The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids in P. putida

FEMS Microbes, vol. 4, pp. xtac030

Abstract

Abstract The expression of biosynthetic genes in bacterial hosts can enable access to high-value compounds, for which appropriate molecular genetic tools are essential. Therefore, we developed a toolbox of modular vectors, which facilitate chromosomal gene integration and expression in Pseudomonas putida KT2440. To this end, we designed an integrative sequence, allowing customisation regarding the modes of integration (random, at attTn7, or into the 16S rRNA gene), promoters, antibiotic resistance markers as well as fluorescent proteins and enzymes as transcription reporters. We thus established a toolbox of vectors carrying integrative sequences, designated as pYT series, of which we present 27 ready-to-use variants along with a set of strains equipped with unique ‘landing pads’ for directing a pYT interposon into one specific copy of the 16S rRNA gene. We used genes of the well-described violacein biosynthesis as reporter to showcase random Tn5-based chromosomal integration leading to constitutive expression and production of violacein and deoxyviolacein. Deoxyviolacein was likewise produced after gene integration into the 16S rRNA gene of rrn operons. Integration in the attTn7 site was used to characterise the suitability of different inducible promoters and successive strain development for the metabolically challenging production of mono-rhamnolipids. Finally, to establish arcyriaflavin A production in P. putida for the first time, we compared different integration and expression modes, revealing integration at attTn7 and expression with NagR/PnagAa to be most suitable. In summary, the new toolbox can be utilised for the rapid generation of various types of P. putida expression and production strains.

Authors 14

1. Robin Weihmann

   Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH , 52425 Jülich, Germany

   Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

2. Sonja Kubicki

   Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH , 52425 Jülich, Germany

   Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

3. Nora Lisa Bitzenhofer

   Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

4. Andreas Domröse

   Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

5. Isabel Bator Aachen iAMB-Institute of Applied Microbiology

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH , 52425 Jülich, Germany

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

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

6. Lisa-Marie Kirschen

   Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

7. Franziska Kofler

   Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

8. Aileen Funk

   Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

9. Till Tiso Aachen iAMB-Institute of Applied Microbiology

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH , 52425 Jülich, Germany

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

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

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

    Forschungszentrum Jülich · RWTH Aachen University

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH , 52425 Jülich, Germany

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

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

11. Karl‐Erich Jaeger

    Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH , 52425 Jülich, Germany

    Institute of Bio-and Geosciences IBG 1: Biotechnology, Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

    Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

12. Thomas Drepper

    Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH , 52425 Jülich, Germany

    Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

13. Stephan Thies corresponding

    Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH , 52425 Jülich, Germany

    Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany

14. Anita Loeschcke corresponding

    Forschungszentrum Jülich · Heinrich Heine University Düsseldorf

    Affiliation as printed

    Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH , 52425 Jülich, Germany

    Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH , 52428 Jülich, Germany