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.