Fig. 3: \({\widetilde{{\boldsymbol{K}}}}_{{\bf{C}}}\) and \({\widetilde{{\boldsymbol{V}}}}_{{\bf{\max }}}\) can be inferred from fitness across a CO₂ titration.

a, Schematic of rubisco selection in [CO₂] titration and some examples of inferred Michaelis–Menten curves of mutants with varying K_C and V_max. b, Variant fitnesses at different [CO₂]. c, Measured fitnesses at different [CO₂] for two mutants (error bars, s.d. of the mean for N = 3 biological replicates). d, The same data as in c plotted under the assumptions of the Michaelis–Menten equation (error bars, s.d. of the mean for N = 3 biological replicates). e, Individually measured rubisco kinetics for the same two mutants from c and d (points, medians of N = 3 measurements; error bars, s.d.). f, Comparison between rubisco K_C values measured in vitro (spectrophotometric assay) and those inferred from fitness values \(({\widetilde{K}}_{{\rm{C}}})\). ρ is calculated from a Spearman correlation; P value reflects the result of a two-sided permutations test analysis. \({\widetilde{K}}_{{\rm{C}}}\) error bars, inner quartiles of the bootstrap fits (Methods); in vitro K_C error bars, s.d. from N = 3 measurements. g, Heatmap of \({\widetilde{K}}_{{\rm{C}}}\) values for all mutants for which the coefficient of variation is less than 1 (N = 5,687 mutants, 65% of total). Two positions with high-affinity mutations are highlighted in the inset expanded below. Variants for which the \({\widetilde{K}}_{{\rm{C}}}\) fits had a coefficient of variation above 1 are in grey. h, Two-dimensional histogram of mutant \({\widetilde{K}}_{{\rm{C}}}\) and \({\widetilde{V}}_{\max }\) values from g with hexagonal bins. Dashed lines, WT values.