d'Anjou M C, Daugulis A J
Department of Chemical Engineering, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Biotechnol Bioeng. 2001 Jan 5;72(1):1-11. doi: 10.1002/1097-0290(20010105)72:1<1::aid-bit1>3.0.co;2-t.
A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy.
一种经过基因改造以生产和分泌海鸦抗冻蛋白的Mut(S)毕赤酵母菌株被用作模型系统，以展示一种合理的、基于模型的方法来提高工艺生产力。在5升规模下进行了一组甘油/甲醇混合进料连续搅拌釜式反应器（CSTR）实验，以表征比生长速率与甲醇上的细胞产率、比甲醇消耗速率、比重组蛋白形成速率以及基于分泌蛋白水平的生产力之间的关系。研究的稀释率范围为0.01至0.10 h(-1)，残余甲醇浓度保持在约2 g/L恒定（低于抑制水平）。假设甘油上的细胞产率恒定，在研究范围内，甲醇上的细胞产率从约0.5增加到1.5。在稀释率为0.06 h(-1)时，比甲醇消耗速率达到最大值20 mg/g·h。在所研究的稀释率范围内，基于产物的比产物形成速率和体积生产力持续增加，最大值分别为0.06 mg/g·h和1.7 mg/L·h。因此，在此范围内未观察到甘油抑制的证据，并且在所研究的最高稀释率下操作可使生产力最大化。基于Monod型动力学和从CSTR工作期间收集的数据得出的参数的补料分批质量平衡方程，随后用于预测细胞生长和重组蛋白生产，并开发使用两种碳源的指数补料策略。根据预测的补料策略，在比生长速率为0.03 h(-1)和0.07 h(-1)下进行了两次指数补料分批发酵，以验证模型的准确性。在两次补料分批运行中，细胞生长均得到准确预测；然而，模型低估了重组产物浓度。两次运行的总体积生产力约为2.2 mg/L·h，与启发式补料策略相比，生产力提高了十倍。
