Fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co-substrate sorbitol
Fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co-substrate sorbitol
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DOI：
10.1002/yea.1679
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发表时间：
2009-09-01
期刊：
影响因子：
2.6
通讯作者：
Oliver, Stephen G.
中科院分区：
文献类型：
--
作者：
Celik, Eda;Calik, Pinar;Oliver, Stephen G.
提出在毕赤酵母 (Mut(+)) 甲醇补料分批发酵诱导阶段分批添加山梨醇作为共底物作为一种有益的重组蛋白生产策略，并系统研究了山梨醇存在下对甲醇补料速率的代谢反应。与单独使用甲醇生长相比，向培养基中分批添加山梨醇具有以下优点：(a) 消除细胞的长滞后期，并在过程的 t = 24 小时时达到“高细胞密度生产”（C-X = 70 g CDW/I）； (b) 实现高 1.8 倍的重组人促红细胞生成素 (rHuEPO)（t = 18 小时）； (c) 特异性蛋白酶产量减少 1.2 倍； (d) 消除乳酸积聚期； (e) 将摄氧率降低两倍； (f) 获得高出 1.4 倍的总产量系数。在山梨醇存在的情况下，最大比醇氧化酶活性不受影响，并且观察到同时利用山梨醇和甲醇。因此，在山梨醇存在的情况下，在 I = 24 小时时产生了 130 mg/l rHuEPO，而单独使用甲醇则产生了 80 mg/l rHuEPO（t = 24 小时）。这项工作不仅证明了将山梨醇掺入巴斯德毕赤酵母 Mut(+) 菌株发酵生产任何生物产品的简便性和效率，而且还为甲基营养酵母毕赤酵母的代谢提供了新的见解。版权所有 (C) 2009 John Wiley & Sons, Ltd.
Batch-wise sorbitol addition as a co-substrate at the induction phase of methanol fed-batch fermentation by Pichia pastoris (Mut(+)) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. Adding sorbitol batch-wise to the medium provided the following advantages over growth on methanol alone: (a) eliminating the long lag-phase for the cells and reaching 'high cell density production' at t = 24 h of the process (C-X = 70 g CDW/I); (b) achieving 1.8-fold higher recombinant human erythropoietin (rHuEPO) (at t = 18 h); (c) reducing specific protease production 1.2-fold; (d) eliminating the lactic acid build-up period; (e) lowering the oxygen uptake rate two-fold; and (f) obtaining 1.4-fold higher overall yield coefficients. The maximum specific alcohol oxidase activity was not affected in the presence of sorbitol, and it was observed that sorbitol and methanol were utilized simultaneously. Thus, in the presence of sorbitol, 130 mg/l rHuEPO was produced at I = 24 h, compared to 80 mg/l rHuEPO (t = 24 h) on methanol alone. This work demonstrates not only the ease and efficiency of incorporating sorbitol to fermentations by Mut(+) strains of P. pastoris for the production of any bio-product, but also provides new insights into the metabolism of the methylotrophic yeast P. pastoris. Copyright (C) 2009 John Wiley & Sons, Ltd.