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

Abstract

The biosynthesis of non-ribosomal peptide and polyketide natural products is facilitated by multimodular enzymes that contain domains responsible for the sequential condensation of amino and carboxylic subunits. These conserved domains provide molecular targets for the discovery of natural products from microbial metagenomes. This study demonstrates the application of tagencoded FLX amplicon pyrosequencing (TEFAP) targeting non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes as a method for determining the identity and diversity of natural product biosynthesis genes. To validate this approach, we assessed the diversity of NRPS and PKS genes within the microbiomes of six Australian marine sponge species using both TEFAP and metagenomic whole-genome shotgun sequencing approaches. The TEFAP approach identified 100 novel ketosynthase (KS) domain sequences and 400 novel condensation domain sequences within the microbiomes of the six sponges. The diversity of KS domains within the microbiome of a single sponge species Scopalina sp. exceeded that of any previously surveyed marine sponge. Furthermore, this study represented the first to target the condensation domain from NRPS biosynthesis and resulted in the identification of a novel condensation domain lineage. This study highlights the untapped potential of Australian marine sponges for the isolation of novel bioactive natural products. Furthermore, this study demonstrates that TEFAP approaches can be applied to functional genes, involved in natural product biosynthesis, as a tool to aid natural product discovery. It is envisaged that this approach will be used across multiple environments, offering an insight into the biological processes that influence the production of secondary metabolites.

Chemical-biogeographic survey of secondary metabolism in soil

“…The use of amplicon sequencing to profile soil microbiomes is an attractive approach for assessing the biosynthetic potential of diverse microbiomes to a depth that is still inaccessible via shotgun sequencing. Previously, investigation of natural product biodiversity using amplicon sequencing had only been applied to a small number of marine and terrestrial environments (21)(22)(23)(24)(25). Our study of nearly 100 unique soil samples builds on these earlier small-scale studies to provide a more detailed picture of the distribution of secondary metabolite gene clusters throughout soil microbiomes.…”

Bioprospecting microbial metagenome for natural products

“…NGS can be very useful in sequence-based approach as it has been demonstrated recently by deep sequencing of amplicons obtained using degenerate primers and eDNA or library as a template. This strategy uncovered more than 50% of the unique PKS and NRPS domains that are accessible from metagenome (Reddy et al 2012;Woodhouse et al 2013).…”

Metagenomic small molecule discovery methods

“…Pyrosequencing of ketosynthase and condensation domains from marine sponge metagenomes uncovered hundreds of previously unknown sequences, including several clades that had not been previously observed by extensive Sanger sequencing of these sponge metagenomes. (23) Furthermore, a head-to-head comparison of shotgun sequencing of the same samples demonstrated that PCR-based approaches were often 10 to 100 times more sensitive in identifying unique sequences from a metagenome of interest. (23) Even greater biosynthetic biodiversity was observed in desert soil microbiomes, where 1,000s of unique adenylation domain sequences were detected with only a fraction of them shared among distinct microbiomes.…”

“…(23) Furthermore, a head-to-head comparison of shotgun sequencing of the same samples demonstrated that PCR-based approaches were often 10 to 100 times more sensitive in identifying unique sequences from a metagenome of interest. (23) Even greater biosynthetic biodiversity was observed in desert soil microbiomes, where 1,000s of unique adenylation domain sequences were detected with only a fraction of them shared among distinct microbiomes. A similar pattern was observed for amplicons derived from Type I (ketosynthase) and Type II (ketosynthase alpha) polyketide biosynthesis, suggesting that soil metagenomes are likely to be a rich source of novel bioactive compounds.…”