Cell surface display of a β-glucosidase employing the type V secretion system on ethanologenic Escherichia coli for the fermentation of cellobiose to ethanol

  • Iván Muñoz-Gutiérrez
  • Ricardo Oropeza
  • Guillermo Gosset
  • Alfredo Martinez


We used the autodisplay system AIDA-I, which belongs to the type V secretion system (TVSS), to display the β-glucosidase BglC from Thermobifida fusca on the outer membrane of the ethanologenic Escherichia coli strain MS04 (MG1655 ∆pflB, ∆adhE, ∆frdA, ∆xylFGH, ∆ldhA, PpflB::pdc Zm -adhB Zm ). MS04 that was transformed with the plasmid pAIDABglCRHis showed cellobiase activity (171 U/gCDW) and fermented 40 g/l cellobiose in mineral medium in 60 h with an ethanol yield of 81 % of the theoretical maximum. Whole-cell protease treatment, SDS-PAGE, and Western-blot analysis demonstrated that BglC was attached to the external surface of the outer membrane of MS04. When attached to the cells, BglC showed 93.3 % relative activity in the presence of 40 g/l ethanol and retained 100 % of its activity following 2 days of incubation at 37 °C with the same ethanol concentration. This study shows the potential of the TVSS (AIDA-I) and BglC as tools for the production of lignocellulosic bio-commodities.


Cellobiose Bioethanol Escherichia coli Type V secretion system AIDA-I Thermobifida fusca β-glucosidase 



We thank Dr. Thomas F. Meyer from Max Planck Institute (Infection Biology) for providing pJM7 plasmid, Dr. David B. Wilson (Department of Molecular Biology and Genetics, Cornell University) for providing pNS6 plasmid, and Luz María Martínez, Mercedes Enzaldo, Georgina Hernández, Omar Arriaga and Shirley Ainsworth for technical support. This work was supported by the Mexican Council of Science and Technology (CONACyT) technological innovation grants: PETRAMIN 2010-13879, 2011-154298, and 2012-184417; and from the Universidad Nacional Autónoma de México: grant DGAPA/PAPIIT/UNAM IT200312-2.


  1. 1.
    Amann E, Ochs B, Abel KJ (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69(2):301–315PubMedCrossRefGoogle Scholar
  2. 2.
    Amorim HV, Lopes ML, de Castro Oliveira JV, Buckeridge MS, Goldman GH (2011) Scientific challenges of bioethanol production in Brazil. Appl Microbiol Biotechnol 91(5):1267–1275PubMedCrossRefGoogle Scholar
  3. 3.
    Andrić P, Meyer AS, Jensen PA, Dam-Johansen K (2010) Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes. Biotechnol Adv 28(3):308–324PubMedCrossRefGoogle Scholar
  4. 4.
    Beall DS, Ohta K, Ingram LO (1991) Parametric studies of ethanol production form xylose and other sugars by recombinant Escherichia coli. Biotechnol Bioeng 38(3):296–303PubMedCrossRefGoogle Scholar
  5. 5.
    Dautin N, Bernstein HD (2007) Protein secretion in gram-negative bacteria via the autotransporter pathway. Annu Rev Microbiol 61:89–112PubMedCrossRefGoogle Scholar
  6. 6.
    Dien BS, Nichols NN, O’Bryan PJ, Bothast RJ (2000) Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass. Appl Biochem Biotechnol 84–86(1–9):181–196PubMedCrossRefGoogle Scholar
  7. 7.
    Edwards MC, Henriksen ED, Yomano LP, Gardner BC, Sharma LN, Ingram LO, Peterson JD (2011) Addition of genes for cellobiase and pectinolytic activity in Escherichia coli for fuel ethanol production from pectin-rich lignocellulosic biomass. Appl Environ Microbiol 77(15):5184–5191PubMedCrossRefGoogle Scholar
  8. 8.
    Ferchak JD, Pye EK (1983) Effect of glucose and other sugars on the β-1,4-glucosidase activity of Thermomonospora fusca. Biotechnol Bioeng 25(12):2855–2864PubMedCrossRefGoogle Scholar
  9. 9.
    Fernandez-Sandoval MT, Gosset G, Martinez A (2010) Ethanol production by ethanologenic Escherichia coli using xylose-glucose-acetate mixtures in batch and continuous cultures. 32nd symposium on biotechnology for fuels and chemicals. Society for Industrial Microbiology. Clearwater Beach, Florida, USAGoogle Scholar
  10. 10.
    Geddes CC, Nieves IU, Ingram LO (2011) Advances in ethanol production. Curr Opin Biotechnol 22(3):312–319PubMedCrossRefGoogle Scholar
  11. 11.
    Hahn-Hägerdal B, Galbe M, Gorwa-Grauslund MF, Lidén G, Zacchi G (2006) Bio-ethanol—the fuel of tomorrow from the residues of today. Trends Biotechnol 24(12):549–556PubMedCrossRefGoogle Scholar
  12. 12.
    Huerta-Beristain G, Utrilla J, Hernández-Chávez G, Bolívar F, Gosset G, Martinez A (2008) Specific ethanol production rate in ethanologenic Escherichia coli strain KO11 is limited by pyruvate decarboxylase. J Mol Microbiol Biotechnol 15(1):55–64PubMedCrossRefGoogle Scholar
  13. 13.
    Ingram LO, Aldrich HC, Borges ACC, Causey TB, Martinez A, Morales F, Saleh A, Underwood SA, Yomano LP, York SW, Zaldivar J, Zhou S (1999) Enteric bacterial catalysts for fuel ethanol production. Biotechnol Prog 15(5):855–866PubMedCrossRefGoogle Scholar
  14. 14.
    Jose J, Meyer TF (2007) The autodisplay story, from discovery to biotechnical and biomedical applications. Microbiol Mol Biol Rev 71(4):600–619PubMedCrossRefGoogle Scholar
  15. 15.
    Jose J, Park M, Pyun JC (2010) Escherichia coli outer membrane with autodisplayed Z-domain as a molecular recognition layer of SPR biosensor. Biosens Bioelectron 25(5):1225–1228PubMedCrossRefGoogle Scholar
  16. 16.
    Jose J, Zangen D (2005) Autodisplay of the protease inhibitor aprotinin in Escherichia coli. Biochem Biophys Res Commun 333(4):1218–1226PubMedCrossRefGoogle Scholar
  17. 17.
    Kaessler A, Olgen S, Jose J (2011) Autodisplay of catalytically active human hyaluronidase hPH-20 and testing of enzyme inhibitors. Eur J Pharm Sc 42(1–2):138–147CrossRefGoogle Scholar
  18. 18.
    la Grange DC, den Haan R, van Zyl WH (2010) Engineering cellulolytic ability into bioprocessing organisms. App Microbiol Biotechnol 87(4):1195–1208CrossRefGoogle Scholar
  19. 19.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685PubMedCrossRefGoogle Scholar
  20. 20.
    Lattemann CT, Maurer J, Gerland E, Meyer TF (2000) Autodisplay: functional display of active β-lactamase on the surface of Escherichia coli by the AIDA-I autotransporter. J Bacteriol 182(13):3726–3733PubMedCrossRefGoogle Scholar
  21. 21.
    Martinez A, York SW, Yomano LP, Pineda LP, Davis FC, Shelton JC, Ingram LO (1999) Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli. Biotechnol Prog 15(5):891–897PubMedCrossRefGoogle Scholar
  22. 22.
    Martinez A, Grabar TB, Shanmugam KT, Yomano LP, York SW, Ingram LO (2007) Low salt medium for lactate and ethanol production by recombinant Escherichia coli B. Biotechnol Lett 29(3):397–404PubMedCrossRefGoogle Scholar
  23. 23.
    Martínez Jiménez A, Gosset Lagarda G, Hernández Chávez G, Huerta Beristain G, Trujillo Martínez B, Utrilla Carreri J (2010) Strains of Escherichia coli modified by metabolic engineering to produce chemical compounds from hydrolyzed lignocellulose, pentoses, hexoses and other carbon sources. WO Patent WO2011/016706A2Google Scholar
  24. 24.
    Maurer J, Jose J, Meyer TF (1997) Autodisplay: one-component system for efficient surface display and release of soluble recombinant proteins from Escherichia coli. J Bacteriol 179(3):794–804PubMedGoogle Scholar
  25. 25.
    Maurer J, Jose J, Meyer TF (1999) Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions. J Bacteriol 181(22):7014–7020PubMedGoogle Scholar
  26. 26.
    McBride JE, Zietsman JJ, Van Zyl WH, Lynd LR (2005) Utilization of cellobiose by recombinant β-glucosidase-expressing strains of Saccharomyces cerevisiae: characterization and evaluation of the sufficiency of expression. Enzyme Microb Technol 37(1):93–101CrossRefGoogle Scholar
  27. 27.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428CrossRefGoogle Scholar
  28. 28.
    Moniruzzaman M, Lai X, York SW, Ingram LO (1997) Isolation and molecular characterization of high-performance cellobiose-fermenting spontaneous mutants of ethanologenic Escherichia coli KO11 containing the Klebsiella oxytoca casAB operon. Appl Environ Microbiol 63(12):4633–4637PubMedGoogle Scholar
  29. 29.
    Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 96(6):673–686PubMedCrossRefGoogle Scholar
  30. 30.
    Orencio-Trejo M, Utrilla J, Fernández-Sandoval MT, Huerta-Beristain G, Gosset G, Martinez A (2010) Engineering the Escherichia coli fermentative metabolism. Adv Biochem Eng Biotechnol 121:71–107PubMedGoogle Scholar
  31. 31.
    Orencio-Trejo M, Flores N, Escalante A, Hernández-Chávez G, Bolívar F, Gosset G, Martinez A (2008) Metabolic regulation analysis of an ethanologenic Escherichia coli strain based on RT-PCR and enzymatic activities. Biotechnol Biofuels 1(1):8PubMedCrossRefGoogle Scholar
  32. 32.
    Puente JL, Juárez D, Bobadilla M, Arias CF, Calva E (1995) The Salmonella ompC gene: structure and use as a carrier for heterologous sequences. Gene 156(1):1–9PubMedCrossRefGoogle Scholar
  33. 33.
    Ryu S, Karim MN (2011) A whole cell biocatalyst for cellulosic ethanol production from dilute acid-pretreated corn stover hydrolyzates. App Microbiol Biotechnol 91(3):529–542CrossRefGoogle Scholar
  34. 34.
    Sambrook J, Rusell D (2001) Molecular cloning a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  35. 35.
    Somerville C (2007) Biofuels. Curr Biol 17(4):R115–R119PubMedCrossRefGoogle Scholar
  36. 36.
    Spiridonov NA, Wilson DB (2001) Cloning and biochemical characterization of BglC, a β-glucosidase from the cellulolytic actinomycete Thermobifida fusca. Curr Microbiol 42(4):295–301PubMedGoogle Scholar
  37. 37.
    Tanaka T, Kawabata H, Ogino C, Kondo A (2011) Creation of a cellooligosaccharide-assimilating Escherichia coli strain by displaying active beta-glucosidase on the cell surface via a novel anchor protein. Appl Environ Microbiol 77(17):6265–6270PubMedCrossRefGoogle Scholar
  38. 38.
    Vinuselvi P, Lee SK (2011) Engineering Escherichia coli for efficient cellobiose utilization. Appl Microbiol Biotechnol 92(1):125–132PubMedCrossRefGoogle Scholar
  39. 39.
    Yomano LP, York SW, Zhou S, Shanmugam KT, Ingram LO (2008) Re-engineering Escherichia coli for ethanol production. Biotechnol Lett 30(12):2097–2103PubMedCrossRefGoogle Scholar
  40. 40.
    Zhou S, Yomano LP, Saleh AZ, Davis FC, Aldrich HC, Ingram LO (1999) Enhancement of expression and apparent secretion of Erwinia chrysanthemi endoglucanase (encoded by celZ) in Escherichia coli B. Appl Environ Microbiol 65(6):2439–2445PubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2012

Authors and Affiliations

  • Iván Muñoz-Gutiérrez
    • 1
  • Ricardo Oropeza
    • 2
  • Guillermo Gosset
    • 1
  • Alfredo Martinez
    • 1
  1. 1.Departamento de Ingeniería Celular y Biocatálisis, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  2. 2.Departamento de Microbiología Molecular, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico

Personalised recommendations