The natural product biosynthesis potential of the microbiomes of Earth – Bioprospecting for novel anti-microbial agents in the meta-omics era

As we stand on the brink of the post-antibiotic era, we are in dire need of novel antimicrobial compounds. Microorganisms produce a wealth of so-called secondary metabolites and have been our most prolific source of antibiotics so far. However, rediscovery of known antibiotics from well-studied cultured microorganisms, and the fact that the majority of microorganisms in the environment are out of reach by means of conventional cultivation techniques, have led to the exploration of the biosynthetic potential in natural microbial communities by novel approaches. In this mini review we discuss how sequence-based analyses have exposed an unprecedented wealth of potential for secondary metabolite production in soil, marine, and host-associated microbiomes, with a focus on the biosynthesis of non-ribosomal peptides and polyketides. Furthermore, we discuss how the complexity of natural microbiomes and the lack of standardized methodology has complicated comparisons across biomes. Yet, as even the most commonly sampled microbiomes hold promise of providing novel classes of natural products, we lastly discuss the development of approaches applied in the translation of the immense biosynthetic diversity of natural microbiomes to the procurement of novel antibiotics

SecMet-FISH: Labeling, visualization, and enumeration of secondary metabolite producing microorganisms

Our understanding of the role of secondary metabolites in microbial communities is challenged by intrinsic limitations of culturing bacteria under laboratory conditions and hence cultivation independent approaches are needed. Here, we present a protocol termed Secondary Metabolite FISH (SecMet-FISH), combining advantages of gene-targeted fluorescence in situ hybridization (geneFISH) with in-solution methods (in-solution FISH) to detect and quantify cells based on their genetic capacity to produce secondary metabolites. The approach capitalizes on the conserved nature of biosynthetic gene clusters (BGCs) encoding adenylation (AD) and ketosynthase (KS) domains, and thus selectively targets the genetic basis of non-ribosomal peptide and polyketide biosynthesis. The concept relies on the generation of amplicon pools using degenerate primers broadly targeting AD and KS domains followed by fluorescent labeling, detection, and quantification. Initially, we obtained AD and KS amplicons from Pseuodoalteromonas rubra, which allowed us to successfully label and visualize BGCs within P. rubra cells, demonstrating the feasibility of SecMet-FISH. Next, we adapted the protocol and optimized it for hybridization in both Gram-negative and Gram-positive bacterial cell suspensions, enabling high-throughput single cell analysis by flow cytometry. Ultimately, we used SecMet-FISH to successfully distinguish secondary metabolite producers from non-producers in a five-member synthetic community