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

Autor: Weihmann R; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany.; Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany., Kubicki S; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany.; Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany., Bitzenhofer NL; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany., Domröse A; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany., Bator I; 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., Kirschen LM; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany., Kofler F; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany., Funk A; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany., Tiso T; 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., Blank LM; 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., Jaeger KE; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany.; Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.; Institute of Bio-and Geosciences IBG 1: Biotechnology, Forschungszentrum Jülich GmbH, 52428 Jülich, Germany., Drepper T; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany.; Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany., Thies S; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany.; Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany., Loeschcke A; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf at Forschungszentrum Jülich GmbH, 52428 Jülich, Germany.; Bioeconomy Science Center (BioSC), Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Jazyk: angličtina
Zdroj: FEMS microbes [FEMS Microbes] 2022 Dec 19; Vol. 4, pp. xtac030. Date of Electronic Publication: 2022 Dec 19 (Print Publication: 2023).
DOI: 10.1093/femsmc/xtac030
Abstrakt: 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 att Tn7, 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 att Tn7 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 att Tn7 and expression with NagR/P nagAa 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.
Competing Interests: None declared
(© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)
Databáze: MEDLINE
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