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

doi:10.7506/spkx1002-6630-20190305-049

食品科学 ›› 2020, Vol. 41 ›› Issue (10): 124-130.doi: 10.7506/spkx1002-6630-20190305-049

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

王儒昕，韩琴，陈园园，吴菁，闫达中，刘军，李鑫

（武汉轻工大学生物与制药工程学院，湖北武汉 430023）

出版日期:2020-05-25 发布日期:2020-05-15
基金资助:
湖北省教育厅科学研究计划指导性项目（B2016080）

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

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)

Online:2020-05-25 Published:2020-05-15

摘要/Abstract

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-HLY-pG418-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34 880、45 600 U/mL和50 180 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%。

关键词: 人溶菌酶, 分子伴侣, 转录因子, 共表达, 毕赤酵母

Abstract: 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 34 880, 45 600 and 50 180 U/mL, respectively. As compared to KM71-HLY, KM71-HLY-PG418-PDI and KM71-HLY-PG418-Aft1 increased total secretory protein production by 8.3% and 46.5%, and extracellular lysozyme activity by 30.7% and 43.9%, respectively.

Key words: human lysozyme, chaperone, transcriptional factor, co-expression, Pichia pastoris

中图分类号:

Q819

王儒昕，韩琴，陈园园，吴菁，闫达中，刘军，李鑫. 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[J]. 食品科学, 2020, 41(10): 124-130.

WANG Ruxin, HAN Qin, CHEN Yuanyuan, WU Jing, YAN Dazhong, LIU Jun, LI Xin. Effect of Co-expression of Chaperone PDI and Transcription Factor Aft1 on the Expression of Recombinant Human Lysozyme in Pichia pastoris[J]. FOOD SCIENCE, 2020, 41(10): 124-130.

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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

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