Guarna M M, Lesnicki G J, Tam B M, Robinson J, Radziminski C Z, Hasenwinkle D, Boraston A, Jervis E, MacGillivray R T, Turner R F, Kilburn D G
The Biotechnology Laboratory, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Biotechnol Bioeng. 1997 Nov 5;56(3):279-86. doi: 10.1002/(SICI)1097-0290(19971105)56:3<279::AID-BIT5>3.0.CO;2-G.
The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol.
甲基营养型酵母巴斯德毕赤酵母可用于在甲醇诱导型醇氧化酶1（AOX1）启动子的控制下高水平表达重组基因。精确调节巴斯德毕赤酵母培养物中的甲醇浓度对于维持诱导作用很有必要，同时要防止甲醇积累到细胞毒性水平。我们开发了一种廉价的甲醇传感器，它使用一根浸入培养基中的透气硅橡胶管和一个有机溶剂蒸汽探测器。该传感器用于在生产人转铁蛋白N叶的巴斯德毕赤酵母克隆的补料分批摇瓶培养过程中连续监测甲醇浓度。在培养期间，传感器校准稳定，输出信号准确反映了通过高效液相色谱法离线测定的甲醇浓度。利用该传感器开发了一种闭环控制系统，并用于在培养物中维持0.3%（v/v）的甲醇浓度。使用该系统使体积蛋白生产力比使用传统补料分批方案获得的水平提高了五倍。
