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Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440

Andreas Wittgens1, Till Tiso2, Torsten T Arndt3, Pamela Wenk4, Johannes Hemmerich4, Carsten Müller4, Rolf Wichmann5, Benjamin Küpper5, Michaela Zwick6, Susanne Wilhelm1, Rudolf Hausmann6, Christoph Syldatk6, Frank Rosenau17 and Lars M Blank2*

Author Affiliations

1 Institute for Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, D-52426 Jülich, Germany

2 Institute of Applied Microbiology, RWTH Aachen University, D-52074 Aachen, Germany

3 Laboratory of Chemical Biotechnology, TU Dortmund University, D-44227 Dortmund, Germany

4 m2p-labs GmbH, D-52499 Baesweiler, Germany

5 Laboratory of Biochemical Engineering, Department of Biochemical and Chemical Engineering, TU Dortmund University, D-44227 Dortmund, Germany

6 Institute of Process Engineering in Life Sciences, Section II: Technical Biology, Karlsruhe Institute of Technology, D-76131 Karlsruhe, Germany

7 Institute of Pharmaceutical Biotechnology, Ulm University, D-89069 Ulm, Germany

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Microbial Cell Factories 2011, 10:80  doi:10.1186/1475-2859-10-80

Published: 17 October 2011

Abstract

Background

Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.

Results

Here, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C10:C10). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/gglucose corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.

Conclusions

A functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.

Keywords:
flux analysis; quantitative physiology; metabolic network; biodetergent; non-pathogenic Pseudomonas; biosurfactants; rhamnolipids; off-gas analysis; 13C labeling; BlueSens