Biology subjects

Owen, J. G.

Publications and source records attributed to Owen, J. G..

4 recordsLinked to original sources

bioRxiv2025

DiscERN: An Automated Genome Mining Tool for the Discovery of Evolutionarily Related Natural Products

Targeted genome mining to expand known families of natural products is a powerful strategy for discovering bioactive compounds, yet it remains a significant bioinformatics challenge. While tools exist for de novo biosynthetic gene cluster identification and large-scale unsupervised clustering, dedicated methods for the targeted, hypothesis-driven expansion of user-defined BGC families are lacking. Here, we present DiscERN (Discoverer of Evolutionarily Related Natural products), a userfriendly tool designed to address this gap. DiscERN leverages a multi-modal ensemble method that integrates four complementary algorithms classifying biosynthetic gene clusters based on Pfam content, sequence homology, and predicted product structure. This approach allows users to strategically balance discovery sensitivity with predictive precision to suit diverse research goals. We demonstrate DiscERNs utility by applying it to a large collection of actinomycete genomes and validating its predictive power through the successful isolation of discomycin A, a new calcium-dependent lipopeptide antibiotic, from a silent biosynthetic gene cluster. DiscERN provides a robust and accessible platform that streamlines the path from genomic data to a prioritised list of candidate biosynthetic gene clusters, effectively bridging the gap between in silico prediction and bioactive compound discovery.

bioinformatics↗

bioRxiv2024

Microbial communities associated with marine sponges from diverse geographic locations harbour biosynthetic novelty

Marine sponges are a prolific source of biologically active small molecules, many of which originate from sponge-associated microbes. Identifying the producing microbes is a key challenge in developing sustainable routes for production and isolation of sponge-associated metabolites, and requires application of several computational tools. To facilitate these analyses, we developed MetaSing, a reproducible singularity-based pipeline for assembly, identification of high quality metagenome-assembled genomes (MAGs), and analysis biosynthetic gene clusters (BGCs) from metagenomic short read data. We apply this pipeline to metagenome datasets from 16 marine sponges collected from New Zealand, Tonga and the Mediterranean Sea. Our analysis yielded 643 MAGs representing 510 species. Of the 2,670 BGCs identified across all samples, 70.8% were linked to a MAG, enabling taxonomic characterisation. Further comparison of BGCs to those identified from previously sequenced microbes revealed high biosynthetic novelty in variety of underexplored phyla including Poribacteria, Acidobacteriota and Dadabacteria. Alongside the observation that each sample contains unique biosynthetic potential, this holds great promise for natural product discovery and for furthering the understanding of different sponge holobionts.

microbiology↗

bioRxiv2023

Metagenomic domain substitution for the high-throughput modification of non-ribosomal peptide analogues

Non-ribosomal peptides are a diverse and medically important group of natural products. They are biosynthesised by modular non-ribosomal peptide synthetase (NRPS) assembly-lines in which domains from each module act in concert to incorporate a specific amino acid into a peptide. This modular biosynthesis has driven efforts to generate new peptide analogues by substituting amino acid specifying domains. Rational NRPS engineering has increasingly focused on using evolutionarily favoured recombination sites for domain substitution. Here, we present an alternative approach inspired by evolution, which involves large-scale diversification and screening. By adopting a metagenomic approach of amplifying amino acid specifying domains from metagenomic DNA derived from soil, we were able to substitute over 1,000 unique domains into a pyoverdine NRPS. To identify functional domain substitutions, we employed fluorescence and mass spectrometry screening techniques, followed by sequencing. This comprehensive screening process successfully identified more than 100 functional domain substitutions, resulting in the production of 16 distinct pyoverdines as major products. The significance of this metagenomic approach lies in its ability to shift the focus of engineering non-ribosomal peptide biosynthesis. Instead of relying on a high success rate of individual domain substitution, we have developed effective methods that enable the exploration of a broader range of substitutions. This opens new possibilities for the discovery and production of novel non-ribosomal peptides with diverse biological activities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/543161v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@8ed078org.highwire.dtl.DTLVardef@188a0eorg.highwire.dtl.DTLVardef@10fd38corg.highwire.dtl.DTLVardef@9c651_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme Fatl (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our Fatl strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded Hisg-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved [~]5- fold more effective than the canonical nitroreductase NfsB.

biochemistry↗