Applied Biochemistry and Biotechnology

, Volume 177, Issue 1, pp 226–236 | Cite as

Optimized Transformation of Newly Constructed Escherichia coli-Clostridia Shuttle Vectors into Clostridium beijerinckii

  • Young Hoon Oh
  • Gyeong Tae Eom
  • Kyoung Hee Kang
  • Jae Woo Choi
  • Bong Keun Song
  • Seung Hwan LeeEmail author
  • Si Jae ParkEmail author


Three Escherichia coli-Clostridia shuttle vectors, pKBA411-MCS, pKBE411-MCS, and pKBM411-MCS, which contain p15A, ColE1, and pMB1 origins for replication in E. coli, respectively, along with the pAMB origin for replication in C. beijerinckii, were constructed and examined for their transformation efficiencies into Clostridium beijerinckii NCIMB8052. The transformation condition of pKBM411-MCS, which was optimized by varying resistance, buffer composition, and DNA concentration, was further employed for the transformation of the other plasmids, pKBA411-MCS and pKBE411-MCS into C. beijerinckii. It was found out that transformation efficiency is highly dependent on the origin of replication. The highest transformation efficiency of 7.44 × 103 colony-forming units per microgram of DNA was obtained at 5.0 kV cm−1 field strength, 200 Ω resistance, 270 mM sucrose concentration, 150 ng μg−1, and 3.0 μg DNA using pKBM411-MCS having pMB1 and pAMB origins of replication. The application of the newly constructed vector system was also investigated by introducing the putative alcohol dehydrogenase gene of C. beijerinckii.


Clostridium beijerinckii E. coli-Clostridia shuttle vector Electroporation 



This work was supported by the R & D Program of MOTIE/KEIT (10049674) and the Technology Development Program to Solve Climate Changes (Systems Metabolic Engineering for Biorefineries) (NRF-2012-C1AAA001-2012M1A2A2026556) from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea. Further support from the Energy Efficiency & Resources Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) granted financial resource from the Ministry of Trade, Industry & Energy (MOTIE), Republic of Korea (No. 20132020000170) is appreciated.


  1. 1.
    Dürre, P. (1998). New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation. Applied Microbiology and Biotechnology, 49, 639–648.CrossRefGoogle Scholar
  2. 2.
    Ezeji, T. C., Qureshi, N., & Blaschek, H. P. (2007). Bioproduction of butanol from biomass: from genes to bioreactors. Current Opinion in Biotechnology, 18, 220–227.CrossRefGoogle Scholar
  3. 3.
    Green, E. M. (2011). Fermentative production of butanol—the industrial perspective. Current Opinion in Biotechnology, 22, 1–7.CrossRefGoogle Scholar
  4. 4.
    Kharkwal, S., Karimi, I. A., Chang, M. W., & Lee, D. Y. (2009). Strain improvement and process development for biobutanol production. Recent Patents on Biotechnology, 3, 202–210.CrossRefGoogle Scholar
  5. 5.
    Lee, S. Y., Park, J. H., Jang, S. H., Nielsen, L. K., Kim, J., & Jung, K. S. (2008). Fermentative butanol production by Clostridia. Biotechnology and Bioengineering, 101, 209–228.CrossRefGoogle Scholar
  6. 6.
    Blaschek, H., Annous, B., Formanek, J., & Chen, C. K. (2002). Method of producing butanol using a mutant strain of Clostridium beijerinckii. US patent 6,358,717.Google Scholar
  7. 7.
    Formanek, J., Mackie, R., & Blaschek, H. P. (1997). Enhanced butanol production by Clostridium beijerinckii BA101 grown in semidefined P2 medium containing 6 percent maltodextrin or glucose. Applied and Environmental Microbiology, 63, 2306–2310.Google Scholar
  8. 8.
    Qureshi, N., & Blaschek, H. P. (1999). Production of acetone butanol ethanol (ABE) by a hyper-producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation. Biotechnology Progress, 15, 594–602.CrossRefGoogle Scholar
  9. 9.
    Ezeji, T. C., Qureshi, N., & Blaschek, H. P. (2007). Butanol production from agricultural residues: impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnology and Bioengineering, 97, 1460–1469.CrossRefGoogle Scholar
  10. 10.
    Keis, S., Shaheen, R., & Jones, D. T. (2001). Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov. International Journal of Systematic and Evolutionary Microbiology, 51, 2095–2103.CrossRefGoogle Scholar
  11. 11.
    Qureshi, N., Ezeji, T. C., Ebener, J., Dien, B. S., Cotta, M. A., & Blaschek, H. P. (2008). Butanol production by Clostridium beijerinckii. Part I: use of acid and enzyme hydrolyzed corn fiber. Bioresource Technology, 99, 5915–5922.CrossRefGoogle Scholar
  12. 12.
    Reid, S. J., Rafudeen, M. S., & Leat, N. G. (1999). The genes controlling sucrose utilization in CIostridiunr beuerinckii NCIMB 8052 constitute an operon. Microbiology, 145, 1461–1472.CrossRefGoogle Scholar
  13. 13.
    Shi, Y., Li, Y. X., & Li, Y. Y. (2010). Large number of phosphotransferase genes in the Clostridium beijerinckii NCIMB 8052 genome and the study on their evolution. BMC Bioinformatics, 11(Suppl 11), S9.CrossRefGoogle Scholar
  14. 14.
    Wilkinson, S. R., & Young, M. (1995). Physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome. Journal of Bacteriology, 177, 439–448.Google Scholar
  15. 15.
    Cornillot, E., Nair, R. V., Papoutsakis, E. T., & Soucaille, P. (1997). The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain. Journal of Bacteriology, 179, 5442–5447.Google Scholar
  16. 16.
    Lee, S. Y., Bennett, G. N., & Papoutsakis, E. T. (1992). Construction of Escherichia coli-Clostridium acetobutylicum shuttle vectors and transformation of Clostridium acetobutylicum strains. Biotechnology Letters, 14, 427–432.CrossRefGoogle Scholar
  17. 17.
    Lόpez-Contreras, A. M., Smidt, H., van der Oost, J., Claassen, P. A., Mooibroek, H., & de Vos, W. M. (2001). Clostridium beijerinckii cells expressing Neocallimastix patriciarum glycoside hydrolases show enhanced lichenan utilization and solvent production. Applied and Environmental Microbiology, 67, 5127–5133.CrossRefGoogle Scholar
  18. 18.
    Oultram, J. D., & Young, M. (1985). Conjugal transfer of plasmid pAMβ1 from Streptococcus lactis and Bacillus subtilis to Clostridium acetobutylicum. FEMS Microbiology Letters, 27, 129–134.Google Scholar
  19. 19.
    Oultram, J. D., Loughlin, M., Swinfield, T. J., Brehm, J. K., Thompson, D. E., & Minton, N. P. (1988). Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation. FEMS Microbiology Letters, 56, 83–88.CrossRefGoogle Scholar
  20. 20.
    Quixley, K. W. M., & Reid, S. J. (2000). Construction of a reporter gene vector for Clostridium beijerinckii using a Clostridium endoglucanase gene. Journal of Molecular Microbiology and Biotechnology, 2(1), 53–57.Google Scholar
  21. 21.
    Heap, J. T., Pennington, O. J., Cartman, S. T., Carter, G. P., & Minton, N. P. (2007). The ClosTron: a universal gene knock-out system for the genus Clostridium. Jonrnal of Microbiology Methods, 70, 452–464.CrossRefGoogle Scholar
  22. 22.
    Park, S. J., Lee, T. W., Lim, S. C., Kim, T. W., Lee, H., Kim, M. K., Lee, S. H., Song, B. K., & Lee, S. Y. (2012). Biosynthesis of polyhydroxyalkanoates containing 2-hydroxybutyrate from unrelated carbon source by metabolically engineered Escherichia coli. Applied Microbiology and Biotechnology, 93(1), 273–283.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Young Hoon Oh
    • 1
  • Gyeong Tae Eom
    • 1
  • Kyoung Hee Kang
    • 1
  • Jae Woo Choi
    • 1
  • Bong Keun Song
    • 1
  • Seung Hwan Lee
    • 2
    Email author
  • Si Jae Park
    • 3
    Email author
  1. 1.Center for Bio-based Chemistry, Green Chemistry & Engineering DivisionKorea Research Institute of Chemical TechnologyDaejeonRepublic of Korea
  2. 2.Department of Biotechnology and Bioengineering, College of EngineeringChonnam National UniversityGwangjuRepublic of Korea
  3. 3.Department of Environmental Engineering and EnergyMyongji UniversityYonginRepublic of Korea

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