A small step to discover candidate biological control agents from preexisting bioresources by using novel nonribosomal peptide synthetases hidden in activated sludge metagenomes

Abstract

Biological control agents (BCAs), beneficial organisms that reduce the incidence or severity of plant disease, have been expected to be alternatives to replace chemical pesticides worldwide. To date, BCAs have been screened by culture-dependent methods from various environments. However, previously unknown BCA candidates may be buried and overlooked because this approach preferentially selects only easy-to-culture microbial lineages. To overcome this limitation, as a small-scale test case, we attempted to explore novel BCA candidates by employing the shotgun metagenomic information of the activated sludge (AS) microbiome, which is thought to contain unutilized biological resources. We first performed genome-resolved metagenomics for AS taken from a municipal sewage treatment plant and obtained 97 nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS)-related gene sequences from 43 metagenomic assembled bins, most of which were assigned to the phyla Proteobacteria and Myxococcota. Furthermore, these NRPS/PKS-related genes are predicted to be novel because they were genetically dissimilar to known NRPS/PKS gene clusters. Of these, the condensation domain of the syringomycin-related NRPS gene cluster was detected in Rhodoferax- and Rhodocyclaceae-related bins, and its homolog was found in previously reported AS metagenomes as well as the genomes of three strains available from the microbial culture collections, implying their potential BCA ability. Then, we tested the antimicrobial activity of these strains against phytopathogenic fungi to investigate the potential ability of BCA by in vitro cultivation and successfully confirmed the actual antifungal activity of three strains harboring a possibly novel NRPS gene cluster. Our findings provide a possible strategy for discovering novel BCAs buried in the environment using genome-resolved metagenomics.