Protocols for yTREX/Tn5-based gene cluster expression in Pseudomonas putida.

Area of Science:

• Synthetic biology
• Microbial biotechnology
• Metabolic engineering

Background:

• Bacterial gene clusters are crucial for producing valuable secondary metabolites.
• Activating these clusters in heterologous hosts is essential for accessing novel compounds.
• Existing methods for gene cluster transfer and expression can be inefficient.

Purpose of the Study:

• To present a detailed protocol for the yTREX gene cluster transplantation tool.
• To enable efficient cloning, transfer, and expression of bacterial gene clusters in heterologous hosts.
• To demonstrate the utility of yTREX for natural product biosynthesis.

Main Methods:

• Yeast recombinational cloning (recombineering) to clone gene clusters into the yTREX vector.
• Conjugational transfer of the yTREX vector into bacterial hosts, such as Pseudomonas putida.
• Transposon Tn5-based integration into the host chromosome for stable expression.
• Design of gene cluster expression cassettes and use of lacZ reporter for production levels.

Main Results:

• Successful cloning and transplantation of the phenazine-1-carboxylic acid biosynthetic gene cluster from Pseudomonas aeruginosa into Pseudomonas putida KT2440.
• Demonstration of effective transcription from a chromosomal promoter following integration.
• Validation of the yTREX system for robust gene cluster transfer and activation.
• Utilized lacZ as a reporter to quantify production levels.

Conclusions:

• The yTREX tool provides a robust and versatile platform for bacterial gene cluster transplantation and heterologous expression.
• This protocol facilitates the discovery and production of valuable natural products from diverse bacterial sources.
• yTREX enables efficient activation of secondary metabolite pathways in engineered microbial hosts.