重组毕赤酵母表达猪胰岛素前体代谢参数分析和动力学模型

Metabolic Data Association Analysis and Modeling for Recombinant Porcine Insulin Precursor Expression in Genetically Engineered Pichia pastoris

采用发酵过程参数相关分析理论,分析了毕赤酵母在不同发酵阶段利用不同碳源时的生理代谢参数变化特征。同时,研究了不同比生长速率下重组毕赤酵母表达猪胰岛素前体补料分批发酵过程,建立蛋白表达阶段的细胞生长非结构模型,结果表明:比生长速率和甲醇浓度符合Monod方程,最大比生长速率(μmax)为0.101h-1,基质的半饱和常数(Ks)为0.252g/L,细胞得率系数YX/S=0.386g/g,细胞维持系数m=0.011g/(g·h)。当比生长速率为0.016h-1时猪胰岛素前体(PIP)最大比形成速率(qp,max)达0.098mg/(g·h)。该模型能较好预测毕赤酵母表达阶段细胞生长和PIP表达的发酵趋势,用其控制发酵过程,目标蛋白PIP产量为优化前的1.5倍,达0.976g/L。

Based on the multi-scale data association analysis of a bioprocess, the profile of physiological parameters (such as OUR, CER and RQ) of Pichia pastoris were analyzed at different fermentation phases with different carbon sources as the sole substrate. A kinetic model of recombinant methylotrophic yeast Pichia pastoris expressing porcine insulin precursor was constructed in the quasi-steady state at the induction phase. The model revealed that the relationship between specific growth rate μ and residue methanol concentration was in accordance with Monod equation. The fermentation kinetic parameters are as follows: maximum specific growth rate μmax = 0.101 h-1, and Ks = 0.252 g/L. YX/S = 0.386 g/g, and m = 0.01l g/(g·h). The maximum specific protein production rate qp,max was 0.098 mg/(g·h) when μ was kept at 0.016 h-1. The model can be used to predict the cell density and PIP production during methanol induction phase with different initial cell density. When the model was applied to the control of PIP fermentation process, the yield of PIP reached 0.976 g/L, which was 1.5-fold as that of the control.