Controlling methanol feeding for recombinant protein production by Pichia pastoris under oxidation stress in fed-batch fermentation

Background: Methanol can be used by Pichia pastoris as the sole carbon source and inducer to produce recombinant proteins in high-cell-density fermentations, but also damages cells due to reactive oxygen species (ROS) accumulation from methanol oxidation. Here, we study the relationship between methanol feeding and ROS accumulation by controlling constant methanol feeding rate during the induction phase.Results: Higher methanol feeding rate increased the level of ROS accumulation caused by methanol oxidation. While the cell growth rate was proportional to the rate of methanol feeding rate, but maximum total protein production and highest enzyme activity were achieved at methanol feeding rate 4 mL/(L·h) as compared to that with 5 mL/(L·h). Moreover, oxidative demage induced by over accumulation of ROS in P. pastoris during the methanol induction phase caused cell death and reduced protein expression ability. ROS scavenging system analysis reveals that the higher methanol feeding rate, especially 5 mL/(L·h), resulted in increased intracellular catalase activity and decreased glutathione content significantly. Finally, Spearman's correlation analysis further reveals that the reduced glutathione might be beneficial for maintaining cell viability and increasing protein production under oxidative stress caused by toxic accumulation of ROS accumulation.Conclusion: Our findings suggest an integrated strategy to control the feeding of the essential substrate based on analyzing its response to oxidative stress caused by toxic accumulation of ROS accumulation, as well as develop strategy to optimize fed-batch fermentation.