*3.2. Genome Annotation*

Gene prediction and automated genome annotation were carried out using Rapid Annotation using Subsystem Technology (RAST) v. 2.0 with default parameters [37–39] followed by manual curation of the some annotations by comparing translated sequences with the NCBI non-redundant database, InterPro (https://www.ebi.ac.uk/interpro/), and Pfam (https://pfam.xfam.org/) databases. For more accurate annotation of carbohydrate-active enzymes, their classification into existing CAZy families and identification of a CAZome repertoire of *Zobellia* genus were performed using the dbCAN2 meta server (http://cys.bios.niu.edu/dbCAN2) [43].

## *3.3. Phylogenetic, PhylogenomicAnalyses, and Comparative Genomics*

Phylogenetic analysis of 16S rRNA gene sequences, also members of GHs and AAs from *Zobellia* species, was performed using the NJ [34] method with bootstrap supporting of 1000 replicates in MEGA v.6.06 [69]. Phylogenomic measures were calculated with the JSpecies Web Server [35] to determine ANI values and Tetra signatures. Genome-wide analysis of orthologous clusters and gene ontology analysis among all predicted protein-coding genes was performed using OrthoVenn2 (https://orthovenn2.bioinfotoolkits.net/home) [42].

#### *3.4. Deposition of the Nucleotide Sequence Accession Number*

The whole-gGenome shotgun sequences of *Z. amurskyensis* KMM 3526<sup>Т</sup> and *Z. laminariae* KMM 3676<sup>Т</sup> have been deposited at DDBJ/ENA/GenBank under the accessions RCNR00000000 and RCNS00000000, respectively. The versions described in this paper are RCNR01000000 and RCNS01000000.

## **4. Conclusions**

Today, some of the most eco-friendly methods for obtaining algal polysaccharides and their derivatives are enzyme-based techniques. Therefore, the search for marine bacteria specialized in the degradation of various polysaccharides is of particular interest. The marine flavobacterium *Z. galactanivorans* DsijT is a model organism for polysaccharide degradation investigation among marine flavobacteria. However, little has been known about the genomic basics of hydrolytic potential of the *Zobellia* genus. To determine the CAZyme content at the species and genus taxonomic levels, we performed genome sequencing of two type *Zobellia* strains and comparative genomic analysis. We identified a relatively high proportion of CAZymes in the genomes of five *Zobellia* strains. Our comparative study strongly suggests a specialization of the *Zobellia* genus in the algal polysaccharide degradation. These microorganisms can be used as both strain-degraders and valuable sources of novel enzymes for potential application in biotechnology, food, and medical industries.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/12/661/s1, Table S1: Repertoire of CAZymes of the *Zobellia* genus; Figure S1: Phylogenetic tree of *Zobellia* GH16 proteins; Figure S2: Phylogenetic tree of *Zobellia* GH117 proteins; Figure S3: Multiple amino acid sequence alignment of *S. maltophilia*, *A. tumefaciens*, *S. multivorum*, and *Zobellia* strain AA3 enzymes (GMC-oxidoreductases); Figure S4: Phylogenetic tree of *Zobellia* AA3 proteins and *S. maltophilia*, *A. tumefaciens*, *S. multivorum* FAD-GOs. Table S2: Comparison of amino acid identities (%) of *Zobellia* AA3 enzymes with *S. maltophilia*, *A. tumefaciens*, *S. multivorum* characterized FAD-GOs.

**Author Contributions:** Conceptualization, M.I.; Data curation, N.C. and M.I.; Funding acquisition, M.I.; Investigation, N.C., E.B., I.G. and O.N.; Methodology, A.S. and O.N.; Software, N.C.; Supervision, M.I.; Writing—original draft, N.C. and E.B.; Writing—review & editing, M.I.

**Funding:** This work was partially supported by the Russian Science Foundation under grant 17-14-01065.

**Acknowledgments:** The authors are grateful to Alexandra Litavrina for the revision of the English text and three anonymous reviewers for helping improve this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
