Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida
Abstract The T7 phage RNA polymerase (RNAP) is a widely used expression platform, but its implementation in non-model microbial hosts poses significant challenges due to cytotoxicity. We constructed an optimized phage phi15-based expression system as alternative to the T7 platform for a wide range o...
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| Format: | Article |
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Nature Portfolio
2025-02-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-025-07508-y |
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| author | Eveline-Marie Lammens Daniel C. Volke Alison Kerremans Yannick Aerts Maarten Boon Pablo I. Nikel Rob Lavigne |
| author_facet | Eveline-Marie Lammens Daniel C. Volke Alison Kerremans Yannick Aerts Maarten Boon Pablo I. Nikel Rob Lavigne |
| author_sort | Eveline-Marie Lammens |
| collection | DOAJ |
| description | Abstract The T7 phage RNA polymerase (RNAP) is a widely used expression platform, but its implementation in non-model microbial hosts poses significant challenges due to cytotoxicity. We constructed an optimized phage phi15-based expression system as alternative to the T7 platform for a wide range of applications in Pseudomonas putida. The new system employs the small phi15 RNAP, driving expression from an orthogonal phi15 promoter. By finetuning expression levels of phi15rnap and introducing a phi15 lysozyme mutant that inhibits phi15 RNAP in uninduced conditions, a stringent system was created with 200-fold inducibility. Moreover, by successfully decoupling cell growth and protein production using phi15 gp16, a host RNAP inhibitor, expression levels could be enhanced further (20%). Apart from creating four optimized platform P. putida hosts and a set of Golden Gate-compatible vectors, we demonstrate the extensive flexibility of the phi15 system. A proof-of-concept expression for industrially relevant fluorinase resulted in 2.5- and 5-fold increased yield compared to other widely-adopted expression systems. The system functions well in combination with several inducer systems, and in a variety of vector-based and genomically integrated set-ups. In conclusion, the phi15 RNAP, promoter, lysozyme and growth-decoupler provide a valuable plug-and-play set of genetic parts for the P. putida toolbox. |
| format | Article |
| id | doaj-art-e033227be83243c0a4a712b6c6025f26 |
| institution | Kabale University |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Nature Portfolio |
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| series | Communications Biology |
| spelling | doaj-art-e033227be83243c0a4a712b6c6025f262025-02-09T12:50:50ZengNature PortfolioCommunications Biology2399-36422025-02-018111510.1038/s42003-025-07508-yEngineering a phi15-based expression system for stringent gene expression in Pseudomonas putidaEveline-Marie Lammens0Daniel C. Volke1Alison Kerremans2Yannick Aerts3Maarten Boon4Pablo I. Nikel5Rob Lavigne6Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet, Building 220, 2800 KgsDepartment of Biosystems, Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462Department of Biosystems, Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet, Building 220, 2800 KgsDepartment of Biosystems, Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg 21 box 2462Abstract The T7 phage RNA polymerase (RNAP) is a widely used expression platform, but its implementation in non-model microbial hosts poses significant challenges due to cytotoxicity. We constructed an optimized phage phi15-based expression system as alternative to the T7 platform for a wide range of applications in Pseudomonas putida. The new system employs the small phi15 RNAP, driving expression from an orthogonal phi15 promoter. By finetuning expression levels of phi15rnap and introducing a phi15 lysozyme mutant that inhibits phi15 RNAP in uninduced conditions, a stringent system was created with 200-fold inducibility. Moreover, by successfully decoupling cell growth and protein production using phi15 gp16, a host RNAP inhibitor, expression levels could be enhanced further (20%). Apart from creating four optimized platform P. putida hosts and a set of Golden Gate-compatible vectors, we demonstrate the extensive flexibility of the phi15 system. A proof-of-concept expression for industrially relevant fluorinase resulted in 2.5- and 5-fold increased yield compared to other widely-adopted expression systems. The system functions well in combination with several inducer systems, and in a variety of vector-based and genomically integrated set-ups. In conclusion, the phi15 RNAP, promoter, lysozyme and growth-decoupler provide a valuable plug-and-play set of genetic parts for the P. putida toolbox.https://doi.org/10.1038/s42003-025-07508-y |
| spellingShingle | Eveline-Marie Lammens Daniel C. Volke Alison Kerremans Yannick Aerts Maarten Boon Pablo I. Nikel Rob Lavigne Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida Communications Biology |
| title | Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida |
| title_full | Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida |
| title_fullStr | Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida |
| title_full_unstemmed | Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida |
| title_short | Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida |
| title_sort | engineering a phi15 based expression system for stringent gene expression in pseudomonas putida |
| url | https://doi.org/10.1038/s42003-025-07508-y |
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