共表达分子伴侣PDI和Ero1对灰盖鬼伞过氧化物酶在毕赤酵母中表达的影响

Enhancement of Coprinus cinereus peroxidase in Pichia pastoris by co-expression chaperone PDI and Ero1

来源于灰盖鬼伞长度为1 092 bp的CiP目的基因与AOX1启动子一起整合进酵母染色体基因组中。重组蛋白CiP在酿酒酵母信号肽的引导下成功分泌到胞外,质谱鉴定为目的蛋白,成功在毕赤酵母中表达灰盖鬼伞过氧化物酶(CiP)。将伴侣蛋白内质网氧化还原酶1(Ero1)、二硫键异构酶(PDI)分别单独及同时转入CiP酵母受体菌中,研究它们对CiP在毕赤酵母中表达的影响。结果表明:在摇瓶中,相对于无分子伴侣的菌株,单独整合PDI及同时整合Ero1、PDI菌株的CiP酶活分别提高了2.43和2.62倍,活力达到316 U/m L和340 U/m L。挑选同时整合Ero1、PDI伴侣蛋白的CiP菌株,5 L发酵罐进行高密度发酵,酶活最高达到3 379 U/m L,比摇瓶提高约10倍。本实验结果较目前已报道的1 200 U/m L已是最高水平。

The 1 095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP（rCiP） fused with the α-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase（CiP） was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1（Ero1） and protein disulfide isomerase（PDI） were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Ero1-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/m L and 340 U/m L respectively. The strains co-expressed with Ero1-PDI was used to high density fermentation, and their activity reached 3 379 U/m L, which was higher than previously reported of 1 200 U/m L.