Analysis of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated formaldehyde dehydrogenase promoter (PFLD1)

Abstract

Background Methanol-free high cell density fed-batch cultivation strategies for the P. pastoris expression system have been recently developed by expressing a Rhizopus oryzae lipase (ROL) under the transcriptional control of the PFLD1 [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. Fed-batch fermentations were performed at three different specific growth rates and showed that productivities were strongly correlated with this parameter (i.e. with the cell's physiological state). 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.