Impact of methanol concentration on secreted protein production in oxygen‐limited cultures of recombinant Pichia pastoris

Abstract

The methylotrophic yeast Pichia pastoris is a powerful system for production of recombinant proteins, showing high ability to secrete properly folded proteins. A major plus is the strong AOX1 promoter highly induced by methanol. During growth on methanol, however, oxygen readily becomes limiting. In oxygen-limited cultivations of recombinant Pichia pastoris, the methanol concentration had a strong impact on the production of a single-chain antibody fragment (scFv). High methanol concentrations were required to compensate the lack of oxygen and fully induce recombinant protein production, at the same time reducing gratuitous biomass formation due to a lower biomass yield. Product concentrations of 60, 150, and 350 mg/L were obtained with methanol concentrations of 0.3, 1, and 3% (v/v). Moreover, accumulation of a putative product fragment that cannot be removed during affinity purification was prevented at high methanol concentrations. Cell vitality after 100 h was maintained above 98% and 96% of the culture with 0.3% and 3% methanol, respectively. In cultivations supplemented with oxygen, in contrast, methanol concentration between 0.3% and 3% did not influence the product yield of 300-400 mg/L. Thus, efficient recombinant protein production under oxygen-limitation seems to require high methanol concentrations, enabling product concentration as high as otherwise obtained only with expensive supply of pure oxygen.

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

“…anol or intermediates formed during methanol catabolism (29,41,46). The observation that cells which had previously been exposed to (and simultaneously utilized) methanol are more sensitive to any additional stress factor introduced subsequently is consistent with previous results.…”

“…There is a wide range of fluorescent dyes available, which enable assessment of both changes to the membrane structures and to the physiology of individual cells in different ways. However, staining by PI remains the gold standard for the determination of vitality (3,7,27,41,46,59). The results obtained with using other vitality dyes (cFDA, EB, and BOX) usually correlate with those obtained with using PI.…”

“…Typically, only one or two process strategies for each particular strain investigated are reported in the literature (5,41,59), and conclusions on the combination of factors affecting cell vitality are seldom possible. Although it is thought that the physiological effects attributed to individual factors are cumulative, it was found that a combination of several factors exerts a synergistic effect on cell vitality.…”

Quantifying the Effects of Frequency and Amplitude of Periodic Oxygen-Related Stress on Recombinant Protein Production in Pichia pastoris

“…These are the typical values for other experiments as well. The reduction in the biomass yield in the course of the induction phase was also reported by Jahic et al [19] and reasoned by increased cell death under methanol-limited conditions [20,21]. However, as it can be seen in Figure 2(b), the difference in yields between experiment 1 and 4 was not significant.…”

Optimal fed-batch operation of recombinant cells subject to plasmid instability and death

“…Recombinant DNA technology has helped to produce valuable cell mass or metabolites in many microorganisms [6,12,15,16,27]. Cell concentration, plasmid stability, gene expression efficiency and metabolite yield etc.…”