Supplementary information provided with Murray et al.: Discovery of an Antarctic ascidian-associated uncultivated Verrucomicrobia with antimelanoma palmerolide biosynthetic potential

• Main Authors: Murray, Alison E, Lo, Chien-chi, Daligault, Hajnalka E, Avalon, Nicole E, Read, Robert W, Davenport, Karen W, Higham, Mary L, Kunde, Yuliya, Dichosa, Armand EK, Baker, Bill J, Chain, Patrick SG
• Format: Other/Unknown Material
• Language: unknown
• Published: Zenodo 2021
• Subjects: Antarctic; The Antarctic; Antarc*
• Online Access: https://dx.doi.org/10.5281/zenodo.5586111; https://zenodo.org/record/5586111

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 74 Kbp candidate BGC encoding the multi-modular 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 – 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-producing 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. : The GFF File needs to be read in a genome editing program. Funding provided by: National Institutes of Health Crossref Funder Registry ID: http://dx.doi.org/10.13039/100000002 Award Number: CA205932Funding provided by: National Science Foundation Crossref Funder Registry ID: http://dx.doi.org/10.13039/100000002 Award Number: OPP-0442857Funding provided by: National Science Foundation Crossref Funder Registry ID: http://dx.doi.org/10.13039/100000001 Award Number: ANT-0838776Funding provided by: National Science Foundation Crossref Funder Registry ID: http://dx.doi.org/10.13039/100000001 Award Number: PLR-1341339 : See the publication for details (main text and supplemental methods section). Briefly, we used a metagenomic approach to sequence a microbial community from an Antarctic ascidian, Synoicum adareanum . From this we assembled a nearly complete metagenome assembled genome (MAG) that encodes a candidate palmerolide A biosynthetic gene cluster of a verrucomicrobium affiliated new bacterium named in this manuscript as Candidatus Synoicihabitans palmerolidicus. The supplemental material provided in this data set includes (i) Figure S1 which provides correlation coefficients derived between real time PCR data (methods described in the manuscript) and amplicon sequence variant (ASV) occurrences across a data set of 63 samples. (ii) Figure S2 displays the taxonomic classification of predicted proteins in the assembled MAG derived from an annotation pipeline called MetaERG. (iii) The annotation of the Ca. S. palmerolidicus genome, as a GFF file formatted file.