuBisCO carboxylation kinetics and can be confounded by toxicity of the RuBisCO substrate RuBP as well as differences in expression between RuBisCO homologs. In this presentation, I will discuss our efforts to develop a genetically encoded system to accurately and rapidly assess RuBisCO-dependent CO2 fixation kinetics in cells in a viability-independent manner. This approach combines organisms that insulate RuBisCO-derived products from host metabolism, engineered enzymes that improve gene circuit tolerance, and biosensors to monitor intracellular RuBisCO abundance and catalysis. Our strategy is compatible with RuBisCOs from all clades, and can be readily used to improve the carboxylation kinetics of RuBisCO. This study highlights the combined power of metabolic engineering and retro-biosynthesis, and provides a novel resource for the discovery of RuBisCOs with improved CO2 fixation capabilities alongside traditional viability-driven selections.