重组毕赤酵母发酵生产猪!干扰素(pIFN-!)过程性能不稳定,高密度细胞培养阶段的性能指标直接影响到甲醇诱导期的猪!干扰素表达水平。测定分析甲醇代谢关键酶基因的转录水平以及酶活,发现甘油流加培养末期乙醇的大量(超过6 g/L)和长期积累(超过4h)不可逆转地抑制醇氧化酶(AOX)的启动,是导致猪!干扰素无法正常表达、发酵生产不稳定的主要原因。为此,提出了基于乙醇在线测量的改良型甘油流加控制策略,该策略可以在保证细胞快速生长的同时、将乙醇浓度有效控制在2 g/L以下的水平。与传统DO-Stat甘油流加策略(策略A)相比较,改良型甘油流加策略(策略B)可以强化猪!干扰素的诱导表达性能、确保其发酵生产的稳定:1)相同诱导条件下(甲醇/山梨醇培养基流加体积比1∶1),两个发酵批次大猪!干扰素浓度比使用策略A时优批次的大值分别提高75.5%和29.8%;2)在合理改变诱导条件时(甲醇/山梨醇培养基流加体积比4∶1),大猪!干扰素浓度仍比使用策略A时优批次的大值提高38.9%。

The performance of porcine interferon-α( pIFN-α) fermentative production by Pichia pastoris is not stable.Cultivation performance in high cell density culture phase is closely associated with the subsequent pIFN-α expression level achieved in induction phase.It was found by analyzing specific activities and transcriptional levels of genes encoding the key enzymes in methanol metabolism that,high( more than 6 g/L) and historic ethanol accumulation in the late glycerol feeding cultivation stage irreversibly deteriorated alcohol oxidase( AOX) promoter,leading to the instability in pIFN-α production.An improved DO-Stat glycerol feeding strategy based on ethanol on-line measurement was thus proposed,to maintain quick cell growth and to control ethanol concentration below 2 g/L simultaneously.Compared with the traditional DO-Stat based glycerol feeding strategy( A),the improved strategy( B) enhanced pIFN-α expression and stabilized fermentation performance,which were showed as follows: 1) under the same induction condition,in two runs,maximal pIFN-α concentration increased 75.5% and 29.8% respectively than that obtained under the best batch using strategy A; 2) reasonably changing induction condition,maximal pIFN-α concentration was still 38.9% higher than that of the best run adopting strategy A.