ArticleMicrobial biotechnology2020
Protocols for yTREX/Tn5-based gene cluster expression in Pseudomonas putida.
Article in Microbial biotechnology, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed.
- Transposon mutagenesis in bacterial natural product discovery.Essays in biochemistry · 2026Review
- Engineering a phi15-based expression system for stringent gene expression in Pseudomonas putida.Communications biology · 2025Article
- Assessing the Orthogonality of Phage-Encoded RNA Polymerases for Tailored Synthetic Biology Applications inInternational journal of molecular sciences · 2023Article
- Production of tailored hydroxylated prodiginine showing combinatorial activity with rhamnolipids against plant-parasitic nematodes.Frontiers in microbiology · 2023Article
- Chemical structure, properties and potential applications of surfactin, as well as advanced strategies for improving its microbial production.AIMS microbiology · 2023Review
- The modular pYT vector series employed for chromosomal gene integration and expression to produce carbazoles and glycolipids inFEMS microbes · 2023Article
- SEVAtile: a standardised DNA assembly method optimised for Pseudomonas.Microbial biotechnology · 2022Article
- Integration of Genetic and Process Engineering for Optimized Rhamnolipid Production UsingFrontiers in bioengineering and biotechnology · 2020Article
- Protocols for yTREX/Tn5-based gene cluster expression in Pseudomonas putida.Microbial biotechnology · 2020Article
- Marine Biosurfactants: Biosynthesis, Structural Diversity and Biotechnological Applications.Marine drugs · 2019Review
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Authors and funding
5 authors.
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Abstract
Bacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX 'gene cluster transplantation tool': Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5-based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX-based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine-1-carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putidaKT2440 as well as the use of β-galactosidase-encoding lacZ as a reporter of production levels.
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