甲醇/山梨醇共混流加控制溶氧改善毕赤酵母表达猪圆环病毒Cap蛋白的发酵性能

Promoting porcine circovirus Cap protein expression in Pichia pastoris through controlling DO by methanol and soribitol co-feeding

研究在重组毕赤酵母(GS115,Mut+)表达猪圆环病毒Cap蛋白的发酵过程中,甲醇毒害作用以及溶氧波动影响目的蛋白的正常表达。基于对甲醇和山梨醇代谢途径的分析,将C源流加的手动调节与反馈控制相结合,提出了1种新型的甲醇/山梨醇共混诱导策略,能够将溶氧稳定地控制于某一设定值,同时避免甲醇毒害作用。使用该策略将溶氧控制于20%的批次,C源(甲醇和山梨醇)添加过少,导致Cap蛋白表达量较低(54 mg/L);而将溶氧控制于10%的批次,C源流加速率适宜,Cap蛋白表达量达到198 mg/L,表达水平明显高于采用传统甲醇诱导策略(0 mg/L)和DO-stat诱导策略的批次(121 mg/L)。

In the fermentation producing of porcine circovirus Cap protein by recombinant Pichia patoris（GS115,Mut^＋）,methanol toxicity and dissolved oxygen fluctuation deteriorated expression of target protein. A novel methanol /sorbitol co-feeding induction strategy was proposed based on the analysis of methanol and sorbitol metabolic pathways. Combining artificial and feedback adjustment of methanol and sorbitol feeding rates,dissolved oxygen（ DO） could be steadily controlled at the desired value,avoiding methanol toxicity at the same time. In the batch controlling DO at 20% with the proposed strategy,Cap protein was weakly expressed because of the shortage of carbon substrate（including methanol and sorbitol）,only 54 mg /L; In the batch maintaining DO at 10%,feeding rates of carbon substrates were controlled at proper level,so Cap protein expression could reach 198 mg /L,and it was obviously enhanced compared with the batches with traditional methanol induction strategy（0 mg /L） and DO-stat strategy（121 mg /L）.