共表达PDI1、MDH1和HAC1基因对重组毕赤酵母分泌表达葡糖氧化酶的影响 *

doi:10.13523/j.cb.20191004

中国生物工程杂志

2019, Vol. 39

Issue (10): 24-33 DOI: 10.13523/j.cb.20191004

研究报告

共表达PDI1、MDH1和HAC1基因对重组毕赤酵母分泌表达葡糖氧化酶的影响 ^*

张文玉,魏东升,钱江潮(

)

华东理工大学生物反应器国家重点实验室上海 200237

Effects of Co-expression of PDI1, MDH1 and HAC1 Gene on Secretory Expression of Recombinant Glucose Oxidase in Pichia pastoris

ZHANG Wen-yu,WEI Dong-sheng,QIAN Jiang-chao(

)

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

全文: PDF(1096 KB) HTML

摘要：

葡糖氧化酶(GOD)可催化葡萄糖生成过氧化氢和葡萄糖酸,在工业上被广泛应用。在构建的一株整合多拷贝GOD基因且含有HAC1表达单元的重组毕赤酵母G/GMH1的基础上,共表达分子伴侣二硫键异构酶基因PDI1和苹果酸脱氢酶基因MDH1,考察PDI1、MDH1基因与HAC1共表达后对GOD分泌表达的影响。这些基因共表达后对重组菌的生长无明显影响。PDI1和MDH1分别与HAC1共表达后,重组菌G/GMH1-PDI1和G/GMH1-MDH1的胞外GOD产量达到11 731.9U/g DCW和1 1047.6U/g DCW,比出发菌提高了17.3%和10.4%,前者达到了目前所报道摇瓶培养的最高单位菌体酶活水平。而三基因共表达重组菌G/GMH1-PDI1-MDH1的GOD产量略有下降。在所构建的菌株中,GOD基因的转录水平仅在G/GMH1-PDI1-MDH1中略有提高,与酶产量的提高无直接关系。与出发菌相比,共表达基因的转录水平在新构建的菌中有不同程度的升高,说明这些基因被成功转录。PDI1和MDH1基因转录在G/GMH1-PDI1和G/GMH1-MDH1中分别被上调,可能与GOD产量提高有关。虽然GOD、HAC1、PDI1和MDH1基因在G/GMH1-PDI1-MDH1中均被上调,但并未促进酶产量的提高。

关键词： 重组毕赤酵母; 葡糖氧化酶; HAC1基因; PDI1基因; MDH1基因

Abstract:

Glucose oxidase (GOD) catalyzes the oxidation of glucose to hydrogen peroxide and gluconic acid, and has a broad commercial application. The recombinant Pichia pastoris G/GMH1 containing multi copies of the GOD gene along with the HAC1 expression cassette had been constructed in our previous works, and was used here as the starting strain to further introduce the chaperone disulfide isomerase gene PDI1 or/and malate dehydrogenase gene MDH1, and to investigate the effects of co-expression of the PDI1, MDH1 and HAC1 gene on the secretory production of GOD. No significant change was observed in cell growth of the recombinant strains after co-expression of these genes. The co-expression of HAC1 with PDI1 or MDH1 improved the extracellular specific GOD activity in the corresponding strain G/GMH1-MDH1 and G/GMH1-PDI1 by 10.4% and 17.3%, respectively, reaching 11 047.6U/g DCW and 11 731.9U/g DCW which, to the best of our knowledge, is the highest reported GOD activity of the recombinant P. pastoris cultured in shake flasks. The GOD production decreased slightly in the strain G/GMH1-PDI1-MDH1 with the co-expression of all three genes. Among these constructed strains, the transcription level of the GOD gene was only slightly increased in G/GMH1-PDI1-MDH1, which did not result in the increase in GOD production. The insertion of the additional PDI1 and MDH1 gene led to a varying increase in their relative transcriptional level, which demonstrated that these genes were transcribed and expressed successfully. The increased transcription of PDI1 and MDH1 gene in G/GMH1-PDI1 and G/GMH1-MDH1 might be related to the increase of GOD production. Although the GOD, HAC1, PDI1 and MDH1 gene were all up-regulated in G/GMH1-PDI1-MDH1, GOD production was not enhanced.

Key words: Recombinant Pichia pastoris Glucose oxidase HAC1 gene PDI1 gene MDH1 gene

收稿日期: 2019-03-28 出版日期: 2019-11-12

ZTFLH:

Q819

基金资助: * 中央高校基本科研业务费专项(22221818014)

通讯作者: 钱江潮 E-mail: jiangchaoqian@ecust.edu.cn

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引用本文:

张文玉,魏东升,钱江潮. 共表达PDI1、MDH1和HAC1基因对重组毕赤酵母分泌表达葡糖氧化酶的影响 ^*[J]. 中国生物工程杂志, 2019, 39(10): 24-33.

ZHANG Wen-yu,WEI Dong-sheng,QIAN Jiang-chao. Effects of Co-expression of PDI1, MDH1 and HAC1 Gene on Secretory Expression of Recombinant Glucose Oxidase in Pichia pastoris. China Biotechnology, 2019, 39(10): 24-33.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191004 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I10/24

表1 本研究中所使用的菌株和质粒

表2 本研究中所使用的引物

图1 PDI1、MDH1基因的扩增和重组质粒酶切鉴定

图2 重组菌G/GMH1-PDI1、G/GMH1-MDH1、G/GMH1-PDI1-MDH1的构建过程

图3 重组菌的PCR鉴定

图4 共表达重组菌的生长曲线

图5 共表达重组菌的胞外(a)和胞内(b)GOD产量

表3 168h共表达重组菌的GOD体积产量

图6 168h共表达重组菌胞内外蛋白质电泳

图7 共表达重组菌的GOD、HAC1、PDI1和MDH1基因转录水平

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