共表达HAC1基因对重组毕赤酵母分泌表达葡萄糖氧化酶的影响

Effects of Co-Expression of HAC1 on Glucose Oxidase Production in Recombinant Pichia pastoris

通过增强不可折叠蛋白质响应(UPR)信号途径以及研究不同培养温度下的影响,来提高重组葡萄糖氧化酶在毕赤酵母中的分泌表达。影响毕赤酵母外源蛋白表达的因素,主要为内质网中的折叠速率以及不可折叠蛋白积累造成的胞内胁迫压力。通过共表达HAC1基因对不可折叠蛋白信号通路进行调控,摇瓶中改造菌株PP-G-HAC1胞外酶活达到161 U/m L,相比于原始重组葡萄糖氧化酶菌株提高了34%。进一步研究不同温度下过量表达HAC1基因对菌株的生长和分泌外源蛋白的影响,菌株PP-G-HAC1在3 L发酵罐中28℃培养,酶活达到1 008 U/m L,胞外重组蛋白质达到14.43 g/L,相比原始菌株在相同条件下提高了3.12倍。

In this work,the enhancement of unfolded protein response transcription pathway at different cultivation temperatures were integrated as a novel protein secretion strategy to improve extracellular enzyme concentration and specific productivity of heterologous glucose oxidase（GOD）in Pichia pastoris. Efficient production of heterologous proteins in P. pastoris was often limited by metabolic and intrinsic stresses such as folding rate and ER stress by accumulation of unfolded protein. The results showed that enzymatic activity of engineered secretion strain PP-G-HAC1,co-expression of UPR activating transcription factor Hac1 p from P. pastoris,was up 161 U/m L in shake flasks, 34% higher compared to control strain. Furthermore,the effects of different cultivation temperatures（22、28 ℃） were also investigated. The highest GOD activity and extracellular concentration at 28 ℃ in PP-G-HAC1 were 1,008 U/m L and 14.43 g/L,respectively,3.12-fold increase than that of control strain.