Applied Microbiology and Biotechnology

, Volume 103, Issue 3, pp 1441–1453 | Cite as

Ethanol effects on the overexpression of heterologous catalase in Escherichia coli BL21 (DE3)

  • Hongchen Zheng
  • Zhenxiao Yu
  • Wenju Shu
  • Xiaoping Fu
  • Xingya Zhao
  • Shibin Yang
  • Ming Tan
  • Jianyong Xu
  • Yihan LiuEmail author
  • Hui SongEmail author
Applied microbial and cell physiology


A novel method involving ethanol-induced increase in the heterologous recombinant protein expression in E. coli cells was commonly used in recent studies. However, the detailed mechanism of this method is still to be revealed. This work used comparative transcriptomic analysis and numerous experiments to uncover the mechanism of ethanol effects on the expression of heterologous catalase in the recombinant strain E. coli BL21 (pET26b-katA). The key regulatory genes malK and prpD were found to have the most significant effects on the expression of heterologous catalase. Thus, the maltose ABC transporter and carbon metabolism from propanoate metabolism to citrate cycle were found to be the main regulatory pathways activated by ethanol to enhance the synthesis of heterologous proteins. Based on these mechanisms, a universally applicable E. coli expression host strain for improving the expression of heterologous proteins might be constructed.


Ethanol treatment Escherichia coli Heterologous protein expression Maltose ABC transporter prpD 



We would like to thank Dr. Changhao Bi for CRISPR/cas9 method to knockout target genes from E. coli genome.

Funding information

This study was supported by the foundation (Grant 2017KF005) of Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key Laboratory of Industrial Microbiology (Tianjin University of Science &Technology). And this work was also supported by the National Natural Science Fund of China (Grant 31701534), the Key Deployment Project in Chinese Academy of Sciences (Grant KFJ-STS-ZDTP-016-1, KFZD-SW-211-2), the Tianjin Science & Technology Planning Project (Grant 16YFZCSY00790, 15PTCYSY00020, and 15YFYSSY00040) and Yantai Marine economy innovation development demonstration project (Grant YHCX-SW-L-201703).

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9509_MOESM1_ESM.pdf (1.6 mb)
ESM 1 (PDF 1647 kb)


  1. Babu M, Bundalovic-Torma C, Calmettes C, Phanse S, Zhang Q, Jiang Y, Minic Z, Kim S, Mehla J, Gagarinova A, Rodionova I, Kumar A, Guo H, Kagan O, Pogoutse O, Aoki H, Deineko V, Caufield JH, Holtzapple E, Zhang Z, Vastermark A, Pandya Y, Lai CC, El Bakkouri M, Hooda Y, Shah M, Burnside D, Hooshyar M, Vlasblom J, Rajagopala SV, Golshani A, Wuchty S, J FG, Saier M, Uetz P, T FM, Parkinson J, Emili A (2018) Global landscape of cell envelope protein complexes in Escherichia coli. Nat Biotechnol 36(1):103–112. CrossRefGoogle Scholar
  2. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22(11):1399–1408. CrossRefGoogle Scholar
  3. Boos W, Shuman H (1998) Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol Mol Biol Rev 62(1):204–229Google Scholar
  4. Bordignon E, Grote M, Schneider E (2010) The maltose ATP-binding cassette transporter in the 21st century--towards a structural dynamic perspective on its mode of action. Mol Microbiol 77(6):1354–1366. CrossRefGoogle Scholar
  5. Cao H, Wei D, Yang Y, Shang Y, Li G, Zhou Y, Ma Q, Xu Y (2017) Systems-level understanding of ethanol-induced stresses and adaptation in E. coli. Sci Rep 7:44150. CrossRefGoogle Scholar
  6. Cheng C, Wu S, Cui L, Wu Y, Jiang T, He B (2017) A novel Ffu fusion system for secretory expression of heterologous proteins in Escherichia coli. Microb Cell Factories 16(1):231. CrossRefGoogle Scholar
  7. Chhetri G, Ghosh A, Chinta R, Akhtar S, Tripathi T (2015a) Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae. Bioengineered 6(1):62–66. CrossRefGoogle Scholar
  8. Chhetri G, Kalita P, Tripathi T (2015b) An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli. MethodsX 2:385–391. CrossRefGoogle Scholar
  9. Chhetri G, Pandey T, Chinta R, Kumar A, Tripathi T (2015c) An improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies. Protein Expr Purif 114:71–76. CrossRefGoogle Scholar
  10. Costa S, Almeida A, Castro A, Domingues L (2014) Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Front Microbiol 5:63. Google Scholar
  11. Goff J, Yee N (2017) Tellurate enters Escherichia coli K-12 cells via the SulT-type sulfate transporter CysPUWA. FEMS Microbiol Lett 364(24): fnx241.
  12. Hochberg Y, Benjamini Y (1990) More powerful procedures for multiple significance testing. Stat Med 9(7):811–818CrossRefGoogle Scholar
  13. Horinouchi T, Tamaoka K, Furusawa C, Ono N, Suzuki S, Hirasawa T, Yomo T, Shimizu H (2010) Transcriptome analysis of parallel-evolved Escherichia coli strains under ethanol stress. BMC Genomics 11:579. CrossRefGoogle Scholar
  14. Ingram LO (1986) Microbial tolerance to alcohols - role of the cell-membrane. Trends Biotechnol 4(2):40–44. CrossRefGoogle Scholar
  15. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with bowtie 2. Nat Methods 9(4):357–359. CrossRefGoogle Scholar
  16. Lin XM, Yang MJ, Li H, Wang C, Peng XX (2014) Decreased expression of LamB and Odp1 complex is crucial for antibiotic resistance in Escherichia coli. J Proteome 98:244–253. CrossRefGoogle Scholar
  17. Lochowska A, Iwanicka-Nowicka R, Zaim J, Witkowska-Zimny M, Bolewska K, Hryniewicz MM (2004) Identification of activating region (AR) of Escherichia coli LysR-type transcription factor CysB and CysB contact site on RNA polymerase alpha subunit at the cysP promoter. Mol Microbiol 53(3):791–806. CrossRefGoogle Scholar
  18. Shilton BH (2008) The dynamics of the MBP-MalFGK(2) interaction: a prototype for binding protein dependent ABC-transporter systems. Biochim Biophys Acta 1778(9):1772–1780. CrossRefGoogle Scholar
  19. Sirko A, Zatyka M, Sadowy E, Hulanicka D (1995) Sulfate and thiosulfate transport in Escherichia-Coli K-12 - evidence for a functional overlapping of sulfate-binding and thiosulfate-binding proteins. J Bacteriol 177(14):4134–4136. CrossRefGoogle Scholar
  20. Thomas JG, Baneyx F (1996) Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing heat-shock proteins. J Biol Chem 271(19):11141–11147CrossRefGoogle Scholar
  21. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25(9):1105–1111. CrossRefGoogle Scholar
  22. von Kamp A, Klamt S (2017) Growth-coupled overproduction is feasible for almost all metabolites in five major production organisms. Nat Commun 8:15956. CrossRefGoogle Scholar
  23. Woodruff LB, Pandhal J, Ow SY, Karimpour-Fard A, Weiss SJ, Wright PC, Gill RT (2013) Genome-scale identification and characterization of ethanol tolerance genes in Escherichia coli. Metab Eng 15:124–133. CrossRefGoogle Scholar
  24. Yang J, Zong Y, Su J, Li H, Zhu H, Columbus L, Zhou L, Liu Q (2017) Conformation transitions of the polypeptide-binding pocket support an active substrate release from Hsp70s. Nat Commun 8(1):1201. CrossRefGoogle Scholar
  25. Yu Z, Zheng H, Zhao X, Li S, Xu J, Song H (2016) High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching. Bioresour Technol 214:303–310. CrossRefGoogle Scholar
  26. Zhao D, Yuan S, Xiong B, Sun H, Ye L, Li J, Zhang X, Bi C (2016) Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Factories 15(1):205. CrossRefGoogle Scholar
  27. Zou C, Duan X, Wu J (2014) Enhanced extracellular production of recombinant Bacillus deramificans pullulanase in Escherichia coli through induction mode optimization and a glycine feeding strategy. Bioresour Technol 172:174–179. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinPeople’s Republic of China
  2. 2.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of BiotechnologyTianjin University of Science and TechnologyTianjinPeople’s Republic of China
  3. 3.Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinPeople’s Republic of China

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