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- W2929028131 abstract "In heterologous protein productions by Pichia pastoris, methanol induction is generally initiated when cell density reaches very high level. However, this traditional strategy suffers with the problems of difficulty in DO control, toxic by-metabolites accumulation and low targeted protein titer. Therefore, initiating methanol induction at lower cell concentration is considered as an alternative strategy to overcome those problems. However, the methanol/energy regulation mechanisms of initiating induction at lower concentration are not clear and seldom reported. In this article, with monellin production as a prototype, we analyzed the methanol/energy metabolism in protein expression process using the strategies of initiating induction at both higher/lower cells concentrations. We attempted to interpret the advantages of the alternative strategy, via online measurements of methanol consumption, CO₂ production and O₂ uptake rates. When adopting this alternative strategy and maintaining temperature at 30 °C, carbon flux ratio directing into monellin precursors synthesis reached the highest level of 65%. In addition, monellin synthesis was completely associated with cell growth.重组毕赤酵母生产表达外源蛋白的过程中，一般在细胞达到高密度后开始启动甲醇诱导。也有报道指出，在较低细胞浓度下，启动甲醇诱导可以有效地控制整个发酵过程的溶解氧浓度，缓解毒副产物的积累，促进目标蛋白的表达。但是，该操作策略下，甲醇/能量调控机制不明，相关研究报道很少。文中以生产表达monellin (甜味蛋白) 的重组毕赤酵母为模式菌株，通过在线分析计量甲醇消耗速率、CO₂ 释放速率和O₂ 摄取速率，探讨了不同细胞浓度下启动甲醇诱导和外源蛋白表达体系的甲醇/能量代谢模式。结果表明，在较低细胞浓度 (50 g DCW/L)启动诱导并将温度控制在30 ℃，走向合成monellin 前体物质途径的碳流最大 (65%)，且能与用于ATP 再生的碳流形成最佳匹配；monellin 的比合成速率与细胞比生长速率完全耦联，且耦联系数最大，比生长速率也较高；理论NADH (能量) 利用效率η最高，η在甲醇诱导的绝大部分时段 (89%) 处于高水平 (≥0.8)，可以为monellin 合成提供足够的能量。因此，该操作条件下，monellin 浓度达到2.62 g/L 的最高水平，是高细胞密度 (100 g DCW/L)启动诱导策略下monellin 浓度的2.5−4.9 倍。." @default.
- W2929028131 created "2019-04-11" @default.
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- W2929028131 date "2018-02-25" @default.
- W2929028131 modified "2023-09-25" @default.
- W2929028131 title "[Optimizing carbon/energy metabolism to enhance monellin production by Pichia pastoris]." @default.
- W2929028131 doi "https://doi.org/10.13345/j.cjb.170189" @default.
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