Figure 2 . Modular development of a new biomass accumulation media for P. pastoris
A) Significance of carbon (fructose, glucose, glycerol), nitrogen (urea and ammonium sulfate), and pH choice (5, 5.75, 6.5) in a least square regression model fitted to a full factorial DOE. B) Fructose and glucose were found to result in significantly higher biomass accumulation after 24 hours of outgrowth than glycerol. C) Ammonium sulfate was found to be more pH sensitive than urea, as shown by the JMP sensitivity profiles during fructose feeding. D) Significance of terms in a least square regression model fitted to a full factorial DOE over fructose, urea, potassium phosphate, and YNB concentrations. E) 1-FAAT optimization of fructose and YNB concentration finds optimal outgrowth performance at a fructose concentration of 22.5 g/L and relative insensitivity over a wide range of YNB concentrations (0.15 to 1.2x). F) A media supplementation screen identified 5 beneficial supplements, related to trace element and amino acid supplementation. G) Further screening of beneficial supplement combinations identified synergistic amino acid and trace metal supplementation strategies. H) Comparison of the effect of MEM amino acid concentration on biomass accumulation at different PTM1 salts concentrations. I) Effect of the concentration of PTM1 salts on biomass accumulation in DM1_dev0 medium supplemented with 1x MEM AA. J) Head-to-head comparison of 4 v/v% glycerol BMGY, 4 v/v% glycerol rich defined medium, the initial defined biomass accumulation media (DM1_dev0), and the final biomass accumulation medium obtained after a full optimization cycle (DM1), demonstrates that DM1 leads to superior biomass accumulation.