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Final Report Summary - BLUEPHARMTRAIN (BluePharmTrain)

Introduction and Aim
Marine sponges harbour extremely diverse populations of microbes, and are world record holders for the production of a plethora of bioactive molecules. Previous studies, however, aiming at the growth of sponges or their associated microbes for the production of bioactive compounds to supply biological material for clinical trials, have been largely unsuccessful.
With BluePharmTrain we had the following objectives:
1. Establish routines for the isolation of sponge-specific microorganisms by integrating novel high-throughput cultivation strategies and state-of-the-art genomics and transcriptomics.
2. Establish sponge cell cultures of target species by translating ecology into technology.
3. Develop heterologous expression tools for sponge-derived bioactives.
4. In addition, we aimed to provide a fundamental understanding of the sponge holobiont.
Achievement of these objectives should lead to the development of a technology platform that is applicable for obtaining a wide variety of bioactive compounds from sponges.

Major Results
With BluePharmTrain we have been successful in advancing the state of the art of science and technical capabilities for each of the before mentioned objectives and major results are briefly summarised per objective:
1. Isolating sponge-specific microorganisms
Sponge-associated bacteria are notoriously difficult to culture and especially the majority termed ‘sponge-specific bacteria’ i.e. bacteria only found in sponges. Yet some representatives of the sponge-specific microbes (belonging to the Chloroflexi, Planctomycetes, Gemmatimonadetes) have been brought into culture in BluePharmTrain. This was achieved by deviating from standard agar plate experiments through: (i) using a different gelling agent: gelrite; (ii) liquid cultures; and (iii) cultivation in 3D-matrices: pluronic and collagen (publication in progress) and (iv) co-cultivation setups (Fig. 1). In addition, we have integrated innovative ‘omics’-based methods in bacterial cultivation. Nowadays, genomes of currently uncultivable bacteria can be reconstructed from metagenomes and used to identify specific metabolic needs or resistance against certain antibiotics (See review Gutleben et al., 2018 from outreach section). This information has been used to design selective growth conditions that provide the metabolic needs of sponge-specific bacteria, such as Poribacteria.

2. Establish sponge cell cultures
Scientists have been pursuing the establishment of primary sponge cell cultures since the 1980s, but without lasting success. Although sponge cells are intrinsically highly suitable for the establishment of cell cultures because of the presence of totipotent cells, they quickly decline in culture. One of the aspects that hampers the development of cell cultures is the presence of bacteria, which could quickly overgrow the sponge cells. Therefore we have selected sponges that harbour relatively few bacteria. In addition, it is difficult to maintain sponge cells in culture because their nutritional requirements are unknown; the catch-22 is that the cells need to be growing to sort out what their nutritional requirements are. We have overcome this issue by focusing on cell metabolic activity using the Vybrant Cell Metabolic Assay Kit, rather than using cell division as the output parameter. This has allowed us to optimize growth media components based on a genetic algorithm approach. At the moment, the compounds that are most promising are considered for applying for IPR and are reported here in detail. Subsequently a new growth medium has been designed in which sponge cells are not only metabolically active, but have also undergone cell divisions. Therefore, for the sponge Dysidea etheria sponge cell cultures beyond primary cultures have been achieved.

3. Develop heterologous expression tools
Heterologous expression of the large gene clusters encoding many of the bioactive compounds is considered the Holy Grail for getting access to the riches of sponge-derived bioactives as it allows near infinite possibilities to exploit these molecules. However, it is also still the greatest challenge for exploiting these compounds (see Loureiro, Medema, Van der Oost and Sipkema, 2018). Because of the experimental hurdles related to heterologous expression of these large gene clusters, we focussed first on host selection and searched for free-living bacteria that produce a gene cluster very similar to some of the gene clusters identified from sponges, such as the polytheonamide gene cluster. A free-living bacterium producing a very similar molecule was identified and made genetically accessible. A his-tagged version of the leader sequence was cloned in front of the promoter in pLMB509 and transformed into the free-living bacterial strain. Analysis of the core metabolite produced by LC-MS/MS revealed complete processing, including 21 epimerizations, 1 dehydration, N-methylation of all 5 Asn residues, and 7 C-methylations. The terminal metabolite was isolated and found active in cytotoxicity assays. This expression system was subsequently used to generate highly processed polytheonamide compounds as well as novel variants. Creation of a production system for some of the most complex and rare marine natural products is an important milestone.

4. Understanding of the sponge holobiont
The uncultivability of the large majority of the members of sponge holobionts make traditional reductionist approaches to take apart the whole, study its individual parts and subsequent reconstruction of the whole not feasible. However, to some extent understanding of the capacities, needs and activity of individual members of the holobiont is required to understand the mode of life of these organisms and niche formation inside sponges. We have circumvented the uncultivability of most of the microbial partners by applying sponge tissue for simultaneous extraction of DNA, RNA and proteins. Using a combined omics approach with cutting-edge assembly, binning and mapping tools, we obtained reconstructed genomes and expression profiles for 15 bacterial species residing in the sponge Aplysina aerophoba with estimated completion values between 65 – 95 % based on the presence of single-copy marker genes. In addition, we provide a preliminary genome for a sponge-associated unidentified lineage (SAUL), namely PAUC34f, with 74% estimated completion. As an example of a new insight in the lifestyle of bacteria inside the sponge tissue, we observed high expression of a superoxide dismutase. The current perception is that sponges the inner tissue of sponges is well oxygenated by the water pumping of the sponge. However, these tissues can turn temporarily anoxic during periods of non-pumping. We suggest that adaptation to oxic/anoxic transitions in the sponge tissue may result in highly expressed superoxide dismutase, an enzyme used for protection against oxidative stress, which is known to be induced more frequently in a symbiotic lifestyle.

The preparation of novel drugs based on our research has not yet been achieved, but it should also be noted that our objectives were largely technical, and a number of milestones related to solving the supply problem for sponge-derived bioactives was achieved, rather than developing new medicine within the time frame of BluePharmTrain. This implies that our current results are likely to be most attractive to further development by academics and Biotech start-up companies rather than by large Pharma companies. Some of these Biotech start-up companies were associated Partners to BluePharmTrain: ArcticMass, Porifarma, KliniPharm and Saebyli. At the end of the project BluePharmTrain results were communicated to different stakeholders at the final symposium ( in specific sections on Blue Business, Blue Policy and Blue Science. In addition, we have all together written a book chapter to disseminate the work of BluePharmTrain to the scientific community. The Chapter “BluePharmTrain – Biology and Biotechnology of Marine Sponges” has been accepted for the book Grand Challenges in Marine Biotechnology.

On behalf of BluePharmTrain,

Detmer Sipkema
Associate Professor Marine Microbiology
Wageningen University
Coordinator of BluePharmTrain

PS: it is a pity I cannot upload pictures here in the text and that textual make up is not allowed, because the whole make up changes when I copy it to the EU portal. I think this makes it a lot less easy and attractive to read.