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细履平沙

Abstract: Pichia pastoris, a methylotrophic yeast, is an efficient producer of recombinant proteins in which the heterologous gene is under the control of the methanolinduced 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 methanolsensor, 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.
译:
摘要:毕赤酵母可把甲醇作为营养基质,可在AOX1启动子的调节下高效地表达异源基因重组蛋白,一般认为产物形成包括两个阶段.在第一个阶段,利用甘油,形成生物质;第二阶段利用甲醇,一方面甲醇作为重组蛋白的诱导物,另一方面作为C源消耗.毕赤酵母对甲醇敏感,因此在培养过程中应该逐渐补加.本文采用了三个甲醇补加策略以优化重组蛋白血管内皮抑素的产量.其中两个策略基于酵母的代谢情况,一个使用甲醇传感器来反映甲醇的消耗,另一个与氧浓度关联,这两个策略,甲醇的补加量是不受限的.第三个策略基于预定的指数补加速率,根据比生长速率为0.02h-1计算补加量,这个策略中,甲醇的补加是受限的.整个诱导相,除了补加甲醇外,甘油以1 g /L h−1的速率连续补加.对比三种策略,整个血管内皮抑素的产量是相似的(3L初始培养基的发酵可得到400mg产量),但是在每三个策略中,甲醇补加量和生物质相对较低.单位生物质和单位甲醇的产率是另外两个策略的两倍,说明生长控制策略不仅更有效,而且更适合下游的处理过程.