Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct...

Area of Science:

• Microbiology and Natural Product Discovery
• Synthetic Biology and Metabolic Engineering

Background:

• Microbial natural products (NPs) are crucial sources for new therapeutics, but their isolation and characterization are challenging.
• Biosynthetic gene clusters (BGCs) can be identified bioinformatically, yet accessing the corresponding NPs remains a bottleneck.
• The RNA chaperone Hfq is essential for the production of most microbial NPs.

Purpose of the Study:

• To develop a method for targeted activation and identification of microbial natural products (NPs).
• To overcome the difficulty in accessing NPs from specific biosynthetic gene clusters (BGCs).
• To facilitate the discovery of novel NPs and streamline bioactivity testing.

Main Methods:

• Deletion of the Hfq gene (Δhfq) in microbial strains to reduce background NP production.
• Replacement of native promoters of target BGCs with inducible promoters in Δhfq mutants.
• Utilized the easy Promoter Activated Compound Identification (easyPACId) approach for targeted NP production.

Main Results:

• Achieved almost exclusive production of NPs from targeted BGCs in Photorhabdus, Xenorhabdus, and Pseudomonas.
• Successfully identified several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS).
• Demonstrated that the easyPACId approach minimizes interference from other NPs, simplifying identification.
• Enabled direct bioactivity testing of secreted NPs in culture supernatants without extensive purification.

Conclusions:

• The easyPACId method significantly enhances the discovery and accessibility of microbial natural products (NPs).
• This approach facilitates the identification of novel compounds, including those from uncharacterized NRPS.
• easyPACId streamlines the process from gene cluster identification to bioactivity screening, accelerating drug discovery.