Engineering adipic acid metabolism in Pseudomonas putida

Metabolic Engineering, vol. 67, pp. 29–40

Abstract

Bio-upcycling of plastics is an upcoming alternative approach for the valorization of diverse polymer waste streams that are too contaminated for traditional recycling technologies. Adipic acid and other medium-chain-length dicarboxylates are key components of many plastics including polyamides, polyesters, and polyurethanes. This study endows Pseudomonas putida KT2440 with efficient metabolism of these dicarboxylates. The dcaAKIJP genes from Acinetobacter baylyi, encoding initial uptake and activation steps for dicarboxylates, were heterologously expressed. Genomic integration of these dca genes proved to be a key factor in efficient and reliable expression. In spite of this, adaptive laboratory evolution was needed to connect these initial steps to the native metabolism of P. putida, thereby enabling growth on adipate as sole carbon source. Genome sequencing of evolved strains revealed a central role of a paa gene cluster, which encodes parts of the phenylacetate metabolic degradation pathway with parallels to adipate metabolism. Fast growth required the additional disruption of the regulator-encoding psrA, which upregulates redundant β-oxidation genes. This knowledge enabled the rational reverse engineering of a strain that can not only use adipate, but also other medium-chain-length dicarboxylates like suberate and sebacate. The reverse engineered strain grows on adipate with a rate of 0.35 ± 0.01 h−1, reaching a final biomass yield of 0.27 ± 0.00 gCDW gadipate−1. In a nitrogen-limited medium this strain produced polyhydroxyalkanoates from adipate up to 25% of its CDW. This proves its applicability for the upcycling of mixtures of polymers made from fossile resources into biodegradable counterparts.

Authors 12

1. Yannic Sebastian Ackermann

   Forschungszentrum Jülich

   Affiliation as printed

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

2. Wing‐Jin Li Aachen Institute of Applied Microbiology - iAMB

   RWTH Aachen University

   Affiliation as printed

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

3. Leonie Op de Hipt

   Forschungszentrum Jülich

   Affiliation as printed

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

4. Paul-Joachim Niehoff Aachen Institute of Applied Microbiology - iAMB

   RWTH Aachen University

   Affiliation as printed

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

5. William T. Casey

   University College Dublin

   Affiliation as printed

   Bioplastech Ltd., NovaUCD, Belfield Innovation Park, University College Dublin, Dublin, Ireland

6. Tino Polen

   Forschungszentrum Jülich

   Affiliation as printed

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

7. Sebastian Köbbing Aachen Institute of Applied Microbiology - iAMB

   RWTH Aachen University

   Affiliation as printed

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

8. Hendrik Ballerstedt Aachen Institute of Applied Microbiology - iAMB

   RWTH Aachen University

   Affiliation as printed

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

9. Benedikt Wynands

   Forschungszentrum Jülich

   Affiliation as printed

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

10. Kevin Edward O’Connor

    University College Dublin

    Affiliation as printed

    UCD Earth Institute and School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland; BiOrbic Bioeconomy SFI Research Centre, University College Dublin, Dublin, Ireland

    BiOrbic Bioeconomy SFI Research Centre, University College Dublin, Dublin, Ireland

    UCD Earth Institute and School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland

11. Lars M. Blank Aachen Institute of Applied Microbiology - iAMB

    RWTH Aachen University

    Affiliation as printed

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

12. Nick J. P. Wierckx corresponding

    Forschungszentrum Jülich

    Affiliation as printed

    Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich, Jülich, Germany. Electronic address: n.wierckx@fz-juelich.de

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