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

Abstract

The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.

Keywords: Bioactive compounds isolation; Bioactivity testing; Biosynthetic gene cluster activation; NRPS; Natural product; PKS; Photorhabdus; Promoter exchange; Pseudomonas; Xenorhabdus; easyPACId.

Figure 1.. Schematic overview of the easyPACId workflow

Figure 2.. Schematic overview of the multiple cloning site of pCEP-km and pCEP_xabA.

Vector map of pCEP-km^R (a) and pCEP_xabA (b), highlighting the empty multiple cloning site (MCS) and the restriction sites for the selected enzymes NdeI and PstI (c) and the inserted gene fragment xabA (d).

Figure 3.. Schematic picture of “easyPACId mutant” Xenorhabdus_∆hfq_pCEP_xyz.

The general verification primer VpCEP-fw (depicted in blue) binds to the integrated pCEP backbone, and the individual verification primer Vxyz-rv (depicted in red) binds in the genome, downstream of the homologous region.

Figure 4.. Different steps of conjugation.

Fresh (a) and 24 h incubated (b) mating plaque of Xenorhabdus_∆hfq + E. coli ST18-pCEP_xyz, which will be scraped off and dissolved in LB (according to the protocol step D1). (c) Promoter exchange mutants Xenorhabdus_∆hfq_pCEP_xyz after plating on LB-km agar and 48 h of incubation at 30 °C. (d) Master plate on LB-km agar of selected mutants from plate depicted in (c).

Figure 5.. Extracted ion chromatograms (EICs) for xenoamicin (m/z 658.2 [M+2H]²⁺).

Depicted are chromatograms from cultures in LB medium from X. doucetiae WT (green), X. doucetiae∆hfq (blue), and easyPACId mutant X. doucetiae∆hfq_pCEP_xabA (red) after induction with 0.2% L-arabinose. Cell-free supernatant was extracted with methanol according to the protocol step E6.