Overproduction of secondary metabolites in Photorhabdus noenieputensis through rpoB mutations.

Abstract

Specific mutations of the rpoB gene, which encodes the β subunit of bacterial RNA polymerase, can enhance the production of secondary metabolites in bacteria such as actinomycetes. Entomopathogenic bacteria Photorhabdus and Xenorhabdus species produce a variety of secondary metabolites. Recently, these genera have attracted attention as a promising source for novel antibiotics. In this study, the effect of rpoB mutations on secondary metabolite production in Photorhabdus noenieputensis DSM 25462, a known producer of the antituberculosis antibiotic evybactin, was evaluated. Spontaneous rifampicin-resistant mutants, frequently carrying rpoB mutations, were generated by plating cells on agar medium containing four times the minimum inhibitory concentration (MIC) of rifampicin and evaluated their antibacterial production using Escherichia coli WO153 as a test strain. Among 190 spontaneous rifampicin-resistant mutants of P. noenieputensis, strain designated R191, which harbors the rpoB Q148K mutation (C442A), displayed higher antibacterial activity than that of the parental strain DSM 25462. The real-time quantitative RT-PCR analysis of 20 putative secondary metabolite biosynthetic gene clusters (BGCs) identified using antiSMASH revealed that seven of these BGCs were overexpressed in the strain R191. Furthermore, comparative high-pressure liquid chromatography (HPLC) analysis of the metabolite profile indicated that the strain R191 produced several compounds that were not detectable in the DSM 25462 culture. These findings suggest that the introduction of rpoB mutations into Photorhabdus strains is an effective strategy for enhancing secondary metabolite production and may lead to the discovery of novel antibiotics.