Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris
Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris
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DOI：
10.1002/bit.10587
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发表时间：
2003-05-20
影响因子：
3.8
通讯作者：
Shiloach, J
中科院分区：
文献类型：
--
作者：
Trinh, LB;Phue, JN;Shiloach, J
毕赤酵母（Pichia pastoris）是一种甲基营养酵母，是重组蛋白的高效生产者，其中外源基因受甲醇诱导的AOX1启动子的控制。因此，公认的生产过程分为两个阶段：第一阶段，酵母利用甘油，积累生物质；在第二阶段，酵母利用甲醇，甲醇既用作重组蛋白表达的诱导剂，又用作碳源。由于酵母对甲醇浓度很敏感，所以甲醇是逐渐供应给生长中的培养物的。对三种甲醇添加策略进行了评价，以优化重组内皮抑素的生产。两种策略基于酵母代谢；其中一个使用甲醇传感器响应甲醇消耗，另一个响应氧气消耗。在这两种策略中，甲醇的供应是无限的。第三种策略是基于预定的指数进料速率，将生长速率控制在0.02 h(-1)，在这种策略中甲醇供应是有限的。在整个诱导阶段，除甲醇外，还以1 g L h（-1）的速率连续添加甘油。在所有三种策略中，内皮抑素的总产量相似（从3l初始体积中获得400 mg），但在预定速率方法中添加的甲醇量和产生的生物质量较低。这使得单位生物量和单位甲醇的内皮抑素比预定率比其他两种方法高2倍，使生长控制策略不仅更有效，而且更方便下游加工。(C) 2003 Wiley期刊有限公司
Pichia pastoris, a methylotrophic yeast, is an efficient producer of recombinant proteins in which the heterologous gene is under the control of the methanol-induced AOX1 promoter. Hence, the accepted production procedure has two phases: In the first phase, the yeast utilizes glycerol and biomass is accumulated; in the second phase, the yeast utilizes methanol which is used both as an inducer for the expression of the recombinant protein and as a carbon source. Since the yeast is sensitive to methanol concentration, the methanol is supplied gradually to the growing culture. Three methanol addition strategies were evaluated for the purpose of optimizing recombinant endostatin production. Two strategies were based on the yeast metabolism; one responding to the methanol consumption using a methanol sensor, and the other responding to the oxygen consumption. In these two strategies, the methanol supply is unlimited. The third strategy was based on a predetermined exponential feeding rate, controling the growth rate at 0.02 h(-1), in this strategy the methanol supply is limited. Throughout the induction phase glycerol, in addition to methanol, was continuously added at a rate of 1 g L h(-1). Total endostatin production was similar in all three strategies, (400 mg was obtained from 3 L initial volume), but the amount of methanol added and the biomass produced were lower in the predetermined rate method. This caused the specific production of endostatin per biomass and per methanol to be 2 times higher in the predetermined rate than in the other two methods, making the growth control strategy not only more efficient but also more convenient for downstream processing. (C) 2003 Wiley Periodicals, Inc.