Effect of Dilution Rate and Methanol-Glycerol Mixed Feeding on Heterologous Rhizopus oryzae Lipase Production with Pichia pastoris Mut1 Phenotype in Continuous Culture

The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris.Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production ofRhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol-glycerol feeding mixtures in fast metabolizingmethanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of bothdilution rate and methanol-glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04-0.06 h(-1) , while ROL yield decreased along the whole dilution rate range evaluated (0.03-0.1 h(-1) ). Compared to production level achieved with methanol-only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g-biomass(-1) h(-1) ), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut(+)phenotype for heterologous protein production.