A new methanol-feeding strategy for the improved production of β-galactosidase in high cell-density fed-batch cultures of Pichia pastoris Mut+ strains

Developing novel methanol-feeding strategies for the improved production of heterologous proteins in high cell-density fed-batch cultures of Pichia pastoris has been of great interest during recent years. In this study, a recombinant P. pastoris strain (GS115/His(+) Mut(+)) producing beta-galactosidase (beta-Gal) was used to investigate conventional feeding strategies and to develop a new strategy to increase the recombinant protein production during fedbatch cultures on methanol. Three types of conventional methanol-feeding strategies, including mu-stat, dissolved oxygen-stat (DO-stat) and constant methanol concentration were investigated and compared with respect to alcohol oxidase (AOX), formate dehydrogenase (FDH) and beta-gal activities, and cell dry weight (CDW), methanol, and formaldehyde concentration variations during the production phase. Methanol feeding with mu-stat 0.025/h exhibited the highest beta-gal activity. Supplementing ammonium and magnesium in mu-stat 0.025/h did not affect the cell growth or methanol or formaldehyde concentrations throughout the fermentation but did improved the maximum beta-gal activity from 148.2 to 158.1 kU/mL. A new three-step methanol-feeding strategy was developed based on the results obtained from conventional feeding strategies, which started with mu-stat 0.025/h for 5 h, then mu-stat 0.030/h, and finally, was switched to DO-stat when maintaining the DO above 20% air saturation became difficult. Implementation of this new feeding strategy resulted in a CDW of 107.2 +/- 0.7 g/L, AOX specific activity of 0.1890 +/- 0.0030 U/mg CDW, and beta-gal activity of 173.5 +/- 2.1 kU/mL after 29 h of fermentation, which shows a 5.6, 29.1, and 15.7% increase in CDW, AOX, and beta-gal activity, respectively, compared to that of mu-stat at 0.025/h.