In Vivo Directed Evolution of an Ultra-Fast Rubisco from a Semi-Anaerobic Environment Imparts Oxygen Resistance.

Carbon dioxide (CO(2)) 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 (kcatCO2) and is competitively inhibited by oxygen (O(2)). These traits impose limitations on photosynthetic efficiency, making Rubisco a compelling target for improvement. Interest in Form II Rubisco from Gallionellaceae bacteria, which comprise a dimer or hexamer of large subunits, arises from their nearly 5-fold higher kcatCO2 than the average Rubisco enzyme. As well as having a fast kcatCO2 (25.8 s (-1) at 25 °C), we show that Gallionellaceae Rubisco (GWS1B) is extremely sensitive to O(2) inhibition, consistent with its evolution under semi-anaerobic environments. We therefore 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 by subduing oxygen affinity, leading to 25% (E40K), 11% (T29A) and 8% (R337C) enhancements in carboxylation efficiency under ambient O(2) at 25 °C. 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.