Interpreting Microbial Biosynthesis in the Genomic Age: Biological and Practical Considerations

Abstract

Genome mining has become an increasingly powerful, scalable, and economically accessible tool for the study of natural product biosynthesis and drug discovery. However, there remain important biological and practical problems that can complicate or obscure biosynthetic analysis in genomic and metagenomic sequencing projects. Here, we focus on limitations of available technology as well as computational and experimental strategies to overcome them. We review the unique challenges and approaches in the study of symbiotic and uncultured systems, as well as those associated with biosynthetic gene cluster (BGC) assembly and product prediction. Finally, to explore sequencing parameters that affect the recovery and contiguity of large and repetitive BGCs assembled de novo, we simulate Illumina and PacBio sequencing of the Salinispora tropica genome focusing on assembly of the salinilactam (slm) BGC.

Horizontal gene transfer to a defensive symbiont with a reduced genome amongst a multipartite beetle microbiome

“…As part of the binning procedure, genome completeness was estimated based on the presence of 139 single-copy marker genes (Rinke et al, 2013) (Dataset S1C). However, as some complete genomes of genome-reduced symbionts have low apparent completeness by this measure (Miller et al, 2017, 2016b), this figure cannot be used alone to determine the size of an incompletely assembled genome. Conversely, even the drastically-reduced genomes of intracellular obligate insect symbionts have been found to almost universally maintain certain genes that we refer to here as ‘core genes’, involved in replication, transcription, protein folding/stability, tRNA modification, sulfur metabolism, RNA modification and translation (McCutcheon and Moran, 2012).…”

Autometa: Automated extraction of microbial genomes from individual shotgun metagenomes

“…This reference-free approach is moving the field of microbiology from phylogenetic profiling of communities and aggregate interpretation of metagenomic data to a higher resolution perspective of which organisms play what role in a given ecosystem. Such information can be invaluable in a diverse array of biotechnological applications, such as identifying the source of bioactive secondary metabolites in complex marine invertebrate communities (Piel et al , 2004;Miller et al , 2017;Lopera et al , 2017) , or antibiotic resistance mechanisms (Ashton et al , 2015;Shore et al , 2011;Moss et al , 2017;Loman et al , 2013;van der Helm et al , 2017) in uncultured clinical samples (Grumaz et al , 2016;Doughty et al , 2014) . However, despite the advances stemming from this paradigm shift in metagenomic analysis, a number of challenges remain.…”

“…Many available automated binning programs require the use of multiple samples in order to bin contigs into genome bins based on differential coverage profiles. However, this type of sample collection strategy is often not possible for marine invertebrate communities with dynamic compositions (Miller, Weyna, et al , 2016) , and can be too costly for exploratory sequencing studies (Miller et al , 2017) . Furthermore, these techniques typically rely on the use of co-assemblies, a strategy whereby reads from multiple samples are pooled prior to assembly.…”

“…The necessity for genome binning ultimately stems from the underlying shortcomings of modern sequencing technology (Sangwan et al , 2016;Miller et al , 2017) , especially in regards to the trade off between read length, accuracy, and sequencing depth (Loman and Pallen, 2015) . Thus, short read sequencing technologies, such as Illumina, are the only platforms currently capable of delivering sufficient sequencing depth and per-read accuracy to effectively assemble low abundance genomes directly from host-associated metagenomes without physical or chemical enrichment of bacterial DNA (Quince et al , 2017) , which can introduce unforseen sampling bias.…”

Coculture of Marine Invertebrate-Associated Bacteria and Interdisciplinary Technologies Enable Biosynthesis and Discovery of a New Antibiotic, Keyicin

“…27 Hence, genomic approaches, although an excellent way to identify BGCs and their putative products, are, in the absence of other complementary technologies, insufficient to achieve broadly successful bioinformatics-guided NP identification goals for some biosynthetic classes. 31, 32 However, when used in combination with proteomics and metabolomics, genomics becomes an increasingly powerful tool linking molecules with their biosynthetic genes. It is becoming increasingly evident that by triangulating genomic, proteomic and metabolomic information for microorganisms one becomes better able to address two key questions in current natural products chemistry: i) How does one identify interesting organisms when it comes to antimicrobial NP biosynthesis, and ii) How can cryptic BGCs be activated in an otherwise unproductive organism?…”