以葡萄糖为生长相基质的重组毕赤酵母表达植酸酶发酵

Production of recombinant phytase by Pichia pastoris using glucose as the growth substrate

甲醇营养型毕赤酵母表达外源蛋白是在醇氧化酶(alcohol oxidase,AOX)启动子(P_(aox1))严格调控下进行的,然而这种启动子在转录水平受到葡萄糖的阻遏。本文研究了毕赤酵母在葡萄糖替代甘油为生长相碳源时表达重组植酸酶蛋白的发酵特征。结果表明:初始葡萄糖浓度为20g/L的细胞得率高,为0.39g[DCW]/g。通过基于实时参数(溶氧和呼吸商)调控的葡萄糖补料策略,生长相40 h后细胞密度达到100 g[DCW]/L,甲醇诱导100 h后植酸酶产量达到2200 FTUphytase/mL,甲醇得率系数为0.25 FTU phytase/g methnol。因此,在毕赤酵母高表达重组蛋白培养中葡萄糖能够用作生长相基质,并能实现重组蛋白的高效表达。

The recombinant protein was expressed by the methylotrophic yeast Pichia pastoris with the control of alcohol oxiddase promoter （PAOXI）- However, PAOXI was strongly repressed by glucose at the transcription level. Thus, a fexmentation strategy with glucose as the growth substrate instead of glycerol was developed based on on-line parameters dissolved oxygen （DO） and respiratory quotient （RQ）. The results showed that a higher cell yield coefficient of 0.39 g [DCW]/g was achieved with a lower initial glucose concentration and biomass of 100 g [DCW]/L was reached at 40 h. Simultanteously, high phytase production level of 2 200 FTU phytase/mL） and phytase yield on methanol of 0.25 FTU/g were obtained after 100 h methanol induction. Therefore, for recombinant protein/enzyme production, glucose could be used as a carbon source during the growth phase of Pichia system.