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.

Keywords: bioactivity testing; easyPACId; natural products; proteobacteria; simplified production.

Figure 1

Schematic overview showing the outcome of promoter exchange for a desired biosynthetic gene cluster (BGC) in wild type (top) and Δhfq mutants (bottom) using integrative pCEP plasmids.10

Figure 2

Promoter exchange in Δhfq results in specific NP production. a) Culture supernatants (top) and XAD‐16 extracts (bottom) of wild type (wt) and Δhfq mutants of P. laumondii TTO1 and X. szentirmaii. HPLC/MS analysis of P. laumondii TTO1 (b) and X. szentirmaii (c) wt and Δhfq mutants are shown as base peak chromatograms (BPC). For better visualization extracted ion chromatograms (EICs) representing major derivatives of all known NP classes in both strains (Supplementary Table 1, Supplementary Figure 1) are shown at the bottom. For 14 and 26, the production titer was still very low compared to other NPs that only EICs of induced (red) and non‐induced Δhfq mutants (black) are shown. Both compounds were not detected in the wt. d) Structures of identified new NPs from X. szentirmaii (14, 15), Photorhabdus PB45.5 (23, 24 a), Xenorhabdus KJ12.1 (25) and Pseudomonas entomophila L48 (32, 33). The chiral centers of hydroxyl groups and amino acid residues in new NPs were predicted by analyzing the corresponding BGCs (for details see Supplementary Information A.5 and Supplementary Figure 3).

Figure 3

Bioactivity of cell‐free culture supernatants enriched in desired NPs (top) derived from Δhfq‐P_BAD‐xy mutants against wild type (WT) or Δhfq alone against different organisms and in vitro assays. Bioactivities are shown for none (white) to highest activity (red) in the different assays (Supplementary Table 5). For NP data and structures see Supplementary Figure 1 and Supplementary Table 1.