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 formaldehyde through maintaining DO at the sufficient level and switching energy regeneration pattern from formaldehyde 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.

甲醇是驱动巴斯德毕赤酵母重组蛋白表达的唯一诱导剂补料分批发酵。然而，当采用纯甲醇进料并将其浓度控制在最佳水平（5-10 g/L）时，由于其高燃烧焓，溶解氧浓度（DO）会降低到极限条件，导致甲醛的严重积累。和有限的重组蛋白表达。为了解决这些问题，借助山梨糖醇的较低燃烧焓，以莫内林生产为原型，提出了一种高效的甲醇/山梨糖醇在线共供策略。该策略的方案是通过 ON-OFF 控制方式控制甲醇的进料，通过控制甲醇浓度在 5-10 g/L 左右来保证甲醇的诱导强度，而山梨糖醇的共同进料是通过改进的 DO-stat 模式来确定的，通过将 DO 维持在 5-15% 的足够水平来抑制甲醛的积累。采用这种策略时，1) DO 保持在足够的水平，因此甲醛积聚严重降低到接近零的水平；2) 维持细胞的甲醇代谢通量可以保持在最低水平（0.011 1/h vs 0.018–0.026 1/h）；3) 蛋白酶对莫内林的降解减弱；4）促进莫内林生物合成的主要能量再生模式从甲醛（甲醇）异化途径转变为TCA循环；5) 获得了2.45 g/L的最大莫内林表达滴度，与仅使用甲醇喂养获得的相比是2.35-4.53倍。综上所述，