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

doi:10.13523/j.cb.20191004

China Biotechnology

2019, Vol. 39

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

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

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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

Received: 28 March 2019 Published: 12 November 2019

ZTFLH:

Q819

Corresponding Authors: Jiang-chao QIAN E-mail: jiangchaoqian@ecust.edu.cn

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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.

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

Table 1 Strains and plasmids used in this study

Table 2 Primers used in this study

Fig.1 PCR amplification of PDI1, MDH1 and verification of recombinant plasmids by digestion (a)Lane M: DNA maker (2 000bp); Lane 1,2: PDI1 and MDH1 genes amplification (b)Lane M: DNA maker (15 000bp); Lane 1,2: pAOX-UH-PDI1 and pAOX-UH-MDH1 digested by NotI/XbaI (c)Lane M: DNA maker (15 000bp); Lane 1: pAOX-UH-PDI1-MDH1 digested by SpeI

Fig.2 Construction process of recombinant strains G/GMH1-PDI1, G/GMH1-MDH1, G/GMH1-PDI1-MDH1 X represents the PDI1, MDH1 and PDI1-AOXTT-pAOX-MDH1 sequences, respectively

Fig.3 Verification of recombinant strains by PCR (a)Lane M: DNA maker (2 000bp); Lane1-4:Verification of G/GMH1, G/GMH1-PDI1, G/GMH1-MDH1, G/GMH1-PDI1-MDH1(upstream sites) (b)Lane M: DNA maker (2 000bp); Lane1-4:Verification of G/GMH1, G/GMH1-PDI1, G/GMH1-MDH1, G/GMH1-PDI1-MDH1(downstream sites)

Fig.4 Growth curve of co-expression recombinant strains

Fig.5 Extracellular (a) and intracellular (b) specific GOD activity of co-expressing recombinant strains

Table 3 GOD activity of the co-expressing recombinant strains at 168h

Fig.6 Extracellular and intracellular SDS-PAGE of co-expressing recombinant strains at 168h Lane M: Protein maker (200kDa;); Lane 1,2: G/GMH1; Lane 3,4: G/GMH1-PDI1; Lane 5,6: G/GMH1-MDH1; Lane 7,8: G/GMH1-PDI1-MDH1; Lane 1, 3, 5, 7: Extracellular protein; Lane 2, 4, 6, 8: Intracellular protein

Fig.7 The GOD, HAC1, PDI1 and MDH1 gene transcription levels in co-expression recombinant strains


[1]	Pluschkell S, Hellmuth K, Rinas U . Kinetics of glucose oxidase excretion by recombinant Aspergillus niger. Biotechnology & Bioengineering, 2015,51(2):215-220.

[2]	Parpinello G P, Chinnici F, Versari A , et al. Preliminary study on glucose oxidase-catalase enzyme system to control the browning of apple and pear purées.LWT-Food Science and Technology, 2002,35(3):239-243.

[3]	Cui C, Chen H, Chen B , et al. Genipin cross-linked glucose oxidase and catalase multi-enzyme for gluconic acid synthesis. Applied Biochemistry and Biotechnology, 2017,181(2):526-535.

[4]	Rico-Rodriguez F, Serrato J C, Montilla A , et al. Impact of ultrasound on galactooligosaccharides and gluconic acid production throughout a multienzymatic system. Ultrason Sonochem, 2018,44:177-183.

[5]	Tzanov T, Costa S A, Gübitz.G M , et al. Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching. Journal of Biotechnology, 2002,93(1):87-94.

[6]	Qi L, Hu Q, Kang Q , et al. Fabrication of liquid-crystal-based optical sensing platform for detection of hydrogen peroxide and blood glucose. Analytical Chemistry, 2018,90(19):11607-11613.

[7]	Ho J A, Wu L C, Fan N C , et al. Development of a long-life capillary enzyme bioreactor for the determination of blood glucose. Talanta, 2007,71(1):391-396. doi: 10.1016/j.talanta.2006.04.023

[8]	Tu T, Wang Y, Huang H , et al. Improving the thermostability and catalytic efficiency of glucose oxidase from Aspergillus niger by molecular evolution. Food Chemistry, 2019,281 : 163-170.

[9]	Sukacheva M V, Davydova M E, Netrusov A . Production of Penicillium funiculosum 433 glucose oxidase and its properties. Applied Biochemistry & Microbiology, 2004,40(1):25-29.

[10]	Yang Z, Zhang Z . Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: a review. Biotechnology Advances, 2018,36(1):182-195.

[11]	Gu L, Zhang J, Liu B , et al. High-level extracellular production of glucose oxidase by recombinant Pichia pastoris using a combined strategy. Applied Biochemistry & Biotechnology, 2015,175(3):1429-1447.

[12]	刘虎军, 罗玮, 范新蕾 , 等. 黑曲霉中葡萄糖氧化酶基因的克隆及其在毕赤酵母中的表达. 食品与生物技术学报, 2013,32(6):615-621.

[12]	Liu H J, Luo W, Fan X L , et al. Cloning and heterologous expression of glucose oxidase gene from Aspergillus niger PCTC in Pichia pastoris. Journal of Food Science & Biotechnology, 2013,32(6):615-621.

[13]	Qiu Z, Guo Y, Bao X , et al. Expression of Aspergillus niger glucose oxidase in yeast Pichia pastoris SMD1168. Biotechnology & Biotechnological Equipment, 2016,30(5):998-1005.

[14]	Bankefa O E, Wang M, Zhu T , et al. Enhancing the secretion pathway maximizes the effects of mixed feeding strategy for glucose oxidase production in the methylotrophic yeast Pichia pastoris. Bioresources & Bioprocessing, 2018,5(1):25.

[15]	Emmanuel B O, Meiyu W, Taicheng Z , et al. Enhancing the secretion pathway maximizes the effects of mixed feeding strategy for glucose oxidase production in the methylotrophic yeast Pichia pastoris. Bioresources & Bioprocessing, 2018,5(1):25.

[16]	高庆华, 董聪, 王玥 , 等. 共表达分子伴侣PDI和Ero1对葡萄糖氧化酶在毕赤酵母中表达的影响. 生物技术通报, 2018,34(7):174-179.

[16]	Gao Q H, Dong C, Wang Y , et al. Enhancement of glucose oxidase in Pichia pastoris by co-expressing chaperone PDI and ero1. Biotechnology Bulletin, 2018,34(7):174-179.

[17]	Gu L, Zhang J, Du G , et al. Multivariate modular engineering of the protein secretory pathway for production of heterologous glucose oxidase in Pichia pastoris. Enzyme & Microbial Technology, 2015,68 : 33-42.

[18]	Nocon J, Steiger M G, Pfeffer M , et al. Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production. Metabolic Engineering, 2014,24 : 129-138.

[19]	钱江潮, 魏东升, 王泽建 , 等. 分泌表达葡萄糖氧化酶的方法、重组菌及其应用:中国, CN107475212A. 2017-12-15[2019-03-24]. 分泌表达葡萄糖氧化酶的方法、重组菌及其应用:中国, CN107475212A. 2017-12-15[2019-03-24]. .

[19]	Qian J C, Wei D S, Wang Z J , et al. Secreting glucose oxidase expression of recombination strain comprises providing yeast cells, introducing exogenous coding gene of glucose oxidase or gene in yeast cells and introducing exogenous PDI1 encoding gene,CN107475212A. 2017-12-15[2019-03-24]. Secreting glucose oxidase expression of recombination strain comprises providing yeast cells, introducing exogenous coding gene of glucose oxidase or gene in yeast cells and introducing exogenous PDI1 encoding gene,CN107475212A. 2017-12-15[2019-03-24].

[20]	钱江潮, 魏东升, 王泽建 , 等. 基于代谢工程优化进行葡萄糖氧化酶分泌表达的方法、重组菌及其应用:中国, CN107460175A. 2017-12-12[2019-03-24]. 基于代谢工程优化进行葡萄糖氧化酶分泌表达的方法、重组菌及其应用:中国, CN107460175A. 2017-12-12[2019-03-24]. .

[20]	Qian J C, Wei D S, Wang Z J , et al. Expressing glucose oxidase using yeast cells, involves introducing exogenous glucose oxidase encoding gene into yeast cells, introducing e.g. malate dehydrogenase 1 encoding gene exogenously, and culturing yeast cell. China, CN107460175A2017-12-12[2019-03-24]. Expressing glucose oxidase using yeast cells, involves introducing exogenous glucose oxidase encoding gene into yeast cells, introducing e.g. malate dehydrogenase 1 encoding gene exogenously, and culturing yeast cell. China, CN107460175A2017-12-12[2019-03-24]. .

[21]	Gao Z, Li Z, Zhang Y , et al. High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization. Biotechnology Letters, 2012,34(3):507-514. doi: 10.1007/s10529-011-0790-6

[22]	顾磊, 张娟, 堵国成 , 等. 共表达HAC1基因对重组毕赤酵母分泌表达葡萄糖氧化酶的影响. 食品与生物技术学报, 2016,35(2):113-122.

[22]	Gu L, Zhang J, Du G C , et al. Effects of co-expression of HAC1 on glucose oxidase production in recombinant Pichia pastoris. Journal of Food Science & Biotechnology, 2016,35(2):113-122.

[23]	Wilkinson B, Gilbert H F . Protein disulfide isomerase. Biochimica Et Biophysica Acta, 2004,1699(1-2):35-44.

[24]	Selinski J, Konig N, Wellmeyer B , et al. The plastid-localized NAD-dependent malate dehydrogenase is crucial for energy homeostasis in developing Arabidopsis thaliana seeds. Molecular Plant, 2014,7(1):170-186. doi: 10.1093/mp/sst151

[25]	Holland J T, Harper J C, Dolan P L , et al. Rational redesign of glucose oxidase for improved catalytic function and stability. PLoS One, 2012,7(6):e37924.

[26]	Ning X, Zhang Y, Yuan T , et al. Enhanced thermostability of glucose oxidase through computer-aided molecular design. International Journal of Molecular Sciences, 2018,19(2):425.

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[2]	LI Rui-rui, LIU Dian-lei, YANG Qing, HAO Qiong, JIANG Kai-kai, LI Pi-wu. *Optimization of Fermentation Conditions for Glucose Oxidase Production by Aspergillus niger* using Response Surface Method**[J]. China Biotechnology, 2013, 33(10): 111-116.

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