Figure 3 . Modular development of a media for heterologous
protein production in P. pastoris
A) Initial full-factorial screen of nitrogen source choice and buffer pH
demonstrates that urea is preferred over ammonium sulfates and high
buffer pH is preferred over lower values. B) A full-factorial
concentration optimization identified methanol as the most concentration
dependent variable. Other components in the base media were predicted to
affect productivity with much lower levels of significance. C)
Evaluation of the effect of methanol concentration on P[8] titer,
using two different base media (urea, buffer, and YNB concentrations):
the biomass accumulation base medium and the optimal base media
composition predicted by our concentration DOE. D) Ranking of
supplements according to their effect on P[8] titer. Supplements
related to membrane fluidity or lipid metabolism ranked highly. E)
Evaluation of combinations of lipid and surfactant supplements confirmed
that cholesterol supplementation leads to the greatest improvement in
P[8] titer. F) Concentration optimization of cholesterol
demonstrated low concentration dependence, with similar performance
observed over a 40-fold range (0.2-8x). G) Comparing cholesterol-free
and cholesterol-supplemented cultures fed at various concentrations
demonstrates that cholesterol supplementation results in a significant
~25% improvement in P[8] titers
(p<0.001). H) No significantly beneficial supplements were
observed when repeating the supplementation screen. I) Screening
supplementation of 20 g/L of co-fed substrates individually or in 1:1
combinations by mass identified sorbitol supplementation as highly
beneficial to P[8] titer. J) Examination of the effect of co-feed
ratio and total carbon concentration on titer in DM2_dev1 supplemented
media. K) Comparison of P[8] titer obtained with DM2 to previous
iterations and other common P. pastoris media demonstrates a
~2x improvement in P[8] titer, relative to 1 v/v%
methanol RDM and 1 v/v% methanol BMMY.