High-Density P. pastoris Fermentation

The fermentation was carried out in a 5 L fermentor with 2 L BSM supplemented containing 8 mL/L PTM1. High cell density fermentation of P. pastoris was applied based on the Pichia Fermentation Process Guidelines (Invitrogen). The pH and the temperature were set at 6 and 28 °C, respectively. During the cell growth phase, the cells were grown until the glycerol were exhausted, which indicates the increase in dissolved oxygen (DO) level. When the glycerol was exhausted around 12 h, the feeding medium containing 50% (w/v) glycerol and 12 mL/L PTM1 solution was pumped in according to a predetermined DO level (10–20%). When the OD₆₀₀ reached 360, the glycerol feed was stopped, which raised DO after about 30 min. At that time, pure methanol containing 12 mL/L PTM1 solution was fed to induce the targeted gene expression, and the fermentation temperature were adjusted to 25 °C, with the DO being set to 10–20%. Culture samples were taken every 12 h to determine the OD₆₀₀, dry weight of the cells, and enzyme activity.