A Novel Auxiliary Agarolytic Pathway Expands Metabolic Versatility in the Agar-Degrading Marine Bacterium Colwellia echini A3.

Marine microorganisms encode a complex repertoire of carbohydrate-active enzymes (CAZymes) for the catabolism of algal cell wall polysaccharides. While the core enzyme cascade for degrading agar is conserved across agarolytic marine bacteria, gain of novel metabolic functions can lead to the evolutionary expansion of the gene repertoire. Here, we describe how two less-abundant GH96 α-agarases harbored in the agar-specific polysaccharide utilization locus (PUL) of Colwellia echini strain A3 facilitate the versatility of the agarolytic pathway. The cellular and molecular functions of the α-agarases examined by genomic, transcriptomic, and biochemical analyses revealed that α-agarases of A3 create a novel auxiliary pathway. α-Agarases convert even-numbered neoagarooligosaccharides to odd-numbered agaro- and neoagarooligosaccharides, providing an alternative route for the depolymerization process in the agarolytic pathway. Comparative genomic analysis of agarolytic bacteria implied that the agarolytic gene repertoire in marine bacteria has been diversified during evolution, while the essential core agarolytic gene set has been conserved. The expansion of the agarolytic gene repertoire and novel hydrolytic functions, including the elucidated molecular functionality of α-agarase, promote metabolic versatility by channeling agar metabolism through different routes. A3 is an example of how the gain of gene(s) can lead to the evolutionary expansion of agar-specific polysaccharide utilization loci (PUL). A3 encodes two α-agarases in addition to the core β-agarolytic enzymes in its agarolytic PUL. Among the agar-degrading CAZymes identified so far, only a few α-agarases have been biochemically characterized. The molecular and biological functions of two α-agarases revealed that their unique hydrolytic pattern leads to the emergence of auxiliary agarolytic pathways. Through the combination of transcriptomic, genomic, and biochemical evidence, we elucidate the complete α-agarolytic pathway in A3. The addition of α-agarases to the agarolytic enzyme repertoire might allow marine agarolytic bacteria to increase competitive abilities through metabolic versatility.