Marine Microverse Institute, LLC

Raising $75k to develop a metagenomics pipeline to accelerate a natural product discovery in a national marine sanctuary

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PROJECT TITLE: METAGENOMICS TO ACCELERATE A NATURAL PRODUCT DISCOVERY PIPELINE IN A NATIONAL MARINE SANCTUARY

[CONFIDENTIAL | MARINE MICROVERSE INSTITUTE, MMI]

OVERVIEW
Marine Sediments are an untapped potential for novel bacterial species and bioactive compound diversity. The explosion in metagenome sequencing and the development of bioinformatics algorithms, which allow for systematic mining of genomes for specific genetic features. We have shown that using these tools, microbial genomes indeed harbor a wealth of biosynthetic gene clusters (BGCs) for which the corresponding chemistry is not currently known. However, our work has just begun and we are at the stage where we need partners to continue our progress. We welcome investor input into the decision-making process. This exciting opportunity has led to the expansion of genome sequencing of cultured microorganisms directly from the environment without disturbing the habitat. The products of our innovative “bioprospecting” method will be a novel metagenomic database of sediment microbes within a national marine sanctuary and a repository of antibiotic, anticancer, and antiviral candidate compounds to be further studied by end-users. Customers include research laboratories, universities, the healthcare industry who have a stake in novel marine-derived bacteria with significant potential for medicinal therapies. The stakeholders in this “blue biotechnology” sector can build on our efforts in this drug discovery pipeline. The next steps include clinical trials to evaluate efficacy against tumor and viral agents utilizing the candidate molecules. Our partners can scale up and implement their resources and expertise in high throughput techniques for in-depth structural determination.

BACKGROUND
There is increasing global recognition of the inter-relationship between ocean ecology and human health. We are requesting funding for a Phase I project to document the significant knowledge gap of the sediment microbial biodiversity within marine protected areas (MPAs). There are two main goals that we aim to achieve. 
·   First, we will complete a baseline database to characterize the sediment microbial biodiversity in the Stellwagen National Marine Sanctuary (SBNMS) (https://pubmed.ncbi.nlm.nih.gov/31680056/). 
·   Second, we will further develop a drug discovery pipeline to metagenomically bio-prospect natural products from bacterial strains with high medicinal value. 
We have accumulated a fair amount of data through field collection, bioinformatics, and molecular bioassays to identify BGCs that naturally produce with antibiotic, anticancer, and antiviral drug candidates with medicinal properties. The outcomes will raise awareness of how valuable these habitats are to human health and protect marine food webs. 
 
Our vision is to transform the marine natural product pipeline into one that has a field-based testing component, which will significantly scale up the novel strain-level screening of bioactive compounds that has limited this area for decades. 
 
National marine sanctuaries can serve as a natural laboratory to better understand the biodiversity in our oceans. We have begun the construction of a metagenomics microbial library from four years of sampling in the SBNMS, which is a unique MPA as it is a working sanctuary that allows commercial fishing in designated sections. A 1992 SBNMS boundary designation is managed by NOAA as part of the Marine Protection, Research and Sanctuaries Act (Title III).  We are excited to report that in our ongoing research (https://pubmed.ncbi.nlm.nih.gov/31680056/), we have identified several unique strains of bacteria. We recently shared our project’s progress at the 2020 American Society of Microbiology Conference (ASM_2020_Bucci_Heinrichs_Aytur ePoster.pdf). 

Molecular biology has entered an age where marine biologists have powerful tools to sequence an organism’s genome directly from the environment without culturing samples in the laboratory. The advantages include the rapid genomic screening of functional gene characteristics. Microbial organisms account for most of the biomass in the oceans and a large portion resides in the surface sediment. However, less than 1% of bacteria can be cultured in laboratories and as result a significant portion is unknown.[1][2] To overcome the barriers of laboratory base culturing, novel high-throughput sequencing and bioinformatics facilitate a more comprehensive evaluation of species biodiversity and genomic analyses.[3][4] Historically, antibiotics such as penicillin, discovered in 1924 were identified from a terrestrial-based natural product, ascomycetous fungi. This discovery has made history for medicine and led to the discovery that ecologists know far less about the biodiversity of microbial communities than realized. Since that time, it has been shown that marine-derived bacterial organisms harbor a significant potential to produce what is known as biosynthetic gene clusters (BGCs) that are clustered in various regions of their genome.

The vast unexplored sediment microbes in the ocean have a high potential as a source for novel bioactive molecules, which have evolved over time from extreme conditions with unique chemical defenses and genetic adaptations for survival.[5][6] Natural products are comprised of secondary metabolites, which are organic compounds that are not directly involved in an organism’s growth, development, and reproduction. Secondary metabolites produced by marine bacteria harbor unique BGCs with the ability to produce medicinal compounds. Species Streptomyces, in particular, produce a significant quantity of secondary metabolites used for the antibiotic, chemotherapeutic and antiviral drugs. The increase in antibiotic-resistant pathogens coupled with the occurrence of global pandemics such as COVID-19 has necessitated a renewed search for bioactive compounds with medicinal properties. Streptomyces hosts a significant number of BGC genes within secondary metabolites and well known as a prolific source (> 80%) of known secondary metabolite.[7][8] 

OUR COMPANY
Marine Microverse Institute (MMI, (https://marinemicroverse.com/) founded in 2015 is a Maine-based marine biotechnology startup focused on developing innovative molecular methods and bioassays to improve the marine ecosystem and human health. We believe these two goals can coexist to build the blue economy. 
 
TEAM AND CREDENTIALS
Our group of scientists has over 40 years of experience in research and industry. The MMI team is led by John P Bucci, Ph.D., founder, and marine molecular scientist (https://www.researchgate.net/profile/John_Bucci). Dr. Bucci has over 25 years of experience in applied and basic genomic research that has yielded numerous peer-reviewed publications and reports. Dr. Bucci’s background is in biological oceanography and molecular ecology and an affiliate assistant professor in the School of Marine Science at the University of New Hampshire (UNH). Another member of MMI’s team is Robert Valentini, MD, Ph.D., a life science executive and biotechnology science board member, Bryan Barney, Ph.D. a bioinformatics scientist, and Kent Hubbard, field project manager.

ACADEMIC PARTNERS
Semra Aytur, Ph.D., MPH, is an Associate Professor in the Department of Health Management and Policy at UNH, and a senior consultant at MMI. She has over 15 years of experience in public health, epidemiology/biostatistics, stakeholder engagement, and environmental policy. She has published over 40 scientific manuscripts (https://www.ncbi.nlm.nih.gov/myncbi/1-SU56Dyu2LA9/bibliography/public/). Dr. Aytur has led several large team science grants focused on evidence-based policy at the nexus of human and environmental health.  On this project, she will be partnering with MMI and the NOAA Stellwagen Bank National Marine Sanctuary Research Team (https://stellwagen.noaa.gov/). Dr. Kelley Thomas, Ph.D. also serves on our Scientific Advisory board who is an endowed Chair at the UNH, Hubbard Genomic Center Director, and Professor of Molecular Biology. He is a leader in comparative and environmental genomics with over 50 articles published.

A link to one of our undergraduate researcher’s presentations, highlighting part of the research that we are conducting (Streptomyces hygroscopicus in the Stellwagen Bank National Marine Sanctuary: Discovery of Biosynthetic Gene Clusters with Potential to Produce Chemotherapeutic Compounds - UNH Media).

Objectives Phase I
1. Produce a comprehensive database of the sediment microbial biodiversity within the SBNMS, including those closed and open to fishing.
2.  Complete a culture-independent natural product pipeline to select the highest quality BGCs and secondary metabolites of species such as Streptomyces.
3.   Validate target strains identified at our sites with the highest potential for producing medicinal compounds. We have identified multiple strains of scabrisporus and hygroscopicus.
4.   Develop bioassays from cultured strains and sequence those genomes that have been identified as having a high potential for harboring medicinal compounds.
5.   Conduct heterologous gene expression experiments to isolate and purify high-value candidate compound structures and biologically active natural products.
 
Our business plan is to market a patented marine natural products library of drug candidate compounds that can be sold to companies for clinical drug trials and development. This invention includes the unique pipeline that we have developed from environmental sample collection to potential natural products.

Phase I, 1-2 yrs: Complete biodiversity study at designates sites.  Complete our segment for a natural product pipeline, including BGC screening and Streptomyces-focused culture-independent screening of viable compounds. Private and investor financial support ($200k)

Phase II 3-5 yrs: Refinement of our custom natural product discovery methods to validate novel antibiotics, anticancer and antiviral agents. This work will be laboratory-based and include culturing and cloning strains as well heterologous gene expression to produce compounds. A benefit is that the native strain-level isolates discovered within the sanctuary can be procured. We will partner with industry, and medicinal chemists to analyze candidates for extraction, isolation, and characterization of bioactive compounds. Investor and Industry partner financial support ($750k)

MMI is working on a provisional patent with the U.S. Patent and Trademark Office on its custom bioinformatics and drug discovery pipeline.

Our project will increase the knowledge of marine biodiversity and increase the quantity of novel marine-based natural products for commercialization. However, a potential bottleneck is the limited availability of stock cultures for the Streptomyces strains we have discovered exist at our sites. and the knowledge of the. This is both an exciting opportunity and a potential challenge to rigorously test the viable compounds they can produce. Fortunately, we have begun the process to test protocols needed from culture-independent analysis, before embarking on pure culture bioassay experiments. MMI will seek to work with international partners such as biobanks and research institutions to characterize the microbial communities in unsampled areas in national marine sanctuaries, which are likely to be highly productive sources of new bioactive compounds. 

OCEAN CONSERVATION BENEFIT
This project will propel the public’s interest in ocean science and provide in-depth knowledge of the biodiversity in marine sanctuaries that serves multiple functions for the ocean and human health. Our objectives support the U.N. Sustainable Development Goals (SDG # 14), which emphasize sustainable use of the ocean’s resources by raising awareness for the importance of managing marine sanctuaries worldwide. This work supports a critical “new era” of transdisciplinary capacity for sustaining MPAs. Results will elucidate the health of the sediment microbial food web and benthic habitat while discovering medically relevant biosynthetic gene clusters that may be present within MPAs. We are working directly with NOAA’s SBNMS branch and the Conservation Law Foundation to ensure ethical practices. These outputs directly address the needs expressed in NOAAs 2020 report [9] by protecting the abundance, distribution, and health of food web organisms that sustain fisheries. 
Literature cited

Literature cited
[1] Pace NR, Stahl DA, Lane DJ, Olsen GJ. 1986. The analysis of natural microbial populations by ribosomal RNA sequences. Adv. Microb. Ecol. 9:1-55. https://doi:10.1007/978-1-4757-0611-6.[2] Kaeberlein et al. 2002. Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science. 296:1127-9.[3] Riesenfeld CS, Schloss PD, Handelsman J. 2004. Metagenomics: Genomic analysis of microbial communities. Annual Review of Genetics. 38(1):525-552.[4] Polinski JM, Bucci JP, Gasser M, Bodnar AG. 2019.Targeted metagenomic assessment of biodiversity across prokaryotic and eukaryotic taxa in sediments from the Stellwagen Bank National Marine Sanctuary. Scientific Reports, Nature. 9: Article number: 14820. https://doi.org/10.1038/s41598-019-51341-3.[5] Dalisay DS, Williams DE, Wang XL, Centko R, Chen J, Andersen RJ. 2013. Marine sediment-derived streptomyces bacteria from British Columbia Canada are a promising microbiota resource for the discovery of antimicrobial natural products. PLoS ONE 8(10): e77078.[6] Manivasagan P, Venkatesan J, Sivakumar K, Kim SK. 2013. Marine actinobacterial metabolites: Current status and future perspectives. Microbiological Research 168(6):311-332. https://doi.org/10.1016/j.micres.2013.02.002.[7] Heinrichs L, Aytur SA, Bucci JP. 2020. Whole metagenomic sequencing to characterize the sediment microbial community within the Stellwagen Bank National Marine Sanctuary and preliminary biosynthetic gene cluster screening of Streptomyces scabrisporus. Marine Genomics. 50. https://doi.org/10.1016/j.margen.2019.100718.[8] Liu R, Deng Z, Liu T 2018. Streptomyces species: Ideal chassis for natural product discovery and overproduction. Metab. Eng. 50:74-84.[9] NOAA Stellwagen Bank National Marine Sanctuary 2020 Report. (https://sanctuaries.noaa.gov/science/condition/sbnms/2020-report-content-summaries.html). 

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