Pathway analysis of recombinant Escherichia coli during protein production process under simulated microgravity

doi:10.11949/j.issn.0438-1157.20150471

Abstract

Abstract:

Microgravity effects were reported to affect the physiological characteristics and perturbations at the molecular levels of the microorganisms. Our previous works reported that the recombinant β-glucuronidase (PGUS) expressing strain Escherichia coli BL21 (DE3)/pET28a-pgus grew faster and the efficiency of recombinant protein production was also enhanced under simulated microgravity (SMG) as compared with the normal gravity (NG). In this study, a quantitative proteomic analysis of triple stable isotope dimethyl labeling using mass spectrometry method to investigate the effects of SMG on the strain and the during recombinant protein production process was performed. A total of 124 differentially expressed proteins under SMG were identified as compared with NG. Among them, 92 proteins were significantly up-regulated and 6 proteins were drastically down-regulated under SMG. The majority of the up-regulated proteins were involved with the metabolism of cell stress response, amino acid metabolism, translation, transcription and carbohydrate metabolic process. The results indicated that the differentially expressed levels of these proteins under SMG were beneficial to the synthesis of the heterologous protein expression of the recombinant Escherichia coli.

Key words: biological engineering, enzyme, metabolism, Escherichia coli, simulated microgravity, proteomics

摘要：

空间微重力对航天医学、材料学等领域发展起到极其重要的推动作用,然而在生物化工领域的研究尚处于起步阶段。微生物在微重力环境中培养时,其生长代谢和基因转录表达都与地球重力场中截然不同。前期研究表明表达重组β-葡萄糖醛酸苷酶的大肠杆菌在模拟微重力(simulated microgravity,SMG)环境中菌体生长和外源蛋白质表达量均比正常重力对照(normal gravity,NG)环境要高。为深入分析SMG环境效应对菌株在外源蛋白质表达过程中的影响,对该重组大肠杆菌菌株在SMG和NG培养过程中加入IPTG诱导外源蛋白质表达4 h 后的全细胞蛋白质酶解产物, 通过三重稳定同位素二甲基标记方法并结合质谱分离鉴定技术,在肽段水平上标记后进行差异定量蛋白质组学分析。结果表明:与NG对照相比,SMG条件下共有124个蛋白质表达水平发生显著变化,其中92个蛋白质上调,6个蛋白质下调。上调蛋白质集中在细胞压力胁迫、氨基酸代谢、翻译转录和碳代谢等途径,这些相关途径的蛋白质表达水平变化可以影响大肠杆菌生理代谢及外源蛋白质表达效率。

关键词: 生物工程, 酶, 代谢, 大肠杆菌, 模拟微重力, 蛋白质组学

CLC Number:

TQ936.2

HUANGFU Jie, LI Qian, LI Jun, LI Chun. Pathway analysis of recombinant Escherichia coli during protein production process under simulated microgravity[J]. CIESC Journal, 2015, 66(12): 4960-4965.

皇甫洁, 李茜, 李珺, 李春. 模拟微重力下影响重组Escherichia coli外源蛋白质表达途径分析[J]. 化工学报, 2015, 66(12): 4960-4965.

References 23

[1]	Dragosits M, Stadlmann J, Albiol J, Baumann K, Maurer M, Gasser B, Sauer M, Altmann F, Ferrer P, Mattanovich D. The effect of temperature on the proteome of recombinant Pichia pastoris [J]. Proteome. Res., 2009, 8: 1380-1392.
[2]	Dragosits M, Stadlmann J, Graf A, Gasser B, Maurer M, Sauer M, Kreil D P, Altmann F, Mattanovich D. The response to unfolded protein is involved in osmotolerance of Pichia pastoris [J]. BMC Genomics., 2010, 11: 1-16.
[3]	Gao H, Ayyaswamy P S, Ducheyne P. Dynamics of a microcarrier particle in the simulated microgravity environment of a rotating wall vessel [J]. Microgravity Sci. Technol., 1997, 9: 154-165.
[4]	Wolf D A, Schwartz R P. Analysis of gravity-induced particle motion and fluid perfusion flow in the NASA-designed rotating zero-head-space tissue culture vessel [R]. NASA Tech. Paper 3143, 1991.
[5]	Mitteregger R, Vogt G, Rossmanith E, Falkenhagen D. Rotary cell culture system (RCCS): a new method for cultivating hepatocytes on micro carriers [J]. Int. J. Arti. Organs., 1999, 22 (12): 816-822.
[6]	Wilson J W, Ott C M, Ramamurthy R, Porwollik S, McClelland M, Pierson D L, Nickerson C A. Low-shear modeled microgravity alters the Salmonella enterica serovar Typhimurium stress response in an RpoS-independent manner [J]. Appl. Environ. Microbiol., 2002, 68 (11): 5408-5416.
[7]	Nickerson C A, Ott C M, Wilson J W, Ramamurthy R, LeBlanc C L, Hönerzu B K, Hammond T, Pierson D L. Low-shear modeled microgravity: a global environmental regulatory signal affecting bacterial gene expression, physiology, and pathogenesis [J]. J. Microbiol. Methods, 2003, 54 (1): 1-11.
[8]	Arnold D, Fang A. Secondary metabolism in simulated microgravity [J]. Chem. Rec., 2001, 1: 333-346.
[9]	Tash J S, Bracho G E. Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility [J]. FASEB J., 1999, 13: S43-S54.
[10]	Xiang L, Qi F, Dai D, Li C, Jiang Y. Simulated microgravity affects growth of Escherichia coli and recombinant β-D-glucuronidase production [J]. Appl. Biochem. Biotechnol., 2010, 162 (3): 654-661.
[11]	Qi F, Imdad K, Lü B, Guo X, Li C. Enhancement of recombinant β-D-glucuronidase production under low-shear modeled microgravity in Pichia pastoris [J]. J. Chem. Technol. Biot., 2011, 86 (4): 505-511.
[12]	Boersema P J, Raijmakers R, Lemeer S, Mohammed S, Heck A J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics [J]. Nat. Protoc., 2009, 4 (4): 484-494.
[13]	Wang F, Chen R, Zhu J, Sun D, Song C, Wu Y, Ye M, Wang L, Zou H. A fully automated system with online sample loading, isotope dimethyl labeling and multidimensional separation for high-throughput quantitative proteome analysis [J]. Anal. Chem., 2010, 82 (7): 3007-3015.
[14]	Mortensen P, Gouw J W, Olsen J V, Ong S E, Rigbolt K T, Bunkenborg J, Cox J, Foster L J, Heck A J, Blagoev B, Andersen J S, Mann M, Quant M S. An open source platform for mass spectrometry-based quantitative proteomics [J]. J. Proteome. Res., 2010, 9 (1): 393-403.
[15]	Baker P W, Meyer M L, Leff L G. Escherichia coli growth under modeled reduced gravity [J]. Microgravity Sci. Technol., 2004, 15 (4): 39-44.
[16]	Raja V, Eric M, Laura L. Changes in gene expression of Escherichia coli under conditions of modeled reduced gravity [J]. Microgravity Sci. Technol., 2008, 20: 41-57.
[17]	Arunasri K, Adil M, Venu Charan K, Suvro C, Himabindu R S, Shivaji S. Effect of simulated microgravity on Escherichia coli K12 MG1655 growth and gene expression [J]. PLoS One, 2013, 8 (3): e57860.
[18]	Tucker D L, Ott C M, Huff S, Fofanov Y, Pierson D L, Willson R C, Fox G E. Characterization of Escherichia coli MG1655 grown in a low-shear modeled microgravity environment [J]. BMC Microbiol., 2007, 7: 15.
[19]	Huang B, Liu N, Rong X, Ruan J, Huang Y. Effects of simulated microgravity and spaceflight on morphological differentiation and secondary metabolism of Streptomyces coelicolor A3(2) [J]. Appl. Microbiol. Biotechnol., 2015, 99 (10): 4409-4422.
[20]	Mairhofer J, Scharl T, Marisch K, Cserjan-Puschmann M, Striedner G. Comparative transcription profiling and in-depth characterization of plasmid-based and plasmid-free Escherichia coli expression systems under production conditions [J]. Appl. Environ. Microbiol., 2013, 79 (12): 3802-3812.
[21]	Marisch K, Bayer K, Scharl T, Mairhofer J, Krempl P M, Hummel K, Razzazi-Fazeli E, Striedner G. A comparative analysis of industrial Escherichia coli K-12 and B strains in high-glucose batch cultivations on process-, transcriptome- and proteome level [J]. PLoS One, 2013, 8(8): e70516.
[22]	Vogl T, Thallinger G G, Zellnig G, Drew D, Cregg J M, Glieder A, Freigassner M. Towards improved membrane protein production in Pichia pastoris: general and specific transcriptional response to membrane protein overexpression [J]. J. Biotechnol., 2014, 31 (6): 538-552.
[23]	Huangfu Jie, Qi Feng, Liu Hongwei, Zou Hanfa, Muhammad Saad Ahmed, Li Chun. Novel helper factors influencing recombinant protein production in Pichia pastoris based on proteomic analysis under simulated microgravity [J]. Appl. Microbiol. Biotechnol., 2015, 99(2): 653-665.