The work performed in this project encompasses multiple interdisciplinary techniques. First, the sponge DNA was sequenced using short-read, Illumina sequencing. Next, high molecular weight DNA was extracted from the sponges and sequenced using an in-house Nanopore MinION. The resulting data was hybrid assembled to yield five sponge metagenomes. Those metagenomes were binned to create hundreds of metagenome assembled genomes (MAGs). From this preliminary data, biosynthetic gene clusters (BGCs) were identified and networked for similarity. Several BGCs were studied more in depth to prioritize which would be good candidates for heterologous expression. Adding the Nanopore reads resulted in more high quality MAGs and, in some cases, more complete BGCs. Once the BGCs were annotated, using software called antiSMASH, BGC networking was done to identify similarities between the BGCs, using software called BiG-SCAPE. Very few of the mesophotic sponge symbiont BGCs networked with already known BGCs. One BGC family did network closely with an already identified BGC for ether lipid production in bacteria, the first of its kind. While ether lipids have been identified in sponges, the exact producers were unknown. Based on our analysis, both Acidobacteria and Poribacteria harbor putative ether lipid-producing BGCs. In the lab, we cloned on of these BGCs into two vectors, one with an inducible promotor system, and measured transcription of the genes in the BGC by reverse-transcriptase PCR. Both constructs showed expression of most genes, but the inducible promotor system showed expression of all genes, so work is continuing on this construct to determine if ether lipids can be made by the heterologous host. In addition to identifying canonical BGCs, we set out to look for BGCs that could produce halogenated compounds, as many of the sponges studied harbor interesting halogenated molecules. For this endeavor, we used a halogenase as an anchor to identify BGCs that aren't easily identified with antiSMASH. In this endeavor, we identified a group of BGCs with a halogenase and multiple other biosynthetic genes. This BGC was also cloned into an expression vector and integrated into Streptomyces hosts, with further investigations ongoing. All these results are being written up to be disseminated as three manuscripts: one on the overall dataset of 60 mesophotic sponges, their symbionts, BGCs, and metabolic capacities; a second on the putative ether lipid producing BGC; and a third on the cryptic halogenase-containing BGC. Aside from the planned manuscripts, four master’s students worked on these data, each disseminating their results as part of their final presentation. These results will be disseminated at an upcoming Sponges Conference in October 2022 and other conferences in the future.
