Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria

Abstract

The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.Uncultured bacteria are predicted to be a significant reservoir of novel small-molecule biosynthetic machinery (19,34). One means by which to access the biosynthetic potential contained within the genomes of uncultured bacteria is functional metagenomics (19). This approach involves cloning DNA directly from naturally occurring microbial populations (environmental DNA [eDNA]) and screening the resulting clone libraries for phenotypes traditionally associated with the production of secondary metabolites. A major limitation of functional metagenomics is its reliance on a foreign host to facilitate the expression of eDNA-derived genes and gene clusters (17). Codon bias, missing substrates, and the inability to recognize foreign regulatory elements, including promoters and ribosomal binding sites, are just some of the obstacles that are likely to limit the success of expression-dependent studies with any single host organism. Circumventing these obstacles through an expansion of the collection of hosts available for functional metagenomic studies should increase the efficacy of this approach.Soil ecosystems are rich in bacterial diversity, and the majority of soil-dwelling bacteria remain recalcitrant to standard microbial culture methods (33, 39). Large-scale metagenomic sequencing studies indicate that soil microbiomes are often dominated by five bacterial phyla: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Acidobacteria, and Actinobacteria (14). Bacteria from these common phyla are therefore appealing hosts for use in functional metagenomic studies of soil-derived eDNA libraries.In this study, six unique bacterial hosts [Agrobacterium tumefaciens (Alphaproteobacteria), ...

Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes of Australian marine sponges

“…Traditional methods, involving exhaustive extractions of sponge, do not reveal the chemical diversity produced by these rare biosphere representatives, or pathways that are inactive within the native host (Chiang et al, 2010). Harnessing this potentially novel chemistry is, therefore, dependent on the ability of molecular approaches to identify where this diversity exists (Fieseler et al, 2007;Hochmuth et al, 2010;Trindade-Silva et al, 2012), to provide access to the genetic basis of biosynthesis (Piel et al, 2004a;Fisch et al, 2009;Banik and Brady, 2010;Brady et al, 2010) and ultimately to facilitate production of these molecules through heterologous expression within a suitable host (Fu et al, 2008;Craig et al, 2010).…”

Functional metagenomic discovery of bacterial effectors in the human microbiome and isolation of commendamide, a GPCR G2A/132 agonist

“…In addition, it couples each observed phenotype to a single fragment of cloned metagenomic DNA, making it possible to simultaneously identify specific effector molecules and the specific effector genes that encode these molecules. Functional metagenomics has been used to isolate small molecules and proteins from soil metagenomes (15)(16)(17)(18)(19)(20)(21)(22)(23)(24); however, its application to the human microbiome has to date been very limited (16,25,26). In this study, a set of arrayed, large-insert cosmid libraries hosted in Escherichia coli was created from DNA isolated from the stool of three patients who, based on their phenotype, were predicted to have different commensal bacteria cohorts.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…A plasmid RK2-based broad-host range vector (pRS44/pTA44) was used to transfer metagenomic libraries to a variety of bacterial species like E. coli, Pseudomonas fluorescens and Xanthomonas compestris (Aakvik, Degnes et al 2009). In a more recent and comprehensive study, broad-hostrange cosmid environmental DNA libraries were screened in parallel for small molecules across six different proteobacterial hosts (Craig, Chang et al 2010). In examples presented here, there was no or very little overlap of phenotypes and clones produced by different hosts expressing the same metagenomic library, suggesting the use of alternate host-vector systems for increasing the success rate in metagenomic functional screenings.…”