共表达分子伴侣PDI和转录因子Aft1对毕赤酵母表达人溶菌酶的影响  

Effect of Co-expression of Chaperone PDI and Transcription Factor Aft1 on the Expression of Recombinant Human Lysozyme in Pichia pastoris

作　　者：王儒昕 韩琴 陈园园 吴菁 闫达中 刘军 李鑫 WANG Ruxin;HAN Qin;CHEN Yuanyuan;WU Jing;YAN Dazhong;LIU Jun;LI Xin(College of Biology and Pharmaceutical Engineering,Wuhan Polytechnic University,Wuhan 430023,China)

机构地区：[1]武汉轻工大学生物与制药工程学院,湖北武汉430023

出　　处：《食品科学》2020年第10期124-130,共7页Food Science

基　　金：湖北省教育厅科学研究计划指导性项目(B2016080)。

摘　　要：通过共表达分子伴侣二硫键异构酶(protein disulfide isomerase,PDI)和毕赤酵母转录因子Aft1,提高重组人溶菌酶(human lysozyme,HLY)在毕赤酵母中的分泌表达水平。将构建的分子伴侣表达载体pG418-PDI和毕赤酵母转录因子Aft1表达载体pG418-Aft1线性化后,电击转化重组毕赤酵母KM71-HLY细胞,用含G418的平板筛选阳性转化子,得到共表达分子伴侣PDI和HLY,及共表达毕赤酵母转录因子Aft1和HLY的工程菌株KM71-HLYpG418-PDI和KM71-HLY-pG418-Aft1。摇瓶发酵分析共表达PDI和Aft1对HLY表达水平的影响。结果表明,工程菌KM71-HLY、KM71-HLY-pG418-PDI和KM71-HLY-pG418-Aft1经甲醇诱导均产生14.7 kDa HLY,发酵上清液在含溶壁微球菌平板上出现抑菌圈。在诱导表达前期,KM71-HLY-pG418-PDI、KM71-HLY-pG418-Aft1和KM71-HLY生长量无明显差别;70 h后,KM71-HLY-pG418-PDI菌株和KM71-HLY-pG418-Aft1菌株生物量少于KM71-HLY,发酵最终生长量分别为KM71-HLY的92.8%与84.1%。KM71-HLY、KM71-HLY-pG418-PDI和KM71-HLY-pG418-Aft1经168 h甲醇诱导,胞外分泌总蛋白量分别为324.02、350.87 mg/L和474.8 mg/L,发酵液酶活力分别为34880、45600 U/mL和50180 U/mL。与KM71-HLY相比,KM71-HLY-pG418-PDI和KM71-HLY-pG418-Aft1发酵产胞外总蛋白分别增加了8.3%和46.5%;KM71-HLY-pG418-PDI和KM71-HLY-pG418-Aft1所产胞外溶菌酶总酶活力较KM71-HLY分别增加了30.7%和43.9%。This study attempted to improve the expression level of recombinant human lysozyme in Pichia pastoris by co-expression of the chaperone protein disulfide isomerase(PDI)and the transcriptional activator of ferrous transport-1(Aft1)from P.pastoris.The constructed expression plasmids,pG418-PDI and pG418-Aft1,were linearized and integrated into the genome of P.pastoris KM71-HLY through homologous recombination by electroporation.The positive transformants were selected based on G418 resistance,yielding two recombinant strains,KM71-HLY-PG418-PDI and KM71-HLY-PG418-Aft1.Shake flask fermentation was performed to analyze the expression level of HLY in the engineered yeasts.Experimental results showed that KM71-HLY,KM71-HLY-pG418-PDI and KM71-HLY-pG418-Aft1 all produced human lysozyme(14.7 kDa)with inhibitory effects on Micrococcus lysodeik under the induction of methanol.These recombinant strains exhibited only a slight difference in the cell growth at the early stage of induction.However,the biomasses of KM71-HLY-PG418-PDI and KM71-HLY-PG418-Aft1 were lower than that of KM71-HLY after 70 h and their optical density values(OD600 nm)at the end of fermentation were 92.8%and 84.1%as compared to KM71-HLY,respectively.When induced by methanol for 168 h,the yields of total secretory protein of KM71-HLY,KM71-HLY-PG418-PDI and KM71-HLY-PG418-Aft1 were 324.02,350.87 and 474.8 mg/L,and the lysozyme activities of the fermentation supernatants were 34880,45600 and 50180 U/mL,respectively.As compared to KM71-HLY,KM71-HLY-PG418-PDI and KM71-HLYPG418-Aft1 increased total secretory protein production by 8.3%and 46.5%,and extracellular lysozyme activity by 30.7%and 43.9%,respectively.

关 键 词：人溶菌酶 分子伴侣 转录因子 共表达 毕赤酵母 

分 类 号：Q819[生物学—生物工程]