“低甲醇浓度-高溶解氧浓度”策略诱导毕赤酵母高效表达HSA-GCSF^m及其转录组学机理分析

Enhanced HSA-GCSF^m production by Pichia pastoris under “low methanol concentration-high dissolved oxygen” induction strategy and its transcriptome analysis

甲醇/山梨醇共混诱导毕赤酵母(Mut^+,GS115)表达人血清白蛋白-人粒细胞集落刺激因子突变体融合蛋白(HSA-GCSFm)的过程中,甲醇消耗与O_2消耗相互偶联,难以将甲醇浓度和溶解氧浓度(DO)同时维持在高水平。该文研究探讨了"低甲醇浓度-高DO"和"高甲醇浓度-低DO"诱导策略下HSA-GCSFm的表达性能。结果表明,"低甲醇浓度-高DO"诱导策略策略下的HSA-GCSF^m浓度达到587.5 mg/L,而"高甲醇浓度-低DO"诱导策略下的HSA-GCSF^m浓度仅达到106 mg/L。利用转录组学技术分析上述2种诱导策略引起HSA-GCSF^m表达水平差异的分子机制,结果表明:"低甲醇浓度-高DO"条件下甲醇代谢途径、TCA循环和过氧化物酶体相关的部分基因上调,而与蛋白水解作用相关的部分基因下调,表明该条件下甲醇和山梨醇的代谢活性更高,细胞抗高DO冲击的能力更强,错误折叠的蛋白减少。

In the process of producing HSA-GCSFm with methanol/sorbitol co-induction by Pichia pastoris, the consumptions of methanol and 02 couple with each other, making it difficult to maintain methanol concentration and dissolved oxygen （DO） at high level simultaneously. In this study, induction strategies of ＂high methanol concentration-low DO＂ and ＂low methanol concentration-high DO＂ were adopted, and the performances of HSA-GCSFm pro- duction were investigated. As a result, the titer of HSA-GCSF^m under ＂high methanol concentration-low DO＂ and ＂low methanol concentration-high DO＂ strategies were 587.5 mg/L and 106 rag/L, respectively. The molecular mechanism of the two induction strategies causing different HSA-GCSFm expression level was revealed by transcriptome analysis. Under the ＂low methanol concentration-high DO＂ strategy, the partial genes correlated with methanol metabolic pathway, TCA cycle and peroxisome were up-regulated, while the partial genes involved in proteolysis were down-regulated. That indicated the higher metabolic activities of methanol and sorbitol, the enhanced resistance to the shock of high DO and reduced protein misfolding.