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High Yield Protein Production from Pichia pastoris Yeast: A Protocol for Benchtop Fermentation

). However, these procedures can be adapted to other size fermentors thereby making the protocols scalable. In the authors laboratories, P. pastoris fermentations are run as multi-stage fed-batch processes with oxygen supplementation (Table 1). Here, oxygen is supplied automatically to meet the dissolved oxygen requirements for highdensity cell growth.


Method for a Typical Culture
A frozen vial of 1 ml P. pastoris sample was inoculated into a 1 L shake flask with 150 mL Yeast Nitrogen Base (YNB)-glycerol medium. A variety of genetically engineered P. pastoris strains were used, many of which are slow growing on methanol (muts) and engineered to produce proteins of interest. The culture was incubated at 30C, 240 rpm, for 14 hours in an environmental incubator shaker (New Brunswick Scientific). The entire 150 mL volume of inoculum was transferred to a 3.3 L fermentor vessel (total volume) containing 1.5 L of basal salts medium (see media components, Table 2) plus 4.4 mL/L trace metal solution (4). The temperature was controlled at 30C. The dissolved oxygen was set at 30% and pH is at 5.0. Ammonium hydroxide solution (30%) was used as the base solution to adjust the pH. After 20 hours of batch culture, the optical density (OD) reaches 42. The glycerol fed-batch process was then initiated. The feeding medium consisted of 50% glycerol and 12 mL/L of trace metal solution. The feed rate was 24 mL/L/h, which was adjusted automatically based on the DO reading. DO control was maintained by the proportional integral derivitive (PID) cascade controller, which changes the speed of agitation. Pure oxygen was automatically supplied to the fermentor to keep the DO level at the setpoint after the agitation speed reached the maximum allowable setpoint. After the growth phase, a half-hour carbon-source starvation period was establish
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