乙醇胁迫抑制毕赤酵母表达外源蛋白的转录组学分析

高鹏1 丁健1 张许1 赵玥1 张猛1 高敏杰1 吴剑荣1 詹晓北1

(1.江南大学生物工程学院工业生物技术教育部重点实验室, 江苏无锡 214122)

【摘要】在5 L发酵罐中进行毕赤酵母发酵表达猪干扰素的实验,发现甘油培养末期乙醇的积累会抑制外源蛋白的表达。从转录组学角度系统分析不同浓度乙醇胁迫条件下,毕赤酵母甘油培养期和甲醇诱导期细胞的生理状态变化。研究结果表明,在甘油培养期,乙醇胁迫使得毕赤酵母细胞中的545个基因发生了显著差异表达(265个基因表达上调,280个基因表达下调),这些差异表达基因的功能主要涉及蛋白质合成、能量代谢、细胞周期和过氧化物酶代谢。乙醇胁迫增加了蛋白质错误折叠的情况,降低了核糖体和线粒体的结构完整性,使得甘油培养末期无法得到大量具有健全功能的酵母细胞。在甲醇诱导期,与甲醇代谢、蛋白质加工合成、氨基酸代谢等途径相关的294个基因发生了显著差异表达(171个基因表达上调,123个基因表达下调),导致内质网胁迫不能被及时解除,破坏了细胞内的氨基酸正常代谢。

【关键词】 毕赤酵母; 外源蛋白; 乙醇胁迫; 转录组学;

【DOI】

【基金资助】 国家自然科学基金(No.31301408) National Natural Science Foundation of China(No.31301408) 江苏省自然科学基金(No.BK20130122) Natural Science Foundation of Jiangsu Province(No.BK20130122) 中国博士后科学基金(No.2014M551501)资助 China Postdoctoral Science Foundation(No.2014M551501)

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    References

    [1]Cereghino GPL,Cereghino JL,Ilgen C,et al.Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris.Curr Opin Biotechnol,2002,13(4):329–332.

    [2]Liang SL,Wang B,Pan L,et al.Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing.BMC Genomics,2012,13:738.

    [3]Gao MJ,Dong SJ,Yu RS,et al.Improvement of ATP regeneration efficiency and operation stability in porcine interferon-αproduction by Pichia pastoris under lower induction temperature.Korean J Chem Eng,2011,28(6):1412–1419.

    [4]Gao MJ,Li Z,Yu RS,et al.Methanol/sorbitol co-feeding induction enhanced porcine interferon-αproduction by P.pastoris associated with energy metabolism shift.Bioproc Biosyst Eng,2012,35(7):1125–1136.

    [5]Zhu TC,Hang HF,Chu J,et al.Transcriptional investigation of the effect of mixed feeding to identify the main cellular stresses on recombinant Pichia pastoris.J Ind Microbiol Biotechnol,2013,40(2):183–189.

    [6]Baumann K,Carnicer M,Dragosits M,et al.A multi-level study of recombinant Pichia pastoris in different oxygen conditions.BMC Syst Biol,2010,4:141.

    [7]Gao MJ,Zheng ZY,Wu JR,et al.Improvement of specific growth rate of Pichia pastoris for effective porcine interferon-αproduction with an on-line model-based glycerol feeding strategy.Appl Microbiol Biotechnol,2012,93(4):1437–1445.

    [8]Wei C,Zhou XS,Zhang YX.Improving intracellular production of recombinant protein in Pichia pastoris using an optimized preinduction glycerol-feeding scheme.Appl Microbiol Biotechnol,2008,78(2):257–264.

    [9]Catala M,Tremblay M,SamsonÉ,et al.Deletion of Rnt1p alters the proportion of open versus closed r RNA gene repeats in yeast.Mol Cell Biol,2008,28(2):619–629.

    [10]Baumann K,Maurer M,Dragosits M,et al.Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins.Biotechnol Bioeng,2008,100(1):177–183.

    [11]Yu RS,Dong SJ,Zhu YM,et al.Effective and stable porcine interferon-αproduction by Pichia pastoris fed-batch cultivation with multi-variables clustering and analysis.Bioproc Biosyst Eng,2010,33(4):473–483.

    [12]Jin H,Liu GQ,Ye XF,et al.Enhanced porcine interferon-αproduction by recombinant Pichia pastoris with a combinational control strategy of low induction temperature and high dissolved oxygen concentration.Biochem Eng J,2010,52(1):91–98.

    [13]Liang JC,Zeng FL,Guo AZ,et al.Microarray analysis of the chelerythrine-induced transcriptome of Mycobacterium tuberculosis.Curr Microbiol,2011,62(4):1200–1208.

    [14]Tedesco V,Roquet RF,De Mis J,et al.Extinction,applied after retrieval of auditory fear memory,selectively increases zinc-finger protein 268 and phosphorylated ribosomal protein S6 expression in prefrontal cortex and lateral amygdala.Neurobiol Learn Mem,2014,115:78–85.

    [15]Iyer LM,Leipe DD,Koonin EV,et al.Evolutionary history and higher order classification of AAA plus ATPases.J Struct Biol,2004,146(1/2):11–31.

    [16]Dragon F,Gallagher JEG,Compagnone-Post PA,et al.A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis.Nature,2002,417(6892):967–970.

    [17]Kalmar B,Greensmith L.Induction of heat shock proteins for protection against oxidative stress.Adv Drug Deliver Rev,2009,61(4):310–318.

    [18]Danial NN,Korsmeyer SJ.Cell death:critical control points.Cell,2004,116(2):205–219.

    [19]Luo HH,Wong J,Wong B.Protein degradation systems in viral myocarditis leading to dilated cardiomyopathy.Cardiovasc Res,2010,85(2):347–356.

    [20]Twig G,Elorza A,Molina AJA,et al.Fission and selective fusion govern mitochondrial segregation and elimination by autophagy.EMBO J,2008,27(2):433–446.

    [21]Boada J,Roig T,Perez X,et al.Cells overexpressing fructose-2,6-bisphosphatase showed enhanced pentose phosphate pathway flux and resistance to oxidative stress.FEBS Lett,2000,480(2/3):261–264.

    [22]Gorsich SW,Dien BS,Nichols NN,et al.Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1,GND1,RPE1,and TKL1 in Saccharomyces cerevisiae.Appl Microbiol Biotechnol,2006,71(3):339–349.

    [23]Marks VD,Sui SJH,Erasmus D,et al.Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response.FEMS Yeast Res,2008,8(1):35–52.

    [24]Teixeira MC,Raposo LR,Mira NP,et al.Genome-wide identification of Saccharomyces cerevisiae genes required for maximal tolerance to ethanol.Appl Environ Microbiol,2009,75(18):5761–5772.

    [25]Yoshikawa K,Tanaka T,Furusawa C,et al.Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae.FEMS Yeast Res,2009,9(1):32–44.

    [26]Clague MJ,Urbe S.Ubiquitin:same molecule,different degradation pathways.Cell,2010,143(5):682–685.

    [27]Penkner AM,Prinz S,Ferscha S,et al.Mnd2,an essential antagonist of the anaphase-promoting complex during meiotic prophase.Cell,2005,120(6):789–801.

    [28]Liu S,Yang H,Zhao J,et al.NEDD8 Ultimate Buster-1 Long(NUB1L)protein promotes transfer of NEDD8 to proteasome for degradation through the P97UFD1/NPL4 complex.J Biol Chem,2013,288(43):31339–31349.

    [29]Lei XG,Cheng WH,Mc Clung JP.Metabolic regulation and function of glutathione peroxidase-1.Annu Rev Nutr,2007,27:41–61.

This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 05, Pages 584-598

May 2016

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  • 1 材料与方法
  • 2 结果与分析
  • 3 讨论
  • 参考文献