2.5.1. Polysaccharide-Degrading GH Systems

A closer inspection of the CAZyme genomic repertoires for four *Zobellia* species (Figure 5 and Table S1) revealed representatives of some GH families targeting red and brown algal polysaccharides, namely four (*Z. amurskyensis* and *Z. laminariae*) to 14 (*Z. galactanivorans* and *Z. uliginosa*) GH16 enzymes, including β-agarases, β-porphyranases, laminarinases and κ-carrageenases; one (*Z. uliginosa*) to two (*Z. galactanivorans*) GH64 laminarinases; one (*Z. laminariae*) to three

(*Z. galactanivorans* and *Z. uliginosa*) GH82 ι-carrageenases; six to seven (*Z. galactanivorans*, *Z. laminariae*, and *Z. uliginosa*) to eight to nine (strains of *Z. amurskyensis*) GH117 α-1,3-(3,6-anhydro)-L-galactosidases; two to three (*Z. amurskyensis* and *Z. laminariae*) to four (*Z. galactanivorans* and *Z. uliginosa*) GH127 α-1,3-(3,6-anhydro)-D-galactosidases. All five *Zobellia* genomes encode for one GH129 α-1,3-(3,6-anhydro)-D-galactosidase, and only *Z. laminariae* has one enzyme assigned to GH50 β-agarase. No representatives from the other agarolytic enzymes GH86, GH96, GH118, or GH150 were identified.

Previously, *Z. galactanivorans* has been extensively investigated in degradation of various algal polysaccharides through genomic and transcriptomic analysis combined with computer modeling and experimental validation [30,51–54]. Therefore, the majority of key genes of agar, laminarin, and carrageenan utilization systems of *Z. galactanivorans* can serve as reference sequences for the annotation of hydrolytic enzymes from other*Zobellia* genomes. Our analysis showed that the genomes of *Z. galactanivorans* and*Z. uliginosa* shared the largest reservoir of agarolytic genes among*Zobellia* genomes. Their polysaccharide-degrading systems were represented by GH16 enzymes, including four to five β-porphyranases PorA-E, four to five β-agarases AgaA-D, three to four laminarinases LamA-D, and one κ-carrageenases CgkA. Based on the phylogenetic analysis (Figure S1) of the GH16 catalytic module, the enzymatic systems of other *Zobellia* species were represented by only PorD (Zam\_1698, Zmar\_1649, and Zlam\_2939), PorB (Zam\_2877, Zmar\_2570, and absent in *Z. laminariae*), AgaC (Zam\_3480, Zmar\_956, and absent in *Z. laminariae*), AgaB (Zam\_3011, Zmar\_1702, and Zlam\_2991), and LamB (Zlam\_4246, absent in *Z. amurskyensis*). The genome of *Z. laminariae* also codes Zlam\_2677 as a new putative GH16 subfamily, which occupies an intermediate position on the tree, between the branches CgkA and LamA. The orthologues genes for AgaA and AgaD, as well as for PorA and PorC, which encode secreted enzymes responsible for the initial attack on agars and porphyrans [51,55], were absent in both *Z. amurskiensis* and *Z. laminariae* genomes. Therefore, PorD, PorB, AgaC, and AgaB, as well as LamB are the genus-specific GH16 enzymes, potentially possessing broader substrate specificities. It has been recently shown that *Z. galactanivorans* AgaC, defined as a new GH16 subfamily, can hydrolyze not only agarose, but also complex agars [56]. Interestingly, the *Z. uliginosa* genome encodes two strongly different AgaC sequences, classical AgaC Zuli\_2505 and AgaC-like Zuli\_8, which can be of a great biotechnological interest because it is a new β-agarase. Therefore, the *Zobellia* β-agarases, which play a key role in agar depolymerization with the release of a range of neoagarooligosaccharides, are likely to be considered for use in industrial and biotechnological applications.

The *Zobellia* genomes contained the multigenic GH117 family coding exolytic 3,6 anhydro-α-L-galactosidases, which cleave neoagarooligosaccharides and produce L-AHG, and therefore perform a key role in terminal steps of polysaccharide saccharification. Previously, the products of some GH117 genes of *Z. galactanivorans* (Zga\_4663 (ZgAhgA), Zga\_3615, and Zga\_3597) were biochemically and structurally characterized [57]. The multigenic GH117 families consisted of six (*Z. galactanivorans*), seven (*Z. uliginosa* and *Z. laminariae*) or eight (*Z. amurskiensis*) genes. Our phylogenetic analysis (Figure S2) is in agreement with the previously obtained GH117 tree [58], with the exception of the additional clades formed by GH117 of *Z. amurskiensis* strains: Clade 8 (ZamT\_1387 and ZamMar\_2539) and Clade 9 (ZamT\_1385 and ZamMar\_2537). We consider these additional clades of GH117 enzymes to reflect new enzymatic specificities.

Analysis of the genomic regions around GH16 and GH117 genes revealed a number of potential GH2 β-galactosidase genes. Recently, a novel agarolytic GH2 β-galactosidase has been found in the marine bacterium *Vibrio* sp. EJY3 [59]. Therefore, we suggested that these GH2s might be exo-β-1,4-galactosidases, removing galactose at the non-reducing end of agarooligosaccharides. Previously, a similar genomic sequence containing several GH2s, GH16s, and GH117s was identified as a putative agarolytic cluster in the human intestinal bacterium *Bacteroides uniformis* Bu NP1 [60]. It was suggested that the products of GH16s were cyclically degraded into monosaccharides by the coordinated work of GH117B and GH2C, respectively.

Since there is a demand for highly specific agarolytic enzymes, the investigation of multigenic families encoding enzymes with slightly different activities and specificities may be the best solution for production of valuable oligosaccharides and rare monomers with different bioactivities or applications.
