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Chemistry of Sponge Microbiomes

Periodic Reporting for period 1 - COSMos (Chemistry of Sponge Microbiomes)

Periodo di rendicontazione: 2020-05-01 al 2022-04-30

Molecules made by nature, or natural products, inspire the majority of currently approved small molecules for use in medicine. With an increase in widespread antibiotic resistance and the perpetual search for anticancer drugs, new sources of natural products must be discovered. The marine environment is a proven, prolific source of novel bioactive molecules and much of the ocean remains chemically and genetically unexplored. The mesophotic zone, where light barely reaches the ocean floor, is particularly neglected because of the technological difficulty of reaching this twilight zone of the ocean. Shallow water marine sponges have been well documented to have diverse microbial populations and impressive chemical repertoires. Mesophotic sponges, however, are relatively unstudied in terms of their natural product potential. This project aimed to take a deep dive into the metagenomes of mesophotic sponges for their natural product potential. Hybrid sequencing and assembly of metagenomes of five prioritized sponges yielded 435 medium to high quality metagenome assembled genomes (MAGs), each representing the first glimpse into uncultured sponge symbionts and their metabolic capacities. Within the metagenomes and MAGs, thousands of biosynthetic gene clusters (BGCs) were identified, the vast majority of which are novel, with less than 1% of BGC families containing a known BGC from a curated database. BGC networking revealed one BGC family that is highly similar to a characterized ether lipid producing BGC. There are multiple putative ether lipid BGC families in our sponge metagenomes, which are found in both Acidobacteria and Poribacteria MAGs. One of these putative ether lipid BGCs was cloned in E. coli and successful expression of each gene in the BGC was determined using RT-PCR. Current work is exploring whether the heterologous host with the ether lipid-like BGC can actually produce ether lipids. To go beyond easily annotated BGCs, we also used non-standard genome mining methods to identify non-canonical BGCs harboring putative halogenases, resulting in an interesting family of BGCs residing in related Acidobacteria MAGs from shallow, mesophotic and deep sea sponges. Continuing efforts are aimed at heterologously expressing these cryptic BGCs in the lab to confirm our bioinformatic findings. Overall, this project revealed the identity and metabolic capacities of hundreds of uncultured mesophotic sponge symbionts and spurred two heterologous expression efforts.
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.
This project benefitted greatly from a novel group of 60 mesophotic sponges, going beyond the usual shallow-water sponge collections often used in natural product discovery. As the oceans acidify and warm, many of the shallow-water sponge species may disappear or move to deeper depths, so understanding how sponges live and survive at greater depths is imperative. Trawling and other human activities can also have an impact on the little-studied mesophotic zone, so understanding these ecosystems and preserving them through sample collection and genetic sequencing can shed light on their importance and need for conservation. Beyond the inherent value of studying lesser-known species, exploiting their genetic potential in a sustainable way can bring about new molecules for medicine, agriculture, and industry. In this project, we progressed beyond the state of the art for gene cloning by cloning entire biosynthetic pathways. In the case of the putative ether lipid pathway, we have been able to demonstrate transcription of every gene in an inducible promotor system. Although some groups have tried, no one has yet been able to express this pathway in bacteria. We have a good indication that we may succeed in this endeavor due to the transcription we observe in our system. Likewise, we went beyond the state of the art in biosynthetic gene cluster (BGC) mining. We not only used the state of the art software, but also used creative bioinformatic analyses to identify BGCs containing halogenases that the standard software did not identify. One such BGC has been cloned and integrated into a heterologous host, and expression studies are underway. If expression is successful from either BGC, it would represent the first, to our knowledge, expression of a BGC from an uncultured sponge symbiont. This would represent a huge leap in the natural products community’s ability to not only study BGCs from inaccessible microbes, but to do so in a sustainable manner, that only requires the collection of a small sample for genomic sequencing.
Overview of the COSMos MSCA Project