食品与发酵工业 >

2025 , Vol. 51 >Issue 16: 36 - 42

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.041206

研究报告

Ophiostoma piceae胆固醇酯酶在毕赤酵母中的高效表达

  • 郭子妍 ,
  • 毛文刚 ,
  • 林红 ,
  • 高文欣 ,
  • 王月峰 ,
  • 舒正玉
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  • 1(福建师范大学 生命科学学院,工业微生物发酵技术国家地方联合工程研究中心,福建 福州,350117)
    2(山东第二医科大学 生命科学与技术学院,潍坊市呼吸道病原菌及药物治疗重点实验室,山东 潍坊,261053)
第一作者:硕士研究生(舒正玉教授为通信作者,E-mail:shuzhengyu@fjnu.edu.cn)

收稿日期: 2024-10-17

  修回日期: 2024-11-30

  网络出版日期: 2025-08-29

基金资助

国家自然科学基金项目(31370802,31870787);福建省自然科学基金项目(2017J01441,2021J01169)

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High yield expression of Ophiostoma piceae steryl esterase in Pichia pastoris

  • GUO Ziyan ,
  • MAO Wengang ,
  • LIN Hong ,
  • GAO Wenxin ,
  • WANG Yuefeng ,
  • SHU Zhengyu
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  • 1(National Joint Engineering Research Center of Industrial Microbiology and Fermentation Technology, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China)
    2(Weifang Key Laboratory of Respiratory Tract Pathogens and Drug Therapy, School of Life Science and Technology, Shandong Second Medical University, Weifang 261053, China)

Received date: 2024-10-17

  Revised date: 2024-11-30

  Online published: 2025-08-29

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摘要

微生物源甾醇酯酶资源的匮乏,制约了甾醇绿色加工技术的普及推广。Ophiostoma piceae胆固醇酯酶(Ophistoma picae steryl esterase,OPE)是到目前为止,第一个报道的真正微生物源胆固醇酯酶。实现OPE的工业化生产,将有效解决该技术瓶颈。为提高其在毕赤酵母中的表达水平,该研究分别考察了基因拷贝数、分泌信号类型和共表达二硫键异构酶对ope基因在P.pastoris中高效表达的影响。结果表明,上述3种因素均可在一定程度上提高重组OPE产量,其中共表达二硫键异构酶的作用最为显著。筛选到的最高表达量的重组P.pastoris菌株,其胞外OPE产量为(27.4±0.8) U/mL,较优化前提高了161.18倍。高效表达ope基因的重组P.pastoris菌株的构建与筛选,将为后续重组OPE的应用奠定基础。

关键词： Ophiostoma piceae胆固醇酯酶; 毕赤酵母; 基因多拷贝重组; 信号肽; 蛋白质二硫键异构酶; 异源高效表达

本文引用格式

郭子妍 , 毛文刚 , 林红 , 高文欣 , 王月峰 , 舒正玉 . Ophiostoma piceae胆固醇酯酶在毕赤酵母中的高效表达[J]. 食品与发酵工业, 2025 , 51(16) : 36 -42 . DOI: 10.13995/j.cnki.11-1802/ts.041206

Abstract

The scarcity of microbial-derived steryl esterase resources restricts the popularization of green processing technology for phytosteryl modification.Ophistoma picae steryl esterase (OPE) is the first reported microbial-derived steryl esterase.The industrial production of OPE will effectively solve this technological bottleneck.To improve expression level of ope gene in Pichia pastoris, effect of ope gene copy numbers, secretion signal peptide types, and co-expression of protein disulfide isomerase on recombinant OPE yield was investigated in this study.Research results showed that all of the investigated factors played positive effect on OPE yield, and the highest yield of recombinant OPE was from the recombinant P.pastoris/pEESB-11, a coexpression system of protein disulfide isomerase gene with ope gene.The maximum expression level of ope gene from P.pastoris/pEESB-11 was 27.4 U/mL after the fermentation medium was optimized.This research work will greatly promote the application of OPE in the future.

Key words： Ophiostoma piceae steryl esterase; Pichia pastoris; gene multi-copy integration; signal peptide types; protein disulfide isomerase; high level heterogeneous expression

参考文献

[1] ZHENG M M, LIN Z, LIN S J, et al.Chemoenzymatic synthesis of steroidal products:Recent advances[J].European Journal of Organic Chemistry, 2024, 27(5):e202301066.
[2] PAVANI M, SINGHA P, DASH D R, et al.Novel encapsulation approaches for phytosterols and their importance in food products:A review[J].Journal of Food Process Engineering, 2022, 45(8):e14041.
[3] FENG S M, WANG L L, SHAO P, et al.A review on chemical and physical modifications of phytosterols and their influence on bioavailability and safety[J].Critical Reviews in Food Science and Nutrition, 2022, 62(20):5638-5657.
[4] WANG H B, ABE I.Recent developments in the enzymatic modifications of steroid scaffolds[J].Organic & Biomolecular Chemistry, 2024, 22(18):3559-3583.
[5] ZHENG M M, HUANG Q, HUANG F H, et al.Production of novel “functional oil” rich in diglycerides and phytosterol esters with “one-pot” enzymatic transesterification[J].Journal of Agricultural and Food Chemistry, 2014, 62(22):5142-5148.
[6] DA S PEREIRA A, DE SOUZA A H, FRAGA J L, et al.Lipases as effective green biocatalysts for phytosterol esters’ production:A review[J].Catalysts, 2022(1), 12:88.
[7] KONTKANEN H, TENKANEN M, FAGERSTRÖM R, et al.Characterisation of steryl esterase activities in commercial lipase preparations[J].Journal of Biotechnology, 2004, 108(1):51-59.
[8] NAIK S, BASU A, SAIKIA R, et al.Lipases for use in industrial biocatalysis:Specificity of selected structural groups of lipases[J].Journal of Molecular Catalysis B:Enzymatic, 2010, 65(1-4):18-23.
[9] GUTIÉRREZ-FERNÁNDEZ J, VAQUERO M E, PRIETO A, et al.Crystal structures of Ophiostoma piceae sterol esterase:Structural insights into activation mechanism and product release[J].Journal of Structural Biology, 2014, 187(3):215-222.
[10] CALERO-RUEDA O, GUTIERREZ A, DEL RIO J C, et al.Hydrolysis of sterol esters by an esterase from Ophiostoma piceae:Application to pitch control in pulping of Eucalyptus globulus wood[J].International Journal of Biotechnology, 2004, 6(4):367-375.
[11] CEDILLO V B, PRIETO A, MARTÍNEZ M J.Potential of Ophiostoma piceae sterol esterase for biotechnologically relevant hydrolysis reactions[J].Bioengineered, 2013, 4(4):249-253.
[12] MOLINA-GUTIÉRREZ M, HAKALIN N L S, RODRÍGUEZ-SANCHEZ L, et al.Green synthesis of β-sitostanol esters catalyzed by the versatile lipase/sterol esterase from Ophiostoma piceae[J].Food Chemistry, 2017, 221:1458-1465.
[13] LIN H, MU X D, HUANG J Z, et al.Comparative analysis of polyester-hydrolysis activity among three lipolytic enzymes[J].Journal of Chemical Technology & Biotechnology, 2019, 94(8):2522-2528.
[14] CEDILLO V B, PLOU F J, MARTÍNEZ M J.Recombinant sterol esterase from Ophiostoma piceae:An improved biocatalyst expressed in Pichia pastoris[J].Microbial Cell Factories, 2012, 11:73.
[15] THAK E J, YOO S J, MOON H Y, et al.Yeast synthetic biology for designed cell factories producing secretory recombinant proteins[J].FEMS Yeast Research, 2020, 20(2):foaa009.
[16] INGRAM Z, PATKAR A, OH D, et al.Overcoming obstacles in protein expression in the yeast Pichia pastoris:Interviews of leaders in the Pichia field[J].Pacific Journal of Health, 2021, 4(1):2.
[17] YANG X Q, ZHANG Y M.Expression of recombinant transglutaminase gene in Pichia pastoris and its uses in restructured meat products[J].Food Chemistry, 2019, 291:245-252.
[18] MATHEWS D H, DISNEY M D, CHILDS J L, et al.Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure[J].Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(19):7287-7292.
[19] XIONG A S, YAO Q H, PENG R H, et al.High level expression of a recombinant acid phytase gene in Pichia pastoris[J].Journal of Applied Microbiology, 2005, 98(2):418-428.
[20] BARRERO J J, CASLER J C, VALERO F, et al.An improved secretion signal enhances the secretion of model proteins from Pichia pastoris[J].Microbial Cell Factories, 2018, 17(1):161.
[21] AZA P, MOLPECERES G, DE SALAS F, et al.Design of an improved universal signal peptide based on the α-factor mating secretion signal for enzyme production in yeast[J].Cellular and Molecular Life Sciences, 2021, 78(7):3691-3707.
[22] WOLLBORN D, MUNKLER L P, HORSTMANN R, et al.Predicting high recombinant protein producer strains of Pichia pastoris MutS using the oxygen transfer rate as an indicator of metabolic burden[J].Scientific Reports, 2022, 12:11225.
[23] JIAO L C, ZHOU Q H, SU Z X, et al.High-level extracellular production of Rhizopus oryzae lipase in Pichia pastoris via a strategy combining optimization of gene-copy number with co-expression of ERAD-related proteins[J].Protein Expression and Purification, 2018, 147:1-12.
[24] RINNOFNER C, FELBER M, PICHLER H.Strains and molecular tools for recombinant protein production in Pichia pastoris[J].Methods in Molecular Biology, 2022, 2513:79-112.
[25] 李兆丰, 陈福生, 江正强, 等.优质食品酶的催化机制及在高端配料创制方面的应用基础[J].中国食品学报, 2024, 24(5):75-88.
LI Z F, CHEN F S, JIANG Z Q, et al.The catalytic mechanism of high-quality food enzymes and their application in the creation of upscale ingredients[J].Journal of Chinese Institute of Food Science and Technology, 2024, 24(5):75-88.
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