Abstract
Pichia pastoris is an attractive candidate platform for recombinant protein production. Dissolved oxygen is one of the most important factors in the cultivation of P. pastoris. However, the effect of oxygen on triggering productivity led to ambivalent results. In our earlier work, a two-compartment system, consisting of a single reactor coupled with a plug flow reactor (PFR), has been proposed as a tool to improve protein quantity and quality. The goal of this work was to investigate the effects of frequency (the residence time of broth in the PFR) and amplitude (the dissolved oxygen level in the reactor) of the stress on productivity, titer and physiology. A recombinant P. pastoris strain, which expressed horseradish peroxidase, was used as the model system. Thirteen experiments were performed. Multivariate data analysis was done and the results showed that the residence time did not influence titer, productivity and physiology over the range of residence time studied while dissolved oxygen influenced titer and specific productivity in a quadratic function. In other words, an intermediate level of dissolved oxygen (25%) showed the highest specific productivity and titter, irrespective of the residence time in the PFR. In turn, the variation of the residence time and dissolved oxygen did not influence growth physiology, as quantified in biomass and carbon dioxide yields.
Highlights
The methylotrophic yeast Pichia pastoris is an attractive candidate platform system for recombinant protein production as it can secrete properly folded proteins
The phenotype of the strain corresponded to an AOX1-deficient clone which is characterized as MutS and horseradish peroxidase (HRP) was secreted into the fermentation broth
All experiments showed similar trends in the responses on DOE1 experiments (Figure 2): The specific productivity increased in a linear manner in the beginning and stayed in a constant level or even decreased (Figure 2(a))
Summary
The methylotrophic yeast Pichia pastoris is an attractive candidate platform system for recombinant protein production as it can secrete properly folded proteins. It is one of the most effective and versatile systems for expression of heterologous proteins [1] because of these reasons: (1) high growth rate (2) high level of productivity in an almost protein-free medium (3) ease of genetic manipulation (4) and the absence of known human pathogenicity [2]. The promoter is repressed in the presence of glycerol as well as glucose and induced in the presence of methanol [1]. The methanol feed rate is increased to the designed flow rate and the recombinant product is induced [4]
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