Skip to main content
SpringerLink
Log in

Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichia coli

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Enhancement of the cellular exportation of heterologous compounds is an important aspect to improve the product yield in microbial cell factory. Efflux pumps can expel various intra- or extra-cellular substances out of microbial hosts and increase the cellular tolerance. Thus in this study, by using the hydrophobic sesquiterpene (amorphadiene) and diterpene (kaurene) as two model compounds, we attempted to improve isoprenoid production through systematically engineering the efflux pumps in Escherichia coli BL21(DE3). The pleiotropic resistant pumps, AcrAB-TolC, MdtEF-TolC from E. coli and heterologous MexAB-OprM pump from Pseudomonas aeruginosa, were overexpressed, assembled, and finely modulated. We found that overexpression of AcrB and TolC components can effectively enhance the specific yield of amorphadiene and kaurene, e.g., 31 and 37 % improvement for amorphadiene compared with control, respectively. The heterologous MexB component can enhance kaurene production with 70 % improvement which is more effective than TolC and AcrB. The results suggest that the three components of tripartite efflux pumps play varied effect to enhance isoprenoid production. Considering the highly organized structure of efflux pumps and importance of components interaction, various component combinations were constructed and the copy number of key components AcrB and TolC was finely modulated as well. The results exhibit that the combination TolC and TolC and AcrB improved the specific yield of amorphadiene with 118 %, and AcrA and TolC and AcrB improved that of kaurene with 104 %. This study indicates that assembling and finely modulating efflux pumps is an effective strategy to improve the production of heterologous compounds in E. coli.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Reference

  • Ajikumar PK, Xiao WH, Tyo KE, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G (2010) Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science 330(6000):70–74

    Article  PubMed  CAS  Google Scholar 

  • Aono R (1998) Improvement of organic solvent tolerance level of Escherichia coli by overexpression of stress-responsive genes. Extremophiles 2(3):239–248

    Article  PubMed  CAS  Google Scholar 

  • Asako H, Nakajima H, Kobayashi K, Kobayashi M, Aono R (1997) Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli. Appl Environ Microb 63(4):1428–1433

    CAS  Google Scholar 

  • Chang MCY, Eachus RA, Trieu W, Ro DK, Keasling JD (2007) Engineering Escherichia coli for production of functionalized terpenoids using plant P450s. Nat Chem Biol 3(5):274–277

    Article  PubMed  CAS  Google Scholar 

  • Deininger KNW, Horikawa A, Kitko RD, Tatsumi R, Rosner JL, Wachi M, Slonczewski JL (2011) A requirement of tolC and MDR efflux pumps for acid adaptation and GadAB induction in Escherichia coli. PLoS One 6(4):e18960

    Article  PubMed  CAS  Google Scholar 

  • Dunlop M (2011) Engineering microbes for tolerance to next-generation biofuels. Biotechnol Biofuels 4(1):32

    Article  PubMed  CAS  Google Scholar 

  • Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC, Lee TS, Keasling JD, Hadi MZ, Mukhopadhyay A (2011) Engineering microbial biofuel tolerance and export using efflux pumps. Mol Syst Biol 7

  • Hasdemir U (2007) The role of cell wall organization and active efflux pump systems in multidrug resistance of bacteria. Mikrobiyol Bul 41(2):309–327

    PubMed  CAS  Google Scholar 

  • Keeney D, Ruzin A, McAleese F, Murphy E, Bradford PA (2008) MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli. J Antimicrob Chemother 61(1):46–53

    Article  PubMed  CAS  Google Scholar 

  • Li XZ, Nikaido H (2009) Efflux-mediated drug resistance in bacteria: an update. Drugs 69(12):1555–1623

    Article  PubMed  CAS  Google Scholar 

  • Nishino K, Senda Y, Hayashi-Nishino M, Yamaguchi A (2009) Role of the AraC-XylS family regulator YdeO in multi-drug resistance of Escherichia coli. J Antibiot 62(5):251–257

    Article  PubMed  CAS  Google Scholar 

  • Ostash I, Rebets Y, Ostash B, Kobylyanskyy A, Myronovskyy M, Nakamura T, Walker S, Fedorenko V (2008) An ABC transporter encoding gene lndW confers resistance to landomycin E. Arch Microbiol 190(1):105–109

    Article  PubMed  CAS  Google Scholar 

  • Papadopoulos CJ, Carson CF, Chang BJ, Riley TV (2008) Role of the MexAB-OprM efflux pump of Pseudomonas aeruginosa in tolerance to tea tree (Melaleuca alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and alpha-terpineol. Appl Environ Microb 74(6):1932–1935

    Article  CAS  Google Scholar 

  • Peralta-Yahya PP, Zhang F, Del Cardayre SB, Keasling JD (2012) Microbial engineering for the production of advanced biofuels. Nature 488(7411):320–328

    Article  PubMed  CAS  Google Scholar 

  • Poole K (2005) Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 56(1):20–51

    Article  PubMed  CAS  Google Scholar 

  • Qiu J, Zhuo Y, Zhu D, Zhou X, Zhang L, Bai L, Deng Z (2011) Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis. Appl Microbiol Biotechnol 92(2):337–345

    Article  PubMed  CAS  Google Scholar 

  • Ro D-K, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MCY, Withers ST, Shiba Y, Sarpong R, Keasling JD (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440(7086):940–943

    Article  PubMed  CAS  Google Scholar 

  • Sambrook JDWR (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Shah AA, Wang C, Chung YR, Kim JY, Choi ES, Kim SW (2013) Enhancement of geraniol resistance of Escherichia coli by MarA overexpression. J Biosci Bioeng 115(3):253–258

    Article  PubMed  CAS  Google Scholar 

  • Sharff A, Fanutti C, Shi J, Calladine C, Luisi B (2001) The role of the TolC family in protein transport and multidrug efflux. Eur J Biochem 268(19):5011–5026

    Article  PubMed  CAS  Google Scholar 

  • Sulavik MC, Houseweart C, Cramer C, Jiwani N, Murgolo N, Greene J, DiDomenico B, Shaw KJ, Miller GH, Hare R, Shimer G (2001) Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes. Antimicrob Agents Chemother 45(4):1126–1136

    Article  PubMed  CAS  Google Scholar 

  • Tenover FC (2006) Mechanisms of antimicrobial resistance in bacteria. Am J Med 119(6, Suppl 1):S3–S10

    Article  PubMed  CAS  Google Scholar 

  • Tikhonova E, Yamada Y, Zgurskaya H (2011) Sequential mechanism of assembly of multidrug efflux pump AcrAB-TolC. Chem Biol 18(4):454–463

    Article  PubMed  CAS  Google Scholar 

  • Tikhonova EB, Dastidar V, Rybenkov VV, Zgurskaya HI (2009) Kinetic control of TolC recruitment by multidrug efflux complexes. Proc Natl Acad Sci U S A 106(38):16416–16421

    Article  PubMed  CAS  Google Scholar 

  • Tikhonova EB, Zgurskaya HI (2004) AcrA, AcrB, and TolC of Escherichia coli form a stable intermembrane multidrug efflux complex. J Biol Chem 279(31):32116–32124

    Article  PubMed  CAS  Google Scholar 

  • Touzé T, Eswaran J, Bokma E, Koronakis E, Hughes C, Koronakis V (2004) Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efflux system. Mol Microbiol 53(2):697–706

    Article  PubMed  Google Scholar 

  • Tsuruta H, Paddon CJ, Eng D, Lenihan JR, Horning T, Anthony LC, Regentin R, Keasling JD, Renninger NS, Newman JD (2009) High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli. PLoS One 4(2):e4489

    Article  PubMed  Google Scholar 

  • Walsh C (2000) Molecular mechanisms that confer antibacterial drug resistance. Nature 406(6797):775–781

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Basic Research Program of China (“973” Program, grant no. 2012CB721104), the National High Technology Research and Development Program (“863” Program, grant no. 2012AA02A701), the National Natural Science Foundation of China (grants no. 31170101 and 31100073), “Technology Innovation Action Plan” Key Project of Shanghai Science and Technology Commission (grant no. 10dz1910100), and the major Projects of Knowledge Innovation Program of Chinese Academy of Sciences (grant no. KSCX2-EW-J-12).

Author information

Authors and Affiliations

  1. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, People’s Republic of China

    Jian-Feng Wang, Zhi-Qiang Xiong, Shi-Yuan Li & Yong Wang

  2. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People’s Republic of China

    Jian-Feng Wang

Authors
  1. Jian-Feng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhi-Qiang Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shi-Yuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yong Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, JF., Xiong, ZQ., Li, SY. et al. Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichia coli . Appl Microbiol Biotechnol 97, 8057–8067 (2013). https://doi.org/10.1007/s00253-013-5062-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-013-5062-z

Keywords

Search

Navigation