Analysis of single‐chain antibody production in Pichia pastoris using on‐line methanol control in fed‐batch and mixed‐feed fermentations

Abstract

In the last few years the Pichia pastoris expression system has been gaining more and more interest for the expression of recombinant proteins. Many groups have employed fermentation technology in their investigations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range. A large number of heterologous proteins from different sources has been expressed, but the fermentation process technology has been investigated to a lesser extent. A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fermentations of methylotrophic yeasts. In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products. We have used a commercially available methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of a Mut(+) strain of Pichia pastoris. Specific glycerol feed rates in the range of 38-4.2 mg. g(-1). h(-1) (mg glycerol per gram fresh weight per hour) were investigated. Expression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg. g(-1). h(-1). At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation. These results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research.

Enhancement of thermoalkaliphilic xylanase production by Pichia pastoris through novel fed-batch strategy in high cell-density fermentation

“…1a). It meant that P. pastoris cells growth could be supported by glycerol or glucose, however, xylanase gene expression driven by AOX1 promoter can’t be induced in glycerol- or glucose-containing medium, which was in agreement with Hellwig’s report [29]. When 0.5% (v/v) methanol was added at 60 h, the xylanase activity increased in the medium whose initial carbon source was glucose or glycerol, and it was always higher than that in the medium with maltose as initial carbon source.…”

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

“…For both batch and fedbatch cultures, a defined mineral medium adapted from Hellwig et al (2001) was used containing 0.17 g CaSO 4 ·2H 2 O, 2.86 g K 2 SO 4 , 0.64 g KOH, 2.3 g MgSO 4 ·7H 2 O, 0.2 g EDTA, 7.23 g H 3 PO 4 and 0.1 ml of polypropylene glycol (PPG), which were all autoclaved, and 4.35 ml of filter-sterilised PTM1 solution l −1 and 0.87 mg of biotin l −1 , which were added separately. The PTM1 stock solution (Invitrogen 2002) contained 5.0 ml of 69% H 2 SO 4 , 3.84 g CuSO 4 , 0.08 g NaI, 3.0 g MnSO 4 ·H 2 O, 0.2 g Na 2 MoO 4 ·2H 2 O, 0.02 g H 3 BO 3 , 0.92 g CoCl 2 ·6H 2 O, 20.0 g ZnCl 2 and 65.0 g FeSO 4 ·7H 2 O l −1 .…”

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

“…These findings are especially noteworthy because glycerol, glucose, ethanol and acetate have all been shown to support growth of P. pastoris cells without inducing the AOX promoter [16]. For example, Hellwig and colleagues [17] demonstrated glycerol in the culture medium inhibited production of a recombinant single-chain antibody in mixed feed bioreactor cultures, while Inan and Meagher reported that ethanol and acetate repress AOX -driven expression [16]. …”