共表达HAC1和分子伴侣基因对甘露聚糖酶在毕赤酵母中表达的影响

doi:10.13560/j.cnki.biotech.bull.1985.2019-0718

生物技术通报 ›› 2020, Vol. 36 ›› Issue (5): 159-168.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0718

共表达HAC1和分子伴侣基因对甘露聚糖酶在毕赤酵母中表达的影响

闵琪¹, 高子涵¹, 姚银¹, 张华山², 熊海容¹, 张莉¹

1.中南民族大学生命科学学院,武汉 430074;
2.湖北工业大学发酵工程省部共建教育部重点实验室,武汉 430068

收稿日期:2019-08-14 出版日期:2020-05-26 发布日期:2020-06-03
作者简介:闵琪,男,硕士研究生,研究方向：微生物酶工程;E-mail：min51777@163.com
基金资助:
国家自然科学基金青年科学基金项目（31802096）,湖北省技术创新专项重大项目（2018ABA093）

Effect of Co-expression of HAC1 and Molecular Chaperone Genes on the Expression of Mannanase in Pichia pastoris

MIN Qi¹, GAO Zi-han¹, YAO Yin¹, ZHANG Hua-shan², XIONG Hai-rong¹, ZHANG Li¹

1. College of Life Sciences,South-Central University for Nationalities,Wuhan 430074;
2. Key Laboratory of Fermentation Engineering,Ministry of Education,Hubei University of Technology,Wuhan 430068

Received:2019-08-14 Published:2020-05-26 Online:2020-06-03

摘要/Abstract

摘要： 为了提高甘露聚糖酶ManA在毕赤酵母中分泌表达的酶活,选择毕赤酵母内质网未折叠蛋白反应（Unfolded protein response,UPR）激活调控因子HAC1与5种毕赤酵母蛋白折叠相关的分子伴侣ERO1、PDI、PDI1、CPR5、BiP,通过构建pPICZA-HAC1等6种胞内表达重组质粒,分别电转化至分泌表达ManA的毕赤酵母重组菌中胞内共表达,并分析其重组菌摇瓶发酵时ManA表达的影响。结果发现在摇瓶发酵水平,胞内共表达HAC1、ERO1、PDI的重组菌发酵上清液中的ManA酶活力分别提高了26%、15%、20%,其重组菌发酵上清液的酶活力分别达到1 014 U/mL、925 U/mL、965 U/mL。通过对各重组菌上清液酶活力、胞内滞留酶活力、上清液蛋白浓度数据进行分析,进一步选择将HAC1、ERO1、PDI进行两基因或三基因组合,并分别在分泌表达ManA的重组菌胞内共表达,但各共表达重组菌发酵上清液的酶活力都没有进一步的提升。单独共表达HAC1或者分子伴侣ERO1、PDI可以辅助ManA的正确折叠,提高其蛋白表达。

关键词: 甘露聚糖酶, 毕赤酵母, HAC1基因, 分子伴侣, 蛋白表达

Abstract: For the higher activity of expressed mannanase ManA in Pichia pastoris,the unfolded protein response（UPR）activation factor HAC1 and 5 reported endoplasmic reticulum protein folding related chaperones ERO1,PDI,PDI1,CPR5,and BiP in P. pastoris were constructed to have 6 intracellular-expressing recombinant plasmids,then each was electro-transformed into recombinant strain P. pastoris secreting ManA for intracellular co-expression respectively,and the influence of the recombinant strain on the expression of ManA during the shake-flask fermentation were analyzed. The results showed that the enzyme activity of ManA in the supernatant of the recombinant strain with co-expressing HAC1,ERO1 and PDI increased by 26%,15% and 20%,and the enzyme activity reached 1 014 U/mL,925 U/mL and 965 U/mL at the shake-flask fermentation. By analyzing the data of enzyme activity in supernatant,intracellular-retention enzyme activity,and protein concentration in supernatant as well as further selecting HAC1,ERO1,PDI to have 2 genes or 3 genes combinations,which then co-expressed in the recombinant strains secreting and expressing ManA,while the enzyme activity in the fermentation supernatants of each co-expressed recombinant strain was not further increased. Co-expression of HAC1 or molecular chaperone ERO1,and PDI separately enhanced the correct folding of ManA and thus increased the expression of the protein.

Key words: mannanase, Pichia pastoris, gene HAC1, molecular chaperones, protein expression

闵琪, 高子涵, 姚银, 张华山, 熊海容, 张莉. 共表达HAC1和分子伴侣基因对甘露聚糖酶在毕赤酵母中表达的影响[J]. 生物技术通报, 2020, 36(5): 159-168.

MIN Qi, GAO Zi-han, YAO Yin, ZHANG Hua-shan, XIONG Hai-rong, ZHANG Li. Effect of Co-expression of HAC1 and Molecular Chaperone Genes on the Expression of Mannanase in Pichia pastoris[J]. Biotechnology Bulletin, 2020, 36(5): 159-168.

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共表达HAC1和分子伴侣基因对甘露聚糖酶在毕赤酵母中表达的影响

Effect of Co-expression of HAC1 and Molecular Chaperone Genes on the Expression of Mannanase in Pichia pastoris

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