In vivo directed evolution of an ultra-fast RuBisCO from a semi-anaerobic environment imparts oxygen resistance

Carbon dioxide assimilation by the enzyme Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO) underpins biomass accumulation in photosynthetic bacteria and eukaryotes. Despite its pivotal role, RuBisCO has a slow carboxylation rate ( ) and is competitively inhibited by oxygen. These traits impose limitations on photosynthetic efficiency, making RuBisCO a compelling target for improvement. Interest in RuBisCO from Gallionellaceae bacteria, which comprise a dimer or hexamer of large subunits, arises from their nearly 5-fold higher than the average RuBisCO enzyme. Here, a comprehensive kinetic analysis of a Gallionellaceae RuBisCO (GWS1B) reveals the fastest CO ₂ fixation rate measured for this enzyme (25.8 s ^{− 1} ), and exposes an extreme sensitivity to oxygen inhibition, consistent with its evolution under semi-anaerobic environments. We used a novel in vivo mutagenesis-mediated screening pipeline to evolve GWS1B over six rounds under oxygenic selection, identifying three catalytic point mutants with improved ambient carboxylation efficiency; Thr-29-Ala (T29A), Glu-40-Lys (E40K) and Arg-337-Cys (R337C). Full kinetic characterization showed that each substitution enhanced the CO ₂ affinity of GWS1B under oxygenic conditions while simultaneously subduing oxygen affinity, leading to 24% (E40K), 9% (T29A) and 7% (R337C) enhancements in carboxylation efficiency under ambient O ₂ . By contrast, under the near anaerobic natural environment of Gallionellaceae , the carboxylation efficiency of each mutant was impaired ∼16%. These findings demonstrate the efficacy of artificial directed evolution to access novel regions of catalytic space in RuBisCO, and a capacity for rapid evolution of kinetic traits in response to environmental change.