On-line monitoring and control of methanol concentration in shake-flask cultures ofPichia pastoris

Abstract

The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol‐inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas‐permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed‐batch shake‐flask culture of a P. pastoris clone producing the N‐lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off‐line by HPLC. A closed‐loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed‐batch protocol. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 279–286, 1997.

Heterologous protein production using the Pichia pastoris expression system

“…Production of large amounts of heterologous proteins in shake-flask culture is difficult, due to the limitations of volume, oxygen transfer, substrate addition and an inability to monitor these factors efficiently. Methods of monitoring methanol in shake flasks are available, such as organic solvent vapour detectors [40], and the oxygen transfer of these systems can be increased by the use of baffles [114]. On-line measurement of oxygen and carbon dioxide transfer rates in shake flasks has also been demonstrated using a respiration activity monitoring system (RAMOS) [100].…”

“…Protein levels in fermenter culture are typically much higher than in shake-flask cultures, although this may be due to poor optimization of methanol-feeding strategies in shake-flask cultures [40]. For this reason, scale-up from shake flask to bioreactor requires much more reoptimization than scale-up from small to large bioreactors [21].…”

“…So, an accurate picture of what is happening within the bioreactor at any given time during the process cannot be obtained. On-line analysis of methanol concentrations in shake flasks has been demonstrated [40,134] using sensors that detect methanol vapour; however, sequential injection analysis (SIA) has been used for on-line methanol determination in a small bioreactor with 1 l working volume [108]. The SIA analyser worked by removing a sample from the fermenter automatically, treating it and then subjecting the sample to enzymatic analysis.…”

Fermentation strategies for recombinant protein expression in the methylotrophic yeastPichia pastoris

“…Brierley et al [5] observed that the Mut + strain was very sensitive to changes in the residual methanol level. Guarna et al [41] compared limited growth to growth while maintaining methanol at 0.3% (v/v) in shake flasks. Katakura et al [42] determined the specific growth rates on methanol at several levels.…”

Disruption of Pichia pastoris PMR1 gene decreases its folding capacity on human serum albumin and interferon‐α2b fusion protein

“…The discrepancy may be ascribed to a high content of calcium (mM grade) and other nutrition components in yeast extract and tryptone (Farrell et al, 1993). For BMMY medium, it may also reflect a suboptimal induction strategy that resulted in carbon source starvation (Guarna et al, 2000).…”