The potential biotechnological applications for the Ophiostoma piceae sterol esterase (OPE) are conditioned to the availability of high enzyme amounts at low prices. This enzyme is a versatile biocatalyst with different biotechnological applications. In this work a systematic study on its heterologous production in different Pichia pastoris strains and operational strategies is presented. The best results were obtained using an AOX1 defective yeast strain in a fed-batch bioprocess using methanol as inducer substrate at a set point of 2.5 g L-1 and sorbitol as cosubstrate by means of a preprogramed exponential feeding rate at a mu = 0.02 h(-1), reaching 30 U mL(-1) of enzyme and a volumetric productivity of 403.5 U L-1 h(-1). These values are twofold higher than those obtained with a Mut(+) phenotype using methanol a sole carbon source. OPE was the main protein secreted by the yeast, 55% for Mut(s) versus 25% for Mut(+). (C) 2014 American Institute of Chemical Engineers