PAOX1 expression in mixed-substrate continuous cultures of Komagataella phaffii (Pichia pastoris) is completely determined by methanol uptake regardless of the secondary carbon source: a simplifying principle for predicting recombinant protein production

Abstract

AbstractThe expression of recombinant proteins by the AOX1 promoter of Komagataella phaffii is typically induced by adding methanol to the cultivation medium. Since growth on methanol imposes a high oxygen demand, the medium is often supplemented with an additional “secondary” carbon source which serves to reduce the consumption of methanol, and hence, oxygen. Early research recommended the use of glycerol as the secondary carbon source, but more recent studies recommend the use of sorbitol because glycerol represses PAOX1 expression. To assess the validity of this recommendation, we measured the steady state concentrations of biomass, residual methanol, and AOX1 over a wide range of dilution rates (0.02–0.20 h-1) in continuous cultures of the Mut+ strain fed with methanol, methanol + glycerol, and methanol + sorbitol. We find that when the specific AOX1 expression and methanol uptake rates for each of the three feeds are plotted against each other, they collapse into a single hyperbolic curve. The specific AOX1 expression rate is therefore completely determined by the specific methanol uptake rate regardless of the existence (present/absent) and type (repressing/non-repressing) of the secondary carbon source. In particular, cultures fed with methanol + glycerol and methanol + sorbitol that consume methanol at equal rates also express the protein at equal rates and levels. Now, it turns out that the simple unstructured model developed by Egli and co-workers can predict the specific methanol uptake rates of single- and mixed-substrate cultures over a wide range of dilution rates and feed concentrations. By combining this model with our data, we derive simple formulas that predicts the protein expression rates and levels of single- and mixed-substrate cultures over a wide range of conditions.