Molecular and enzymatic verification of the dual agarolytic pathways in a marine bacterium, sp. strain EJY3.

sp. EJY3 is an agarolytic marine bacterium that catabolizes 3,6-anhydro-l-galactose (AHG), a monomeric sugar unit of agarose. While the AHG catabolic pathway in EJY3 has been recently discovered, the complete agarolytic system of EJY3 remains unclear. We have identified five enzymes, namely the β-agarases GH50A, GH50B, GH50C, and GH50D and the α-neoagarooligosaccharide (NAOS) hydrolase GH117, involved in the agarolytic system of EJY3. Based on the characterization of recombinant enzymes and intracellular metabolite analysis, we found that EJY3 catabolizes agarose via two different agarolytic pathways. Among the four EJY3 β-agarases, GH50A, GH50B, and GH50C, were found to be extracellular agarases, producing mainly neoagarotetraose (NeoDP4) and neoagarobiose. By detecting intracellular NeoDP4 in EJY3 grown on agarose, NeoDP4 was observed being taken up by cells. Intriguingly, intracellular NeoDP4 acted as a branching point for the two different downstream agarolytic pathways. First, via the well-known agarolytic pathway, NeoDP4 was depolymerized into monomeric sugars by the exo-type β-agarase, GH50D and α-NAOS hydrolase, GH117. Second, via the newly found alternative agarolytic pathway, NeoDP4 was depolymerized into AHG and agarotriose (AgaDP3) by GH117, and AgaDP3 was then completely depolymerized into monomeric sugars by sequential enzymatic reactions of the agarolytic β-galactosidase (ABG), ABG and GH117. Therefore, by experimentally verifying agarolytic enzymatic activity and transport of NeoDP4 into EJY3 cells, we revealed that EJY3 possesses both the known pathway and a newly discovered alternative pathway that involves α-NAOS hydrolase and ABG. Agarose is the main polysaccharide of red macroalgae and is composed of galactose and 3,6-anhydro-l-galactose. Many marine bacteria possess enzymes capable of depolymerizing agarose into oligomers and then depolymerizing the oligomers into monomers. Here, we experimentally verified that both a well-known and a novel agarolytic pathway exist in a marine bacterium, sp. EJY3. In agarolytic pathways, agarose is depolymerized mainly into 4-sugar-unit oligomers by extracellular enzymes, which are then transported into cells. The imported oligomers are intracellularly depolymerized into galactose and 3,6-anhydro-l-galactose by two different agarolytic pathways, using different combinations of intracellular enzymes. These results elucidate the depolymerization routes of red macroalgal biomass in the ocean by marine bacteria and provide clues for developing industrial processes for efficiently producing sugars from red macroalgae.