Biosynthesis of cell wall polysaccharides from brown algae: screening glycosyl transferase activities – BrownSugar

Brown algae are large biomass producers in coastal regions. Cultivation projects have increased sharply in Europe, essentially of the kelp Saccharina latissima. Carbohydrates are among the top-value added products and major constituents of this biomass. The main polymers are the alginates and the fucose-containing sulfated polysaccharides (FCSPs), including fucans, from the extracellular matrices (ECMs). Alginates are used as texturing agent in the food industry while the FCSPs have extensive interest in biomedical applications. Despite this importance, virtually nothing is known about the biosynthesis of ECM-glycans in brown algae. Glycosyltransferases (GTs) are key enzymes involved in glycan biosynthesis. In eukaryotes most of the GTs are localized in the Golgi apparatus or at the plasma membrane. In brown algae the first genome sequence released in 2010, allowed us to reconstruct the putative routes for the biosynthesis of ECM-glycans. Candidate GTs genes were suggested for the synthesis of sulfated fucans and alginates, likely localized in distinct organelles. Yet the candidate genes still have a putative status. No GT has been purified and/or heterologously expressed to date in brown algae. The Brown Sugar project aims to functionally characterize brown algal GTs for the very first time. A multidisciplinary strategy will be used from gene analyses to protein isolation and activity screening.

In addition to a management task (WP4), the experimental work is structured in three work packages: WP1) we will locate native GTs and their associated proteins at subcellular levels. Fractionations will be made on S. latissima using sucrose gradients to gain distinct organelle fractions. The protein and GT content will be analyzed by proteomics; WP2) we will perform family-wise annotations of GTs based on sequence homology and we will heterologously express some of the candidate genes. This will use extensive genomic/transcriptomic datasets from brown algae, including that from S. latissima. Among our preliminary results we can mention our successful production of a GT64 in its soluble form from a closely related brown alga; WP3) we will determine the first GT activities from brown algae. Dedicated activated nucleotide-sugars will be chemically synthesized. They will bear a reporter to ease subsequent activity detection by a click-chemistry technique. Series of acceptors molecules are already available but this library will be extended. The donor/acceptor molecules will feed the activity tests for both GTs isolated in WP1 (enriched fractions) and WP2 (recombinant proteins).

The work program of this project is designed for 4 years for approximately 3.6 full time permanent researchers in total, further supported by 3 additional scientists (including one PhD student) to be recruited among the 4 Partner labs. Partner 1 (coordinator, WP2 leader) is the Marine Glycobiology group from the marine research center Station Biologique de Roscoff, France. It will coordinate the preparation of the subcellular fractions (WP1, Task1), the recombinant expression of the GTs (WP2, Task2), and the activity tests (WP3, Task2). Partner2 (WP1 leader) is the proteomics core facility Protim, Rennes, France. It will perform the protein identification and characterization of the subcellular fractions prepared by Partner1 (WP1, Task2). Partner 3 is the CAZy team, Marseille, France. It will perform expert annotations of the GTs (WP2, Task1). Partner 4 (WP3 leader) is from the chemistry teaching and research center ISCR, Rennes. It will produce a subset of labelled activated nucleotide sugars and acceptor molecules, with the priority to highlight FucT and ManAT activities (WP3, Task1).

The 1st biochemical characterization of GTs from brown algae will represent a significant breakthrough in the understanding of polysaccharide synthesis in these organisms. The Brown Sugar project could have important benefits in the field of the seaweed-based blue economy.