中国生物工程杂志

In the process of expressing porcine interferon-α (pIFN-α) by methylotrophic Pichia pastoris under high cells density, methanol concentration is generally controlled at relatively high levels of 5-10g/L with the dissolved oxygen concentration (DO) naturally declining to near 0% level. Heterologous proteins induction by P. pastoris is an extremely high aerobic system. If P. pastoris is subject to the environments of high methanol concentration and low DO for a long period, the cells metabolic activity and heterologous protein expression efficiency of P. pastoris would significantly deteriorate; intracellular methanol could severely accumulate, and the intracellular methanol can not be effectively oxidized by/energizing for methanol dissimilation route to synthesize the targeted protein. A novel periodic methanol induction control strategy was proposed and implemented at 20℃. During the induction period, methanol was firstly controlled at the high concentration level of 8-10g/L with DO level naturally staying at about 0% for 7h. Then raising/maintaining DO around 20% by reducing methanol feeding rate. By doing so, methanol concentration naturally reduced to low level of 0-1g/L, and then the environment of high DO/low methanol concentration was created and kept for another 4h. A total of 6 such cycles was repeated. By periodically shifting methanol concentration and DO in between “high/low” and “low/high” environments, the metabolic activity of P. pastoris could be recovered and maintained at higher levels for longer time; intracellular methanol concentration could be reduced to a very low level (≤ 0.003g/g DCW) but without deteriorating pIFN-α induction, the methanol toxicity was greatly relieved. As a result, pIFN-α activity reached the highest level of 3.90×10 ⁷IU/ml, which was 86% higher than that using the traditional constant methanol concentration control strategy. The proposed periodic methanol induction control strategy showed its effectiveness in pIFN-α production and would supply the useful information/reference for other heterologous proteins fermentation processes.