Development and in silico analysis of a new nitrogen-limited feeding strategy for fed-batch cultures of Pichia pastoris based on a simple pH-control system

• A new nitrogen-limited fed-batch strategy is presented.
• Manipulating the pH controller settings for base addition varied growth rate.
• Higher rate of base addition causes increased growth and protein production.
• This feeding strategy was investigated by a genome-scale metabolic model.
• Higher speed of adding base causes higher fluxes of intracellular reactions.

A novel fed-batch strategy based on a pH control system was devised in order to address some of the disadvantages associated with available control strategies for Pichia pastoris cultures. Changing settings of the controller, the tON/tOFF time ratio, adjusted the speed in which the ammonium hydroxide was added for pH control and the sole nitrogen source. Setting the tON/tOFF ratio to 0.5/0.5, 0.5/10 and 0.5/15 resulted in a decrease in the growth rate as the tOFF value increased. Therefore, the design allows running the culture at lower growth rates in a controlled way. Two high cell density cultures with tON/tOFF ratio of 0.5/10 and 0.5/20 were also fed ammonia with the same controller. Setting tON/tOFF at 0.5/10 and 0.5/20 led to an increase in final biomass and protein as the tOFF decreased. A metabolic model assessed the effect of nitrogen-limitation in high cell density cultures on the yeast metabolism. Model results show that fluxes of intracellular reactions such as glycolysis, the tricarboxylic cycle and the pentose-phosphate pathway are higher at tON/tOFF = 0.5/10. Additionally, higher sensitivity of growth and protein production to oxygen at tON/tOFF = 0.5/10 proves that oxygen limitation led to higher protein production in this case.