easyPACId, a Simple Method for Induced Production, Isolation, Identification, and Testing of Natural Products from...

Area of Science:

• Microbiology
• Natural Product Chemistry
• Synthetic Biology

Background:

• Biosynthetic gene clusters (BGCs) encode valuable natural products but are often difficult to activate.
• Entomopathogenic bacteria Xenorhabdus and Photorhabdus produce diverse bioactive compounds.
• Current methods for natural product discovery face challenges in activation, expression, and purification.

Purpose of the Study:

• To present the easyPACId (easy Promoter Activation and Compound Identification) approach for targeted activation of BGCs.
• To demonstrate the utility of easyPACId in entomopathogenic bacteria Xenorhabdus and Photorhabdus.
• To streamline the process of natural product discovery and characterization.

Main Methods:

• Targeted activation of BGCs encoding NRPS, PKS, or hybrid synthetases.
• Exchange of native promoters with the L-arabinose inducible PBAD promoter in ∆hfq mutants.
• Application in Xenorhabdus and Photorhabdus, with adaptation for Pseudomonas entomophila.

Main Results:

• Enrichment of crude extracts with single compounds or compound classes.
• Simplified isolation and identification of natural products due to reduced background in ∆hfq strains.
• Successful application in multiple proteobacteria, facilitating direct testing against target organisms.

Conclusions:

• easyPACId offers an efficient strategy for activating silent or cryptic BGCs.
• The approach circumvents issues associated with heterologous expression and chemical synthesis.
• easyPACId facilitates the discovery and development of novel natural products from microbial sources.