Fig. 2: Adaptive laboratory evolution to improve native methanol assimilation in CEN.PK 113-5D.

a Schematic of the ALE design. Three independent lineages of CEN.PK 113-5D (with pRS416) were grown in baffled shake flasks in condition A or B for 230 generations. Under condition A, cultures were passaged from YNB medium without amino acids and 1% glucose (24 h) to YNB medium without amino acids and 0.1% yeast extract (YE) (48 h). Under condition B, cultures were passaged from YNB medium without amino acids and 1% glucose to YNB medium without amino acids, 2% methanol and 0.1% yeast extract (MeOH + YE) (48 h). Figure was created with BioRender.com. b Schematic of the mutations in YGR067C from the three evolved lineages grown under condition B, and the changes they caused to the protein, all three mutations theoretically led to truncations. c Growth profiles of CEN.PK 113-5D (black squares), the three evolved lineages (A, dark blue upright triangles. B, light blue inverted triangles. C, purple diamonds.) in condition B, and the reconstructed CEN.PK 113-5D strain with the mutation observed in the evolved lineage C (green circles). Strains were grown in liquid YNB medium with 0.1% yeast extract and 2% methanol. Data are from two independent biological replicates with lines representing the mean. Source data are provided as a Source Data file.