EFFECT OF METHANOL CONCENTRATION ON THE RECOMBINANT PICHIA PASTORIS MUT<I>^S</I> FERMENTATION

Abstract

Methanol is the sole carbon and energy source as well as inducer of heterologue protein production in recombinant Pichia pastoris fermentations. The role of methanol on P. pastoris GS115 Mut^S HSA model strain was thoroughly studied in a series of fed-batch cultivations at different constant alcohol levels in bench-top and pilot-scale bioreactors. The results showed that whereas specific growth rate does not show correlation with methanol concentration, specific product formation rate is strongly dependent on substrate level. The fact that the specific growth rate did not decrease with increasing methanol concentrations can be explained by the diminished toxic metabolic compound formation compared to P. pastoris Mut ⁺ cells. While metabolic inhibition was not characteristic in the range of methanol concentration 0.45-8.85 g/L, the product formation was mainly controlled by the initiation of transcription and was favoured by low methanol concentrations. As a result of the relatively high value of maintenance coefficient (0.026 1/h), all fermentations were performed in energy limit which explains that the specific product formation rate was not correlated to the specific growth rate. The highest value of volumetric productivity (0.0187 mg HSA/(L*h)) was measured at 0.45 g/L methanol concentration. The values of specific metabolic quotients as functions of methanol concentration in scaled-up fermentations coincided with the results in bench top reactors when adequate mixing was applied.