Enhancing monellin production in Pichia pastoris fed-batch fermentation through an efficient on-line methanol/sorbitol co-feeding strategy

Methanol is the sole inducer for driving recombinant protein expression in P. pastoris fed-batch fermentation. However, when adopting methanol-only feeding and controlling its concentration at the optimal level (5-10 g/L) would decrease dissolved oxygen concentration (DO) down to limitation conditions due to its high combustion enthalpy, leading to the severe accumulations of formaldehyde and a limited recombinant protein expression. To solve these problems, with help of the lower combustion enthalpy of sorbitol, an efficient on-line methanol/ sorbitol co-feeding strategy was proposed, using monellin production as a prototype. The scheme of this strategy was that the feeding of methanol was controlled by ON-OFF control mode to ensure methanol induction strength via controlling its concentration around 5-10 g/L, while the co-feeding of sorbitol was determined by an improved DO-stat mode to repress the accumulation of formaldehyde via maintaining DO at sufficient levels of 5-15%.When adopting this strategy, 1) DO was maintained at sufficient levels and thus the severe accumulation of formaldehyde was reduced to nearly zero level; 2) the methanol metabolic flux to cells maintenance could be kept at the lowest level (0.011 1/h vs 0.018-0.026 1/h); 3) monellin degradation by proteases was weakened; 4) the major energy regeneration pattern energizing monellin biosynthesis switched from formaldehyde (methanol) dissimilatory pathway to TCA cycle; 5) the maximum monellin expression titer of 2.45 g/L was obtained, which was 2.35-4.53 fold compared with that obtained using methanol-only feeding. In conclusion, the proposed on-line methanol/sorbitol co-feeding strategy could inhibit the severe accumulation of toxic metabolite formalde-hyde through maintaining DO at the sufficient level and switching energy regeneration pattern from formal-dehyde dissimilatory pathway to TCA cycle, while increasing cells metabolic activities reflected by a lower maintenance coefficient and formation rate of proteases etc., which eventually achieved an effective monellin expression in turn.