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

Abstract

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene‐sequence‐similarity‐based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non‐ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

Fabclavine diversity in Xenorhabdus bacteria

“…The general interest on Photorhabdus and Xenorhabdus increased in recent years, not only because of their large number of SMs, but also due to their easy-to-handle cultivation under laboratory conditions in combination with the accessibility for genetic manipulations such as genomic integrations or deletions [14][15][16][17]. Furthermore, recently published studies focused on the possible application of Photorhabdus and Xenorhabdus as biological pest control agents with and without the corresponding nematodes [18,19].…”

Bacterial pathogens: threat or treat (a review on bioactive natural products from bacterial pathogens)

“…While promoter exchange in the wild type strains produces several background peaks, [139][140][141][142] promoter substitution in Dhfq mutants leads to culture supernatants containing only the compounds of interest, thereby enabling direct bioactivity testing, requiring no laborious and time-consuming isolation and purication steps. 151 The promoter exchange strategy resulted in overproducing mutants with signicantly higher production titres relative to the WT strains. 151 In X. szentirmaii-Dhfq, two silent BGCs were activated that encode for the known depsipeptides, xenobactin 152 and szentiamide.…”

“…151 The promoter exchange strategy resulted in overproducing mutants with signicantly higher production titres relative to the WT strains. 151 In X. szentirmaii-Dhfq, two silent BGCs were activated that encode for the known depsipeptides, xenobactin 152 and szentiamide. 153 Additionally, a new oxidized diketopiperazine (DKP), szentirazine 27, and three new shortened PAX-peptides (28a-c) were produced.…”

“…The structures of the lipopeptides (28a-c) were elucidated by detailed MS-MS analysis while szentirazine 27 was isolated from a large-scale culture, and its structure was characterized by NMR spectroscopy. 151 antifouling, antitumor, and antimicrobial. 154 Three molecular species have been reported from S. marcescens, serrawettin W1 (also known as serratamolide A 20a), serrawettin W2 32a, and serrawettin W3.…”

“…Additionally, promoter exchange in Dhfq mutants resulted in the production of desired metabolites for further bioactivity testing. 151 The development of high-throughput next-generation sequencing methods, together with the development of new bioinformatics tools that can assemble nearly complete genomes, will continue to revolutionize microbial "dark matter" exploration. Furthermore, improvements in analytical platforms (mass spectrometry, NMR) coupled with recent advancements in metabolomics enable the detection and identication of compounds in minute quantities from complex biological samples.…”