Transcriptional analysis of protein production and induction of unfolded protein response in Pichia pastoris expressing a Rhizopus oryzae lipase under the FLD1 promoter

Abstract

Background Methanol-free high cell density fed-batch cultivation strategies for the Pichia pastoris expression system have been recently developed by expressing a Rhizopus oryzae lipase (ROL) under the transcriptional control of FLD1 promoter (P FLD1)[1]. These cultivation strategies were based on the use of sorbitol and methylamine as carbon and nitrogen source, respectively, during the induction phase of the cultivation process. The specific growth rate proved to be an important parameter in the productivity of secreted ROL. Moreover, intracellular active product accumulation and a decrease in the specific product secretion rate were observed along the induction phase of the fermentation process. These results suggested the presence of a bottleneck(s) throughout the synthesis and secretion process of the heterologous lipase. Recently [2], flow cytometry analyses of intracellular ROL levels confirmed that a fraction of the product was retained within the cell. Further, this intracellular product accumulation was concomitant with an increase on the BiP protein, a chaperone of the HSP70 class that plays an important role in the unfolded protein response (UPR). Notably, the increase of BiP and ROL content in the cell was detected soon after the beginning of the induction phase. Interestingly, the intracellular BiP and ROL profiles were different depending on the specific growth rate of the cells. In this study, we report the application of a sandwich hybridization assay-based technique [3] for quantification of specific mRNAs levels during the extracellular production of ROL in P. pastoris under the transcriptional control of PFLD1. These studies have been carried out in fed-batch cultures at two different specific growth rates.