Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter.

The yeast Pichia pastoris has been previously used for extracellular expression of a Rhizopus oryzae lipase (Rol). However, limitations in Rol folding and secretion through the cell wall became apparent when producing it in fed-batch cultivations. In this study, we have investigated the effect of combining two cell engineering strategies to alleviate putative bottlenecks in Rol secretion, namely the constitutive expression of the induced form of the Saccharomyces cerevisiae unfolded protein response transcriptional factor Hac1 and the deletion of the GAS1 gene encoding beta-1,3-glucanosyltransglycosylase, GPI-anchored to the outer leaflet of the plasma membrane, playing a key role in yeast cell wall assembly. The performance of these engineered Rol-producing strains has been compared in fed-batch cultivations set at a low specific growth rate of about 0.005 h-(1). It was found that Rol overexpression in a P. pastoris strain expressing constitutively the induced form of S. cerevisiae Hac1 and the deletion of GAS1 resulted in about a 3-fold and 4-fold increase in the overall process specific productivity, respectively, whereas the double mutant HAC1/deltagas1 strain yielded about a 7-fold increase. Overall, these results reflect the multiplicity of physiological bottlenecks at different levels/steps throughout the Rol synthesis, secretion and excretion processes in P. pastoris.