Fig. 1: A deep mutational scan individually characterizes all single-amino acid mutations in rubisco.
a, Summary of the metabolism of Δrpi—the rubisco-dependent strain. b, Δrpi grows with a rate proportional to the flux through rubisco. c, Schematic of library selection. A library of rubisco single-amino acid mutants was transformed into Δrpi then selected in minimal medium supplemented with glycerol at elevated CO2. Samples were sequenced before and after selection and barcode counts were used to determine the relative fitness of each mutant. d, Correspondence between two example biological replicates; each point represents the median fitness among all barcodes for a given mutant. e, Fitness of 77 mutants with measurements in previous studies compared with the rate constants measured in those studies (kcat). The outlier is I190T (see Methods for discussion). Fitness error values are the s.e.m. of nine replicate enrichment measurements; kcat errors are from the literature, where available. f, Variant fitnesses (grey) were normalized between values of 0 and 1, with 0 representing the average of fitnesses of mutations at a panel of known active site positions (red distribution, average is plotted as a red dashed line) and 1 representing the average of wild-type (WT) barcodes (white dashed line). g, Heatmap of variant fitnesses. Conservation by position and sequence logo were determined from a MSA of all rubiscos. Black triangle, G186 (an example of a position with high conservation that is mutationally tolerant); grey triangles, active site positions. Ri5P, ribose 5-phosphate; Ru5P, ribulose-5-phosphate; RuBP, ribulose-1,5-bisphosphate; TIM, triosephosphate isomerase.
