© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. The methylotrophic yeast Pichia pastoris (Komagataella spp.) is widely used as cell factory for recombinant protein production. In the past recent years, important breakthroughs in the systems-level quantitative analysis of its physiology have been achieved. This wealth of information has allowed the development of genome-scale metabolic models, which make new approaches possible for host cell and bioprocess engineering. Nevertheless, the predictive accuracy of the previous consensus model required to be upgraded and validated with new experimental data sets for P. pastoris growing on glycerol or methanol as sole carbon sources, two of the most relevant substrates for this cell factory. In this study, we have characterized P. pastoris growing in chemostat cultures using glycerol or methanol as sole carbon sources over a wide range of growth rates, thereby providing physiological data on the effect of growth rate and culture conditions on biomass macromolecular and elemental composition. In addition, these data sets were used to improve the performance of the P. pastoris consensus genomic-scale metabolic model iMT1026. Thereupon, new experimentally determined bounds, including the representation of biomass composition for these growth conditions, have been incorporated. As a result, here, we present version 3 (v3.0) of the consensus P. pastoris genome-scale metabolic model as an update of the iMT1026 model. The v3.0 model was validated for growth on glycerol and methanol as sole carbon sources, demonstrating improved prediction capabilities over an extended substrate range including two biotechnologically relevant carbon sources.