Discovery of an Antarctic ascidian-associated uncultivated Verrucomicrobia with antimelanoma palmerolide biosynthetic potential

Abstract

The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially-produced hybrid non-ribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74kb candidate BGC encoding the multimodular enzymatic machinery (hybrid Type I-trans-AT polyketide synthase-non-ribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide-gene targets and a single 16S rRNA gene variant (R=0.83 to 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a near-complete genome (10 contigs) of the BGC organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as Candidatus Synoicihabitans palmerolidicus. The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium.

Bioinformatic and mechanistic analysis of the palmerolide PKS-NRPS biosynthetic pathway from the microbiome of an Antarctic ascidian

“…The details of field collections, sample processing and genomic methods (sequencing, assembly, and analysis) used to identify the putative biosynthetic gene clusters are described in detail in two related publications (Murray et al, 2020(Murray et al, , 2021. In brief, Synoicum adareanum samples were collected by SCUBA from the Antarctic Peninsula in the Anvers Island Archipelago and flash frozen.…”

“…The metagenome assembled genome (MAG) sequence analyzed in this study was generated from two S. adareanum samples (Bon-1c-2011 andDel-2b-2011) with high copy numbers of the putative biosynthetic gene cluster sequenced using PacBio technology. The resulting assembly of produced a nearly complete 4.3 MB genome, with five unique contigs and five varying copies of the pal BGC (referred to as pal BGC 1 through pal BGC 5) (Murray et al, 2021). Given the phylogenomic novelty of the MAG, the name Candidatus Synoicihabitans palmerolidicus was proposed for this new genus in the Opitutaceae family (Verrucomicrobia phylum).…”

“…The Ca. Synoicohabitans palmerolidicus MAG reported a candidate hybrid PKS-NRPS biosynthetic gene cluster that was present in multiple, non-identical copies (Murray et al, 2021).…”

“…The D-arabinono-1,4-lactone oxidase (palH) is a FAD-dependent oxidoreductase that likely works in concert with the glycosyltransferase. An ATP-binding cassette (ABC) transporter is encoded between the core biosynthetic genes and the genes for the trans-acting enzymes (Murray et al, 2021). This transporter, which has homology to SryD and contains the key nucleotide-binding domain GGNGSGKST, may be responsible for the translocation of the macrolide out of the cell, since it is housed within the BGC, it is likely under the same regulatory control.…”

“…Next, a persistent cohort of bacteria present across many individual ascidians -a core microbiome -for Synoicum adareanum was identified through analysis of occurrence of distinct amplicon sequence variants (ASV) from iTag sequencing of the Variable 3-4 regions of the bacterial 16S rRNA (Murray et al, 2020). This work ultimately led to the evaluation of the microbiome metagenome and the subsequent assembly of a metagenome assembled genome (MAG) of Candidatus Synoicohabitans palmerolidicus, a verrucomicrobium in the family Opitutaceae (Murray et al, 2021). Contained within the genome are five non-identical copies of a candidate pal BGC.…”