Phylogenomic and multi-omics analysis of Agarivorans sp. D1326 uncovers an agarolytic pathway involving rare GH118 β‑agarase

Agar, a key polysaccharide in marine red algae, offers a sustainable biomass resource. In this study, phylogenetic and phylogenomic analyses classified the agarolytic marine bacterium strain D1326 ^T , isolated from the Yellow Sea, as a member of the genus Agarivorans . This work elucidates the comprehensive agarolytic network of Agarivorans sp D1326 by multi-omics. Genomic analysis revealed a diverse repertoire of agarase genes, including glycoside hydrolase (GH) members from the GH16, GH50, GH86, and GH118 families. Transcriptomic and proteomic profilings demonstrated the strong co-upregulation and predominant extracellular secretion of these enzymes upon agarose induction. Three key secreted β -agarases—GH86 (Aga1002), GH118 (Aga2820), and GH16 (Aga2799)—were identified as primary initiators, showing extraordinary extracellular upregulation (443.97-fold, 303.7-fold, and 299.69-fold, respectively) and likely establishing distinct pathways for producing various neoagarooligosaccharides. The initial cleavage by the Aga2820 into neoagarooctaose/decaose, which then serve as substrates for further degradation by GH16, GH50, and GH86 agarases. GH50-family enzymes, exhibiting dual localization, facilitate supplementary degradation both outside and inside the cell. A notable finding is the extracellular detection of key metabolic enzymes—neoagarobiose hydrolase and the AHG-dehydrogenase/cycloisomerase—despite their lack of signal peptides. This suggests non-classical secretion and points to a previously unrecognized capacity for partial extracellular processing of AHG into 3,6-anhydro-L-galactonate, a central metabolic intermediate. The proposed agarolytic pathway integrates extracellular polysaccharide depolymerization, oligosaccharide transport, and intra- and extracellular sugar metabolism. This work provides the first comprehensive elucidation of a native bacterial agarolytic network incorporating a rare GH118 β -agarase, offering fundamental insights for the enzymatic valorization of agar.