The biosynthetic pathway of laminarin is essentially unknown, but we identified several genes which are likely to be involved in this metabolism in the Ectocarpus genome. This seaweed contains two cytosolic isoforms of UDP-glucose pyrophosphorylase (UGP, Esi0144_0004 and Esi0430_0005), supporting the assumption that UDP-glucose is the activated sugar needed for the production of laminarin. Beta-1,3-glucan synthases fall into two different GT families: the GT2, a polyspecific family which includes bacterial β-1,3-glucan synthases, and the GT48, which only contains eukaryotic β-1,3-glucan synthases (Cantarel et al., 2009). Ectocarpus harbors 11 GT2 homologous to cellulose synthases. Phylogenetic analysis confirms that these enzymes are not significantly related to bacterial β-1,3-glucan synthases (Michel et al., 2010). By contrast, the three members of the GT48 family (Esi0033_0138, Esi0193_0029 and Esi0338_0032) display significant similarities with plant callose synthases (c. 35% sequence identity). The phylogenetic tree of the GT48 family (Fig. 4) is congruent with the currently accepted phylogeny of the Eukaryotes (Fig. 1), with the β-1,3-glucan synthases from plants, fungi, Apicomplexa and Stra-menopiles consistently emerging as distinct clades. The putative β-1,3-glucan synthases of Stramenopiles are further divided into three groups. Clade A, which includes Esi0338_0032, is common to brown algae, diatoms and Oomycetes, suggesting that these glycosyltransferases are responsible for the polymerization of the backbones of laminarin, chrysolaminarin and mycolaminarin, respectively. The β-1,3-glucan synthases of clade B are only found in Phytophthora, and therefore they are likely to be involved in the biosynthesis of the Oomycete cell wall β-1,3-glucans. Clade C is unique to brown algae, but the exact role of Esi0033_0138 and Esi0193_0029 is unclear. These β-1,3-glucan synthases could specifically catalyze the production of laminarin M-series, which is a distinctive feature of brown algae (Read et al., 1996). Alternatively, they might be involved in callose biosynthesis, this molecule having been found in the sieve plates of Laminariales (Parker & Huber, 1965). In addition, Ectocarpus possesses two proteins (Esi0100_0034 and Esi0243_0020) which are homologous to KRE6, a GH16 family transglycosylase involved in β-1,6-branching of cell wall β-1,3-glucans in yeasts (Montijn et al., 1999). Therefore, these two proteins represent good candidates for the synthesis of β-1,6-linked branches of laminarin. This hypothesis is strengthened by the conservation of KRE6-like proteins in diatoms and Oomycetes. Remarkably, the KRE6-like protein PITG_03335 from P. infestans (Haas et al., 2009) is fused to a GT48 family β-1,3-glucan synthase. This GT48 module belongs to the clade A (Fig. 4), the very subgroup that we suggest to be responsible for laminarin polymerization.
The degradation of laminarin is potentially catalyzed by 10 endo-1,3-β-glucanases belonging to three different families (GH16, four genes; GH17, one gene; GH81, five genes), and by two exo-1,3-beta-glucanases (family GH5). These numerous laminarinases have homologs in bacteria (family GH16), fungi (families GH5 and GH81) and plants (family GH17), underlining the complexity of laminarin metabolism in brown algae. Laminarin oligosaccharides would be further hydrolyzed by β-glucosidases of the GH1 (Esi0061_0010, Esi0176_0045 and Esi0212_0019) or GH3 families (Esi0010_0226). The end-product, glucose, would be subsequently phosphorylated by a glucokinase (Esi0000_0270) before entering glycolysis.