Brown seaweed fucoidans : structure, diversity and enzymatic degradation

Marine macroalgae (seaweeds) are vital players in marine ecology and the global carbon cycle and represent a natural renewable resource for humankind. Seaweed consumption is rooted in millenary knowledge and traditions. At present, seaweeds have a variety of industrial applications, and their aquaculture is a developed and growing industry with the potential to contribute to climate change mitigation and adaptation. Globally, seaweeds fixate large amounts of carbon dioxide into various polysaccharides of great ecological, biogeochemical, and technological relevance. Some of these glycans enter the organic carbon pool in the ocean and serve as a source of energy for heterotrophic bacteria and subsequently to higher trophic levels, while other glycan fractions have been found in sinking particles and marine sediments and are thus regarded to be involved in carbon sequestration. From a biorefining perspective, alginates extracted from brown seaweeds, similar for agar and carrageenan extracted from red seaweeds, are used as stabilizing, thickening, and gelling agents in a wide range of food, biomedical, and biotechnology applications and are of great economic importance. Fucoidans, also produced by brown seaweeds, are a broad group of fucose-containing sulfated polysaccharides. They have been linked to carbon sequestration and are regarded as promising therapeutic agents. However, the structural complexity and biogeographical and seasonal variation of fucoidans are crucial factors limiting the knowledge of their role in the carbon cycle and the development of applications. Glycan-modifying enzymes used by marine bacteria to feed on macroalgal polysaccharides represent a valuable toolbox for fucoidan processing and structural characterization of sulfated polysaccharides. However, the characterization of such enzymes remains challenging due to the polymeric, heterogeneous, and relative undefined nature of their substrates. In this thesis, an enzymatic toolbox for processing and characterization of sulfated polysaccharides was established and applied to characterize brown algae fucoidans. Moreover, new methods for fucoidan extraction and characterization were developed. New analytical protocols based on matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) to investigate and characterize sulfated polysaccharides, and their modifying enzymes, were developed. These protocols were optimized and applied to characterize chemically and enzymatically hydrolysed fucoidans (Paper 1, 3, 4) and carrageenan (Paper 2). The development of MALDI-MS is described in detail in Paper 1. In this paper, MALDI-MS was applied as a rapid and sensitive screening method to identify enzymatically produced fucoidan oligosaccharides by GH107 fucanases P5AFcnA and Wv323. Notably, the analysis was compatible directly with unrefined macroalgal biomass and adaptable to MALDI-TOF and MALDI-Orbitrap instruments. Further analytic development consisted in establishing protocols for extraction of fucoidans from brown seaweed biomass. Using mild conditions along the extraction and purification process yielded high quality fucoidans, as indicated by their high molecular weight and sulfate content (Paper 3, 4). Novel enzymes active on sulfated polysaccharides were characterized (Paper 2, 3). Candidate enzymes had been previously identified from a deep-sea hydrothermal vent metagenome (AMOR) and from the genome marine bacteria specialized in fucoidan degradation, Lentimonas spp., and were here screened against a large substrate collection. In Paper 2, a novel thermophilic κ‑carrageenan sulfatase identified in the AMOR metagenome was characterized. This sulfatase, AMOR_S1_16A, belonging to the sulfatase S1_16 subfamily, selectively desulfates the nonreducing end galactoses of κ-carrageenan oligomers in an exo- mode of action. Notably, AMOR_S1_16A represents the first κ-carrageenan sulfatase within the S1_16 subfamily and exhibits a novel enzymatic activity. In Paper 3, seven novel fucanases from families GH107 (2) and GH168 (5) from Lentimonas spp. and from the AMOR metagenome were characterized using different analytical techniques. Using a carefully extracted fucoidan from Macrocystis pyrifera as substrate demonstrated enzymatic activity for all enzyme candidates except for FunA50 (GH168). Further screening on a collection of similarly extracted fucoidans from 21 brown seaweed species across 5 orders (Laminariales, Fucales, Ectocarpales, Dictyotales, Tilopteridales) revealed activity for FunA50, and extended the number of substrates recognized and hydrolysed by these novel enzymes. Finally, fucoidans from several brown seaweed species were characterized. In Paper 3, the GH107 fucanase P5AFcnA was used to digest the fucoidan extracted from M. pyrifera, and the products were characterized by NMR, MALDI-TOF-MS, SEC, and composition analysis. It was shown that P5AFcnA hydrolyses M. pyrifera fucoidan in an endolytic mode, releasing oligosaccharides that are gradually degraded into two sulfated tetramers and a disulfated fucose monomer, indicating that approximately half of the M. pyrifera fucoidan consists of a sulfated fucan with a regular structure. In Paper 4, the analytical MALDI-MS capacity developed in Paper 1 was exploited to investigate the natural variability of brown seaweed fucoidans. Five GH107 fucanases, including P5AFcnA characterized in Paper 1 and 3, as well as Fp277.23 and Fp277.50 newly identified in Paper 3, were employed to digest a broad range of fucoidan samples across different axes of diversity (tissue, taxonomic, seasonal, and geographic), and the resulting hydrolysates were analysed using MALDI-Orbitrap. This approach revealed differences in the composition of fucoidans across tissue, taxonomy, season, and geography. Overall, this thesis presents the characterization of novel enzymes involved in the depolymerization of sulfated polysaccharides from seaweeds, as well as novel analytical protocols and their application to characterized brown seaweed fucoidans.

Marine makroalger (tang og tare) er viktige aktører innen marin økologi og det globale karbonkretsløpet og representerer en naturlig fornybar ressurs for menneskeheten. Tang og tare har blitt anvendt som mat i flere tusen år. I dag har tang og tare en rekke industrielle anvendelser, og akvakultur er nå en utviklet og voksende industri med potensial til å bidra til å redusere og tilpasse oss klimaendringer. Globalt sett fikserer tang og tare store mengder karbondioksid og lager ulike polysakkarider av stor økologisk, biogeokjemisk og teknologisk relevans. Noen av disse glykanene lekker uti havet og tjener som en energikilde for heterotrofe bakterier og deretter til høyere trofiskenivåer, mens andre glykaner er funnet i synkende partikler og marine sedimenter og dermed bidrar til karbonbinding. Fra et bioraffineringsperspektiv brukes alginater ekstrahert fra brunalger, og agar og karragenan ekstrahert fra rødlager som stabilisator, fortyknings- og geleringsmidler i et bredt spekter av mat, biomedisinske og bioteknologiske anvendelser og er av økonomisk betydning. Fucoidaner er en bred gruppe fukose-rike sulfaterte polysakkarider som finnes i brunalger. De er involvert i karbonlagring og blir sett på som lovende terapeutiske karbohydrater. Imidlertid er den strukturelle kompleksiteten og den biogeografiske og sesongmessige variasjonen til fucoidaner utfordrende og mer kunnskap er nødvendig for å bedre forstå deres rolle i karbonsyklusen og for å utvikle anvendelser.