Abstract
Carbon dioxide (CO2) 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 (O2). 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 carboxylation rate than the average RuBisCO enzyme. As well as having a fast carboxylation rate (25.8 s−1 at 25C), we show that Gallionellaceae RuBisCO (GWS1B) is extremely sensitive to O2 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 CO2 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 O2 at 25C. 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Manuscript text was updated, figures were slightly revised, and supplemental files were updated. Main conclusions remain unchanged.
