Uncovering Lasonolide A biosynthesis using genome-resolved metagenomics

Abstract

Invertebrates, in particular sponges, have been a dominant source of new marine natural products. For example, lasonolide A (LSA) is a potential anti-cancer molecule isolated from the marine sponge Forcepia sp., with nanomolar growth inhibitory activity and a unique cytotoxicity profile against the National Cancer Institute 60 cell line screen. Here, we identified the putative biosynthetic pathway for LSA. Genomic binning of the Forcepia sponge metagenome revealed a gram-negative bacterium belonging to the phylum Verrucomicrobia as the candidate producer of LSA. Phylogenetic analysis showed this bacterium, herein named Candidatus Thermopylae lasonolidus, only has 88.78% 16S rRNA identity with the closest relative Pedosphaera parvula Ellin514, indicating it represents a new genus. The lasonolide A (las) biosynthetic gene cluster (BGC) was identified as a trans-AT polyketide synthase (PKS) pathway. When compared with its host genome, the las BGC exhibits a significantly different GC content and penta-nucleotide frequency, suggesting a potential horizontal acquisition of the gene cluster. Furthermore, three copies of the putative las pathway were identified in the candidate producer genome. Differences between the three las repeats were observed including the presence of three insertions, two single-nucleotide polymorphisms and the absence of a stand-alone acyl carrier protein in one of the repeats. Even though the Verrucomicrobial producer shows signs of genome-reduction, its genome size is still fairly large (about 5Mbp) and when compared to its closest free-living relative contains most of the primary metabolic pathways, suggesting that it is in the early stages of reduction. ImportanceWhile sponges are valuable sources of bioactive natural products, a majority of these compounds are produced in small amounts by uncultured symbionts, hampering the study and clinical development of these unique compounds. Lasonolide A (LSA), isolated from marine sponge Forcepia sp., is a cytotoxic molecule active at nanomolar concentrations and causes premature chromosome condensation, blebbing, cell contraction and loss of cell adhesion, indicating a novel mechanism of action and making it a potential anti-cancer drug lead. However, its limited supply hampers progression to clinical trials. We investigated the microbiome of Forcepia sp. using culture-independent DNA sequencing to uncover how an uncultured bacterium produces LSA. This provides future opportunities for heterologous expression and cultivation efforts that may minimize LSAs supply problem.