Vibrio 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 VejGH50A, VejGH50B, VejGH50C, and VejGH50D and the α-neoagarooligosaccharide (NAOS) hydrolase VejGH117, 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, VejGH50A, VejGH50B, and VejGH50C, 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, VejGH50D and α-NAOS hydrolase, VejGH117. Second, via the newly found alternative agarolytic pathway, NeoDP4 was depolymerized into AHG and agarotriose (AgaDP3) by VejGH117, and AgaDP3 was then completely depolymerized into monomeric sugars by sequential enzymatic reactions of the agarolytic β-galactosidase (ABG), VejABG and VejGH117. 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.

IMPORTANCE 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, Vibrio 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.

弧菌属。EJY3是一种可分解​​代谢3,6-脱水-升-半乳糖（AHG），琼脂糖的单体糖单元。尽管最近发现了EJY3中的AHG分解代谢途径，但EJY3的完整琼脂糖分解系统仍不清楚。我们确定了五种酶，即参与EJY3琼脂糖分解系统的β-琼脂糖Vej GH50A，Vej GH50B，Vej GH50C和Vej GH50D和α-新葡糖寡糖（NAOS）水解酶Vej GH117。基于重组酶的表征和细胞内代谢产物分析，我们发现EJY3通过两种不同的琼脂糖分解途径分解琼脂糖。在四个EJY3β-agaras中，Vej GH50A，Vej GH50B和Vej发现GH50C是细胞外琼脂酶，主要产生新琼脂四糖（NeoDP4）和新琼脂糖。通过在琼脂糖上生长的EJY3中检测细胞内NeoDP4，可以观察到NeoDP4被细胞摄取。有趣的是，细胞内NeoDP4充当了两个不同下游琼脂糖分解途径的分支点。首先，通过众所周知的琼脂糖分解途径，通过外切型β-琼脂糖酶Vej GH50D和α-NAOS水解酶Vej GH117将NeoDP4解聚为单体糖。第二，通过新发现的替代途径琼胶降解，NeoDP4被解聚到由AHG和agarotriose（AgaDP3）Vej GH117，和AgaDP3然后完全解聚到由琼胶降解β半乳糖苷酶的（ABG）顺序酶促反应单体糖，Vej ABG和Vej GH117。因此，通过实验验证琼脂糖分解酶的活性和将NeoDP4转运到EJY3细胞中，我们发现EJY3既具有已知途径，又具有涉及α-NAOS水解酶和ABG的新发现的替代途径。

重要信息琼脂糖是红色大型藻类的主要多糖，由半乳糖和3,6-脱水-升-半乳糖。许多海洋细菌拥有能够将琼脂糖解聚为低聚物，然后再将寡聚体解聚为单体的酶。在这里，我们通过实验验证了海洋细菌弧菌（Vibrio sp。）中存在着既已知的又是新型的琼脂糖分解途径。EJY3。在琼脂糖分解途径中，琼脂糖主要通过细胞外酶解聚为4糖单元低聚物，然后转运到细胞中。进口的低聚物在细胞内解聚为半乳糖和3,6-脱水-升-半乳糖通过两种不同的琼脂糖分解途径，使用细胞内酶的不同组合。这些结果阐明了海洋细菌在海洋中红色大藻类生物质的解聚路线，并为开发从红色大藻类高效生产糖的工业工艺提供了线索。