Fig. 2

ALE enhanced final biomass and tolerance to 33 °C of P. pastoris in 0.5% methanol minimal medium. a, The process of ALE; b, Procedure of ALE that was illustrated by enhanced cell growth; c, Automatic growth curves of the starting strain X-33 at 30 or 33 °C and the evolved strains at 33 °C; d, % maximum OD₆₀₀ of the starting strain X-33 and the evolved strains at 33 °C relative to the starting strain X-33 at 30 °C. Three independent colonies of X-33 were transferred to a 250 ml shake flask with 100 mL of 0.5% methanol minimal medium with an initial OD₆₀₀ of 0.25 at 33 °C at 220 rpm. Transferred the evolved strains to fresh growth medium every 24 h, until a high density of cells were obtained. Final evolved strains were spotted on YPD plates for further growth analysis using an automatic microbial growth curve analyzer. Data are average values and standard deviations of triplicate experiments