Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production Using Pseudomonas putida

Frontiers in Bioengineering and Biotechnology, vol. 8, pp. 976

Abstract

Rhamnolipids are biosurfactants produced by microorganisms with the potential to replace synthetic compounds with petrochemical origin. To promote industrial use of rhamnolipids, recombinant rhamnolipid production from sugars needs to be intensified. Since this remains challenging, the aim of the presented research is to utilize a multidisciplinary approach to take a step toward developing a sustainable rhamnolipid production process. Here, we developed expression cassettes for stable integration of the rhamnolipid biosynthesis genes into the genome outperformed plasmid-based expression systems. Furthermore, the genetic stability of the production strain was improved by using an inducible promoter. To enhance rhamnolipid synthesis, energy- and/or carbon-consuming traits were removed: mutants negative for the synthesis of the flagellar machinery or the storage polymer PHA showed increased production by 50%. Variation of time of induction resulted in an 18% increase in titers. A scale-up from shake flasks was carried out using a 1-L bioreactor. By recycling of the foam, biomass loss could be minimized and a rhamnolipid titer of up to 1.5 g/L was achieved without using mechanical foam destroyers or antifoaming agents. Subsequent liquid-liquid extraction was optimized by using a suitable minimal medium during fermentation to reduce undesired interphase formation. A technical-scale production process was designed and evaluated by a life-cycle assessment (LCA). Different process chains and their specific environmental impact were examined. It was found that next to biomass supply, the fermentation had the biggest environmental impact. The present work underlines the need for multidisciplinary approaches to address the challenges associated with achieving sustainable production of microbial secondary metabolites. The results are discussed in the context of the challenges of microbial biosurfactant production using hydrophilic substrates on an industrial scale.

Authors 16

1. Till Tiso corresponding Aachen iAMB-Institute of Applied Microbiology

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

   iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany

   iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany

2. Nina Ihling Aachen Chair of Biochemical Engineering (AVT.BioVT)

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

   Chair of Biochemical Engineering (AVT.BioVT), RWTH Aachen University, Aachen, Germany

3. Sonja Kubicki

   Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

   Institute of Molecular Enzyme Technology, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

4. Andreas Biselli Aachen Fluid Process Engineering (AVT.FVT)

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

   Fluid Process Engineering (AVT.FVT), RWTH Aachen University, Aachen, Germany

5. Andreas Schonhoff

   Forschungszentrum Jülich

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

   Institute of Energy and Climate Research - Systems Analysis and Technology Evaluation (IEK-STE), Forschungszentrum Jülich GmbH, Jülich, Germany

   Institute of Energy and Climate Research – Systems Analysis and Technology Evaluation (IEK-STE), Forschungszentrum Jülich GmbH, Jülich, Germany

6. Isabel Bator Aachen iAMB-Institute of Applied Microbiology

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

   iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany

   iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany

7. Stephan Thies

   Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

   Institute of Molecular Enzyme Technology, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

8. Tobias Karmainski Aachen iAMB-Institute of Applied Microbiology

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

   iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany

   iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany

9. Sebastian Kruth Aachen iAMB-Institute of Applied Microbiology

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

   iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany

   iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany

10. Anna-Lena Willenbrink Aachen Fluid Process Engineering (AVT.FVT)

    Forschungszentrum Jülich · RWTH Aachen University

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

    Fluid Process Engineering (AVT.FVT), RWTH Aachen University, Aachen, Germany

11. Anita Loeschcke

    Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

    Institute of Molecular Enzyme Technology, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

12. Petra Zapp

    Forschungszentrum Jülich

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

    Institute of Energy and Climate Research - Systems Analysis and Technology Evaluation (IEK-STE), Forschungszentrum Jülich GmbH, Jülich, Germany

    Institute of Energy and Climate Research – Systems Analysis and Technology Evaluation (IEK-STE), Forschungszentrum Jülich GmbH, Jülich, Germany

13. Andreas Jupke Aachen Fluid Process Engineering (AVT.FVT)

    Forschungszentrum Jülich · RWTH Aachen University

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

    Fluid Process Engineering (AVT.FVT), RWTH Aachen University, Aachen, Germany

14. Karl‐Erich Jaeger

    Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

    Institute of Bio- and Geosciences IBG 1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany

    Institute of Molecular Enzyme Technology, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany

15. Jochen Büchs Aachen Chair of Biochemical Engineering (AVT.BioVT)

    Forschungszentrum Jülich · RWTH Aachen University

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

    Chair of Biochemical Engineering (AVT.BioVT), RWTH Aachen University, Aachen, Germany

16. Lars M. Blank corresponding Aachen iAMB-Institute of Applied Microbiology

    Forschungszentrum Jülich · RWTH Aachen University

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, Jülich, Germany

    iAMB - Institute of Applied Microbiology, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany

    iAMB – Institute of Applied Microbiology, ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany