A Genetically Encoded System to Quantify and Evolve RuBisCO-Catalyzed Carbon Fixation

Strategies to study and alter the biochemical properties of RuBisCO often couple CO 2 fixation to bacterial growth. However, these viability-coupled strategies are not quantitative and are limited by toxicity of the RuBisCO substrate RuBP, the slow kinetics of RuBisCO, and differences in RuBisCO expression. We report the development of the first genetically encoded system capable of accurately quantifying RuBisCO-dependent CO 2 fixation in cellulo using a tripartite approach that combines bacterial strains which insulate RuBisCO-derived products, an engineered pathway that eliminates RuBP toxicity, and biosensors to concurrently monitor RuBisCO abundance and catalysis. We extended this biosensing strategy to 43 Form II and II/III RuBisCO homologs, finding strong agreement with in vitro -derived enzyme kinetics in living cells for the first time. Finally, we show how this system can be used to rapidly evolve functional RuBisCO enzymes. Our approach overcomes prior limitations by streamlining intracellular RuBisCO analyses and will enable the development of enzymes with improved CO 2 fixation capabilities.