Genome Mining and Functional Characterization of Agarases from a Novel Vibrio Species

Marine bacteria produce diverse carbohydrate-active enzymes (CAZymes), which include agarases that cleave glycosidic bonds in agars (agarans). Agars, sulfated polysaccharides that mostly make up the cell walls and intercellular matrix in many species of red algae, are used in various industrial applications. The discovery of novel agarases is thus of biological and biotechnological interest. In this study, the CAZymes and, in particular, the agarases of a seaweed-associated Vibrio sp. isolate, an agarolytic bacterium isolated from a macroalga, were characterized using genomic and phenotypic approaches. A draft genome assembly of Vibrio sp. A37 was generated using high-throughput short-read sequencing. The draft genome assembly comprised approximately 5 megabases (Mb) organized into 78 contigs, with an N50 of 381 kilobases (kb), GC content of 41%, and a read coverage of 209x. Genome-based comparisons show that strain A37 exhibits substantial divergence from its nearest described species, Vibrio marisflavi, with values of 78.93% ANI, 86.89% AAI, and 22.4% dDDH-well below the thresholds generally accepted for species delineation. Together with corroborating phenotypic data, these observations support the recognition of strain A37 as a novel genomospecies, for which we propose the provisional name 'Vibrio bolinaoensis'. Analysis of the carbohydrate-active enzyme (CAZyme) repertoire revealed diverse CAZyme classes, including five β-agarases-four of which form a single CAZyme gene cluster (CGC). Further genomic analysis highlighted the isolate's potential to degrade and utilize agarose, fucoidan, and alginate. Two β-agarase genes, Ag46 (a member of glycosyl hydrolase family 16 (GH16)) and Ag48 (GH50), were successfully expressed in Escherichia coli using the pCri-18a vector, originally designed for Bacillus subtilis. This led to the serendipitous discovery of the utility of this expression vector to enable secretion and extracellular activity of the agarases, and potentially other CAZymes, in E. coli, underscoring the fundamental mechanistic compatibility between the Sec-dependent translocation pathways of Gram-positive and Gram-negative bacteria.