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Methods for Recombinant Rhamnolipid Production

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Hydrocarbon and Lipid Microbiology Protocols

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

Rhamnolipids are glycolipidic microbial biosurfactants with potential, e.g., as emulsifier or foaming agents in industrial applications. Currently rhamnolipids are produced with pathogenic wild-type strains such as Pseudomonas aeruginosa during growth on hydrophobic substrates (e.g., plant oils). The combination of complex regulation of secondary metabolites in wild-type strains like the quorum sensing system in P. aeruginosa, costly substrates, and low production rates is hindering the market success of rhamnolipids. Challenges include sophisticated fermentation, time-consuming production, and laborious and expensive downstream processing. Recombinant rhamnolipid production is capable of overcoming all these downsides.

Here we present a protocol for the heterologous production of rhamnolipids. This method offers advantages such as (1) easy expression regulation by IPTG induction, (2) glucose as carbon source, and (3) a nonpathogenic host. The most prominent genes responsible for rhamnolipid synthesis, emanating from Pseudomonas or Burkholderia species, are already identified. We describe a method for cloning and expressing the genes rhlA and rhlB from P. aeruginosa in vector pVLT33 allowing for the production of mono-rhamnolipids. Furthermore, we present cultivation methods for the constructed strain, fermentation procedures for upscaled production of the product of interest, qualitative as well as quantitative analytical methods, and finally purification protocols.

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Acknowledgments

The authors express their gratitude to Frank Rosenau and Andreas Wittgens at IMET, HHU Düsseldorf, for carrying out the molecular biology work described. We also thank Rudolf Hausmann and Michaela Zwick (Karlsruhe Institute of Technology) for providing reference material for analytics. Additional thanks go to Wing-Jin Li for providing the picture of the CTAB plate. The “Deutsche Bundesstiftung Umwelt” (DBU) and the Cluster of Excellence “Tailor-Made Fuels from Biomass,” which is funded by the Excellence Initiative of the German federal and state governments to promote science and research at German universities, are gratefully acknowledged for providing financial support.

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Authors and Affiliations

  1. RWTH Aachen University, iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, D-52074, Aachen, Germany

    Till Tiso, Andrea Germer & Lars M. Blank

  2. Department of Bio and Chemical Engineering, Bioprocess Engineering, TU Dortmund, D-44227, Dortmund, Germany

    Benjamin Küpper & Rolf Wichmann

Authors
  1. Till Tiso
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  2. Andrea Germer
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  3. Benjamin Küpper
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  4. Rolf Wichmann
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  5. Lars M. Blank
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Corresponding author

Correspondence to Lars M. Blank .

Editor information

Editors and Affiliations

  1. Dept. Biological Sciences, University of Essex, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Braunschweig, Germany

    Kenneth N. Timmis

  3. Dept. Biologia, Univ de les Illes Balears, Palma de Mallorca, Spain

    Balbina Nogales Fernández

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Tiso, T., Germer, A., Küpper, B., Wichmann, R., Blank, L.M. (2015). Methods for Recombinant Rhamnolipid Production. In: McGenity, T., Timmis, K., Nogales Fernández, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_60

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  • DOI: https://doi.org/10.1007/8623_2015_60

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-49126-3

  • Online ISBN: 978-3-662-49127-0

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