Construction and Application of Cell Lysis Systems in the Expression of Mycotoxin Degrading Enzyme in <i>Escherichia coli</i>

doi:10.13523/j.cb.20190409

China Biotechnology

2019, Vol. 39

Issue (4): 69-77 DOI: 10.13523/j.cb.20190409

Construction and Application of Cell Lysis Systems in the Expression of Mycotoxin Degrading Enzyme in Escherichia coli

Cheng-cheng ZHAO,Chang-po SUN,Xiao-jiao CHANG,Song-ling WU,Zhen-quan LIN(

)

Academy of National Food and Strategic Reserves Administration, Institute of Cereal Processing Science and Technology, Beijing 100037,China

Download:

HTML

PDF(1412KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Objective: Extracellular production of recombinant proteins in Escherichia coli is limited by the inefficiency of inherent secretion system. An inducible cell lysis system was designed and constructed to enhance secretion of intracellular recombinant protein in E.coli.Methods: Considering colicin E7 could promote cell lysis, E.coli cell lysis systems were constructed by co-expressing target protein and colicin E7 lysis to release the recombinant proteins to culture medium.Results: A reporter protein (red fluorescent protein, RFP), as a recombinant protein, was co-expressed with E7 in E.coli to evaluate the cell lysis systems. Expression of recombinant protein was controlled by T7 promoter. While E7 cassette was controlled by two promoters (T7 promoter and araBAD promoter) which determined the expression timing of E7 and therefore determined the timing of cell lysis. Compared to one-step induction by IPTG, the two-step induction by IPTG and L-arabinose was better for the production and secretion of recombinant proteins. The two-step inducible lysis system was also used to express zearalenone (ZEN) degrading enzyme, and high enzyme activity was detectable in the culture supernatant samples. The secreted enzyme could degrade about 5.8μg ZEN in 30minutes at 37℃.Conclusion: The colicin E7 assisted two-step inducible cell lysis system could be potential for expression of recombinant proteins and their secretion to extracellular in E. coli.

Key words： E. coli Cell lysis system Red fluorescent protein Zearalenone degrading enzyme

Received: 21 August 2018 Published: 08 May 2019

ZTFLH:

Q819

Corresponding Authors: Zhen-quan LIN E-mail: zhenquanlin09@gmail.com

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Cheng-cheng ZHAO
	Chang-po SUN
	Xiao-jiao CHANG
	Song-ling WU
	Zhen-quan LIN

Cite this article:

Cheng-cheng ZHAO,Chang-po SUN,Xiao-jiao CHANG,Song-ling WU,Zhen-quan LIN. Construction and Application of Cell Lysis Systems in the Expression of Mycotoxin Degrading Enzyme in Escherichia coli. China Biotechnology, 2019, 39(4): 69-77.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190409 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I4/69

Table 1 Strains and plasmids used in this study

Table 2 Primers used in this study

Fig.1 Schematic illustration of the cell lysis progress in the inducible lytic system of E.coli

Fig.2 Construction of recombinant plasmids containing an expression cassette of target protein controlled by the T7 promoter and a cassette of colicin E7 lysis controlled by the T7 promoter or araBAD promoter P_T7 and P_BAD are short for T7 promoter and araBAD promoter, respectively

Fig.3 Time course of biomass for different recombinant E.coli strains

Fig. 4 SDS-PAGE analysis of culture supernatants from the recombinant E.coli strains expressing RFP T7E7-T7RFP refers to E.coli BL21[pET28a(+)-T7E7-T7rfp], and T7E7 refers to E.coli BL21[pET28a(+)-T7E7]. AraE7-T7RFP refers to E.coli BL21[pET28a(+)-AraE7-T7rfp], and AraE7 refers to E.coli BL21[pET28a(+)-AraE7]. I-0h, I-2h and I-4h refer to culture supernatants before IPTG induction, 2h after IPTG induction and 4h after IPTG induction, respectively. A-0h, A-2h and A-4h refer to culture supernatants before L-arabinose induction, 2h after arabinose induction and 4h after arabinose induction, respectively

Fig.5 Quantification of RFP fluorescence intensity in the culture supernatant of recombinant E.coli strains expressing RFP Time on the abscissa refers to induction time by IPTG for E.coli BL21[pET28a(+)-T7E7-T7rfp] and induction time by L-arabinose for E.coli BL21[pET28a(+)-AraE7-T7rfp], respectively

Fig.6 Cellular morphology of two recombinant strains expressing RFP by microscopy under the conditions with or without induction (a)-(c) Refer respectively to cell shapes of E.coli BL21[pET28a(+)-T7E7-T7rfp] before IPTG induction, 2h after IPTG induction and 4h after IPTG induction (d)-(f) Refer respectively to cell shapes of E.coli BL21[pET28a(+)-T7E7-T7rfp] before L-arabinose induction, 2h after arabinose induction and 4h after arabinose induction

Fig.7 SDS-PAGE analysis of 10-fold concentrated culture supernatants from the recombinant E.coli strains expressing ZENd AraE7-T7ZENd refers to E.coli BL21[pET28a(+)-AraE7-T7ZENd], and AraE7 refers to E.coli BL21[pET28a(+)-AraE7]. I-0h and I-2h refer to culture supernatants before IPTG induction, 2h after IPTG induction, respectively. A-0h, A-1h, A-2h, A-4h and A-6h refer to culture supernatants before L-arabinose induction, 1h after arabinose induction, 2h after arabinose induction, 4h after arabinose induction and 6h after arabinose induction, respectively

Fig.8 Degradation of ZEN by culture supernatants from the recombinant E.coli strains expressing ZENd


[1]	Baneyx F, Mujacic M . Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology, 2004,22(11):1399-1408. doi: 10.1038/nbt1029

[2]	Bell P A . E.coli expression systems//Gerstein A S. Molecular biology problem solver: A laboratory guide. New York: John Wiley and Sons Ltd, 2002: 461-490.

[3]	Mergulhão F J M, Summers D K, Monteiro G A . Recombinant protein secretion in Escherichia coli. Biotechnology Advances, 2005,23(3):177-202. doi: 10.1016/j.biotechadv.2004.11.003 pmid: 15763404

[4]	Gentschev I, Dietrich G, Goebel W . The E.coli α-hemolysin secretion system and its use in vaccine development. TRENDS in Microbiology, 2002,10(1):39-45. doi: 10.1016/S0966-842X(01)02259-4 pmid: 11755084

[5]	Fernández L A, Lorenzo V D . Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway. Molecular Microbiology, 2001,40(2):332-346. doi: 10.1046/j.1365-2958.2001.02410.x pmid: 11309117

[6]	Koster M, Bitter W, Tommassen J . Protein secretion mechanisms in Gram-negative bacteria. International Journal of Medical Microbiology, 2000,290(4):325-331. doi: 10.1016/S1438-4221(00)80033-8 pmid: 11111906

[7]	Humphreys D P, Sehdev M, Chapman A P , et al. High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5'end of the coding sequence. Protein Expression and Purification, 2000,20(2):252-264. doi: 10.1006/prep.2000.1286 pmid: 11049749

[8]	Zamani M, Nezafat N, Negahdaripour M , et al. In silico evaluation of different signal peptides for the secretory production of human growth hormone in E.coli. International Journal of Peptide Research and Therapeutics, 2015,21(3):261-268. doi: 10.1007/s10989-015-9454-z

[9]	Jeong K J, Lee S Y . Excretion of human β-endorphin into culture medium by using outer membrane protein F as a fusion partner in recombinant Escherichia coli. Applied and Environmental Microbiology, 2002,68(10):4979-4985. doi: 10.1128/AEM.68.10.4979-4985.2002 pmid: 126437

[10]	Gray G L, Baldridge J S , McKeown K S , et al. Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable. Gene, 1985,39(2-3):247-254. doi: 10.1016/0378-1119(85)90319-1 pmid: 3912261

[11]	Robbens J, Raeymaekers A, Steidler L , et al. Production of soluble and active recombinant murine interleukin-2 in Escherichia coli: high level expression, Kil-induced release, and purification. Protein Expression and Purification, 1995,6(4):481-486. doi: 10.1006/prep.1995.1064 pmid: 8527934

[12]	Sommer B, Friehs K, Flaschel E . Efficient production of extracellular proteins with Escherichia coli by means of optimized coexpression of bacteriocin release proteins. Journal of Biotechnology, 2010,145(4):350-358. doi: 10.1016/j.jbiotec.2009.11.019 pmid: 19958803

[13]	Hsiung H M, Cantrell A, Luirink J , et al. Use of bacteriocin release protein in E.coli for excretion of human growth hormone into the culture medium. Nature Biotechnology, 1989,7(3):267-271. doi: 10.1038/nbt0389-267

[14]	Kiany J, Zomorodipour A, Raji M A , et al. Construction of recombinant plasmids for periplasmic expression of human growth hormone in Escherichia coli under T7 and lac promoters. Journal of Sciences, Islamic Republic of Iran, 2003,14(4):311-316.

[15]	Yamabhai M, Emrat S, Sukasem S , et al. Secretion of recombinant Bacillus hydrolytic enzymes using Escherichia coli expression systems. Journal of Biotechnology, 2008,133(1):50-57. doi: 10.1016/j.jbiotec.2007.09.005 pmid: 17950946

[16]	Morita M, Asami K, Tanji Y , et al. Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes. Biotechnology Progress, 2001,17(3):573-576. doi: 10.1021/bp010018t pmid: 11386882

[17]	Yang Y G, Tong Q, Hu T S , et al. The application of a novel lytic system to the recovery of recombinant proteins in E .coli. Acta Biochimica et Biophysica Sinica, 2000,32(3):211-216. doi: 10.1016/S0014-5793(00)01482-4 pmid: 12075442

[18]	Lo T M, Tan M H, Hwang I Y , et al. Designing a synthetic genetic circuit that enables cell density-dependent auto-regulatory lysis for macromolecule release. Chemical Engineering Science, 2013,103:29-35. doi: 10.1016/j.ces.2013.03.021

[19]	Chen Y R, Yang T Y, Lei G S , et al. Delineation of the translocation of colicin E7 across the inner membrane of Escherichia coli. Archives of Microbiology, 2011,193(6):419-428. doi: 10.1007/s00203-011-0688-7 pmid: 21387181

[20]	Wang N, Guo X, Ng I S . Simultaneous release of recombinant cellulases introduced by coexpressing colicin E7 lysis in Escherichia coli. Biotechnology and Bioprocess Engineering, 2016,21(4):491-501. doi: 10.1007/s12257-016-0260-z

[21]	Lin L J R, Liao C C, Chen Y R , et al. Induction of membrane permeability in Escherichia coli mediated by lysis protein of the ColE7 operon. FEMS Microbiology Letters, 2009,298(1):85-92. doi: 10.1111/j.1574-6968.2009.01705.x pmid: 19673051

[1]	WU Hong-xuan, YANG Jin-hua, SHEN Pei-jie, LI Qing-chen, HUANG Jian-zhong, QI Feng. *Study on the Production of Indole-3-acetic Acid Using E. coli* Cell Factory**[J]. China Biotechnology, 2021, 41(1): 12-19.

[2]	YAN Wei-huan,HUANG Tong,HONG Jie-fang,MA Yuan-yuan. Recent Advances in Butanol Biosynthesis of Escherichia coli[J]. China Biotechnology, 2020, 40(9): 69-76.

[3]	WEI Yan, WANG Huan-qin, WU Meng, ZHANG Feng-juan, LIANG Guo-dong, ZHU Wu-yang. Construction and Identification of the Cell Line for Detecting Flaviviruses[J]. China Biotechnology, 2015, 35(9): 35-41.

[4]	TIAN Ting, CHANG Jian, ZHANG Xin, JIANG Chen-yu, ZHANG Yun-hai, LIU Xiao-mei, ZHANG Chun. In Vivo Imaging of Near-infrared Fluorescent Protein in Skeletal Muscle of Mice Mediated by Recombinant Adeno-associated Virus[J]. China Biotechnology, 2014, 34(10): 67-72.

Viewed

Full text

Abstract

Cited

Shared

Discussed