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  • ISSN 1006-3080
  • CN 31-1691/TQ

葡萄糖氧化酶在毕赤酵母中的高效分泌表达

魏东升 段广东 钱江潮

魏东升, 段广东, 钱江潮. 葡萄糖氧化酶在毕赤酵母中的高效分泌表达[J]. 华东理工大学学报(自然科学版), 2021, 47(3): 300-307. doi: 10.14135/j.cnki.1006-3080.20200213001
引用本文: 魏东升, 段广东, 钱江潮. 葡萄糖氧化酶在毕赤酵母中的高效分泌表达[J]. 华东理工大学学报(自然科学版), 2021, 47(3): 300-307. doi: 10.14135/j.cnki.1006-3080.20200213001
WEI Dongsheng, DUAN Guangdong, QIAN Jiangchao. Efficient Secretory Expression of Glucose Oxidase in Pichia pastoris[J]. Journal of East China University of Science and Technology, 2021, 47(3): 300-307. doi: 10.14135/j.cnki.1006-3080.20200213001
Citation: WEI Dongsheng, DUAN Guangdong, QIAN Jiangchao. Efficient Secretory Expression of Glucose Oxidase in Pichia pastoris[J]. Journal of East China University of Science and Technology, 2021, 47(3): 300-307. doi: 10.14135/j.cnki.1006-3080.20200213001

葡萄糖氧化酶在毕赤酵母中的高效分泌表达

doi: 10.14135/j.cnki.1006-3080.20200213001
基金项目: 中央高校基本科研业务费专项资助项目 (22221818014)
详细信息
    作者简介:

    魏东升(1990-),男,江苏人,硕士生,研究方向为代谢工程。E-mail:dswecust@163.com

    通讯作者:

    钱江潮,E-mail:jiangchaoqian@ecust.edu.cn

  • 中图分类号: Q815

Efficient Secretory Expression of Glucose Oxidase in Pichia pastoris

  • 摘要: 为了获得高效分泌表达葡萄糖氧化酶(GOD)的毕赤酵母,采用黑曲霉来源的GOD基因序列,按照毕赤酵母的密码子偏好性进行优化,构建AOX1启动子诱导的分泌表达载体pPIC9K-GOD和重组菌G/GOD,通过提高遗传霉素G418浓度筛选到第1代高拷贝高产量重组菌G/GODM,单位细胞干重胞外产酶水平可达5 843.2 U/g, 为G/GOD最高产量的8.2倍。在此基础上,进一步构建第2代高产菌,分别考察了共表达不同辅助折叠因子和强化中心碳代谢途径基因对GOD产量的影响。结果表明,共表达辅助折叠因子PDI1、PDI2和HAC1,可使GOD的胞外产酶产量分别提高32.7%、8.9%和54.4%;强化磷酸戊糖途径的SOL3基因和三羧酸循环的MDH1基因后,GOD的胞外产量提高了6.3%和11.6%。在50 L反应器中,共表达HAC1的高产菌G/GMH1胞外GOD单位菌体酶活(单位细胞干重)可达6 656.6 U/g。

     

  • 图  1  酶切验证质粒pPIC9K-GOD(a)和重组菌G/GOD、G/GODM的PCR验证(b)

    Lane M—DNA marker; Lane 1'—pPIC9K-GOD digested by BamH І and Sal І; Lane 1—P.pastoris GS115 genomic DNA; Lane 2—Genomic DNA of G/GOD; Lane 3—Genomic DNA of G/GODM; Primers GODF/GODM were used

    Figure  1.  Enzyme digestion to confirm plasmid pPIC9K-GOD (a) and verification of recombinant strain G/GOD and G/GODM by PCR analysis (b)

    图  2  不同拷贝重组菌在摇瓶中的生长(a)和胞外GOD比活(b)曲线

    Figure  2.  Growth (a) and extracellular GOD specific activity (b) curve of the recombinant strains containing different copy of Pgod gene in shake flask

    图  3  二代重组菌的构建

    Figure  3.  Construction of second-generation recombinant strains

    图  4  二代重组菌的验证

    1−G/GMP1;2−G/GMMP1;3−G/GMP2;4−G/GMB;5−G/GMS1;6−G/GMH1;7−G/GMZ1;8−G/GMS3;9−G/GMG3; 10−G/GMMD1; M—DNA marker

    Figure  4.  Verification of second-generation recombinant strains

    图  5  诱导120 h时二代重组菌胞内外酶活

    Figure  5.  Extracellular and intracellular GOD specific activity of the second-generation recombinant strains after 120 h of induction

    图  6  二代高产重组菌的生长(a)和胞外酶活(b)

    Figure  6.  Growth (a) and extracellular GOD specific activity (b) of the second-generation recombinant strains

    图  7  5 L发酵罐中重组菌的生长(a)和胞外酶活(b)

    Figure  7.  Cell growth (a) and extracellular GOD specific activity (b) of recombinant strains in the 5 L bioreactor

    图  8  G/GMH1在50 L发酵罐中分批发酵的胞外GOD酶活、细胞干重、DO、OUR、CER、RQ时间曲线(a)和培养上清液的蛋白电泳(b)

    Lane M—Protein marker; Lane 1~7: GOD expression in fermentation supernatant at the induction time of 24, 48, 72, 96, 120, 144 h and 156 h, respectively

    Figure  8.  Time courses of extracellular GOD specific activity, Dry cell weight, DO, OUR, CER, RQ (a) and protein electrophoresis of supernatant solution (b) in the 50 L fedbatch fermentation using the strain G/GMH1

    表  1  本研究中所使用的引物

    Table  1.   Primers used in this study

    Primer namePrimer sequence (5′-3′)
    GODFAGAGAGGCTGAAGCTTACGTAGAATTCTCTAATGGTATTGAAGCATCAT
    GODRTAAGGCGAATTAATTCGCGGCCGCTTGCATTGAAGCGTAGTCTT
    gapdhFTGATGACCACCGTCCACTCCAT
    gapdhRGTTCTACCACCTCTCCAGTCCTTGT
    qGODFGGTGACATTTTCGGTTCTTCAGTCG
    qGODRCTAAAGTTGAACCACCCAAACCATTACC
    RHFGCTGGAAAGGTTTGAGGAGGATCTG
    AOXRGTAATGCGGAGCTTGTTGCATTCG
    CYCTTFCCGCTCTAACCGAAAAGGAAGGAG
    DDKCRGAACCCAACAAATTCAGAGATGCCTC
    下载: 导出CSV

    表  2  所构建重组菌的胞外单位菌体GOD酶活

    Table  2.   Extracellular GOD specific activity of the recombinant strains

    StrainGOD activity/
    (U·g−1)
    Fold change of GOD activity
    (Compared with G/GOD)
    G/GOD714.2±32.61.0
    G/GODM5843.2±42.08.2
    G/GMP17753.6±402.410.9
    G/GMMP12330.6±58.63.3
    G/GMP26363.7±43.68.9
    G/GMB4190.4±120.95.9
    G/GMS14385.5±72.46.1
    G/GMH19022.1±214.712.6
    G/GMZ15248.7±46.07.3
    G/GMS36209.2±338.48.7
    G/GMG33636.9±164.65.1
    G/GMMD16521.2±422.39.1
    下载: 导出CSV
  • [1] 王文婷, 赵伟, 郭美锦, 等. 不同来源葡萄糖氧化酶的分离纯化及其生物催化特性[J]. 华东理工大学学报(自然科学版), 2016, 42(4): 484-491.
    [2] GIBSON Q H, SWOBODA B E, MASSEY V. Kinetics and mechanism of action of glucose oxidase[J]. Journal of Biological Chemistry, 1964, 239(11): 3927-3934.
    [3] RASIAH I A, SUTTON K H, LOW F L, et al. Crosslinking of wheat dough proteins by glucose oxidase and the resulting effects on bread and croissants[J]. Food Chemistry, 2005, 89(3): 325-321. doi: 10.1016/j.foodchem.2004.02.052
    [4] BONET A, ROSELL C M, CABALLERO P A, et al. Glucose oxidase effect on dough rheology and bread quality: A study from macroscopic to molecular level[J]. Food Chemistry, 2006, 99(2): 408-415. doi: 10.1016/j.foodchem.2005.07.043
    [5] TZANOV T, SILGIA A, GUBITZ G M, et al. Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching[J]. Journal of Biotechnology, 2002, 93(1): 87-94. doi: 10.1016/S0168-1656(01)00386-8
    [6] WILSON R, TURNER A P F. Glucose oxidase and ideal enzyme[J]. Biosensors & Bioelectronics, 1992, 7(3): 165-185.
    [7] ETEMADZADEH H, AINAMO J, MURTOMAA H. Plaque growth-inhibiting effects of an abrasive fluoride-chlorhexidine toothpaste and a fluoride toothpaste containing oxidative enzymes[J]. Journal of Clinical Periodontology, 1985, 12(7): 607-616. doi: 10.1111/j.1600-051X.1985.tb01393.x
    [8] CHEN T, BARTON S C. A miniature biofuel cell[J]. Journal of the American Chemical Society, 2001, 123(35): 8630-8631. doi: 10.1021/ja0163164
    [9] WITT S, SINGH M, KALISZ H. Structural and kinetic properties of nonglycosylated recombinant Penicillium amagasakiense glucose oxidase expressed in Escherichia coli[J]. Applied & Environmental Microbiology, 1998, 64(4): 1405-1411.
    [10] KAPAT A, JUNG J K, PARK Y H. Improvement of extracellular recombinant glucose oxidase production in fed-batch culture of Saccharomyces cerevisiae: Effect of different feeding strategies[J]. Biotechnology Letters, 1998, 20(3): 319-323. doi: 10.1023/A:1005354608653
    [11] YAMAGUCHI M, TAHARA Y, NAKANO A, et al. Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris[J]. Protein Expression and Purification, 2007, 55(2): 273-278. doi: 10.1016/j.pep.2007.05.006
    [12] GAO Z W, LI Z F, YAO B, et al. High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization[J]. Biotechnology Letters, 2012, 34(3): 507-514. doi: 10.1007/s10529-011-0790-6
    [13] 顾磊. Aspergillus niger葡萄糖氧化酶的异源分泌表达、分子改造和发酵生产[D]. 江苏无锡: 江南大学, 2014.
    [14] MOUNA G, STEFAN R, PIETER P J, et al. The HAC1 gene from Pichia pastoris: Characterization and effect of its overexpression on the production of secreted, surface displayed and membrance proteins[J]. Microbial Cell Factories, 2010, 9(1): 49-61. doi: 10.1186/1475-2859-9-49
    [15] NOCON J, STEIGER M G, PFEFFER M, et al. Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production[J]. Metabolic Engineering, 2014, 24: 129-138.
    [16] DUAN G D, DING L M, QIAN J C, et al. Screening endogenous signal peptides and protein folding factors to promote the secretory expression of heterologous proteins in Pichia pastoris[J]. Journal of Biotechnology, 2019, 306: 193-202.
    [17] 张莹. 基于转录组分析及碳代谢途径改造优化毕赤酵母合成S-腺苷甲硫氨酸[D]. 上海: 华东理工大学, 2016.
    [18] SANDRA A, KERSTIN K, ASTRID H, et al. Real-time PCR-based determination of gene copy numbers in Pichia pastoris[J]. Journal of Biotechnology, 2010, 5(4): 413-420. doi: 10.1002/biot.200900233
    [19] 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[J]. Molecular Plant, 2014, 7(1): 170-186. doi: 10.1093/mp/sst151
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出版历程
  • 收稿日期:  2020-02-13
  • 网络出版日期:  2020-07-14
  • 刊出日期:  2021-06-30

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