Identification of the enantiomeric nature of 2-keto-3-deoxy-galactonate in the catabolic pathway of 3,6-anhydro-L-galactose.

A novel metabolic pathway of 3,6-anhydro-L-galactose (L-AHG), the main sugar component in red macroalgae, was first discovered in the marine bacterium Vibrio sp. EJY3. L-AHG is converted to 2-keto-3-deoxy-galactonate (KDGal) in two metabolic steps. Here, we identified the enantiomeric nature of KDGal in the L-AHG catabolic pathway via stereospecific enzymatic reactions accompanying the biosynthesis of enantiopure L-KDGal and D-KDGal. Enantiopure L-KDGal and D-KDGal were synthesized by enzymatic reactions derived from the fungal galacturonate and bacterial oxidative galactose pathways, respectively. KDGal, which is involved in the L-AHG pathway, was also prepared. The results obtained from the reactions with an L-KDGal aldolase, specifically acting on L-KDGal, showed that KDGal in the L-AHG pathway exists in an L-enantiomeric form. Notably, we demonstrated the utilization of L-KDGal by Escherichia coli for the first time. E. coli cannot utilize L-KDGal as the sole carbon source. However, when a mixture of L-KDGal and D-galacturonate was used, E. coli utilized both. Our study suggests a stereoselective method to determine the absolute configuration of a compound. In addition, our results can be used to explore the novel L-KDGal catabolic pathway in E. coli and to construct an engineered microbial platform that assimilates L-AHG or L-KDGal as substrates. KEY POINTS: • Stereospecific enzyme reactions were used to identify enantiomeric nature of KDGal • KDGal in the L-AHG catabolic pathway exists in an L-enantiomeric form • E. coli can utilize L-KDGal as a carbon source when supplied with D-galacturonate.

Metabolic and enzymatic elucidation of cooperative degradation of red seaweed agarose by two human gut bacteria.

Various health beneficial outcomes associated with red seaweeds, especially their polysaccharides, have been claimed, but the molecular pathway of how red seaweed polysaccharides are degraded and utilized by cooperative actions of human gut bacteria has not been elucidated. Here, we investigated the enzymatic and metabolic cooperation between two human gut symbionts, Bacteroides plebeius and Bifidobacterium longum ssp. infantis, with regard to the degradation of agarose, the main carbohydrate of red seaweed. More specifically, B. plebeius initially decomposed agarose into agarotriose by the actions of the enzymes belonging to glycoside hydrolase (GH) families 16 and 117 (i.e., BpGH16A and BpGH117) located in the polysaccharide utilization locus, a specific gene cluster for red seaweed carbohydrates. Then, B. infantis extracted energy from agarotriose by the actions of two agarolytic ß-galactosidases (i.e., Bga42A and Bga2A) and produced neoagarobiose. B. plebeius ultimately acted on neoagarobiose by BpGH117, resulting in the production of 3,6-anhydro-L-galactose, a monomeric sugar possessing anti-inflammatory activity. Our discovery of the cooperative actions of the two human gut symbionts on agarose degradation and the identification of the related enzyme genes and metabolic intermediates generated during the metabolic processes provide a molecular basis for agarose degradation by gut bacteria.

Characterization of BpGH16A of Bacteroides plebeius, a key enzyme initiating the depolymerization of agarose in the human gut.

Seaweeds have received considerable attention as sources of dietary fiber and biomass for manufacturing valuable products. The major polysaccharides of red seaweeds include agar and porphyran. In a marine environment, marine bacteria utilize agar and porphyran through the agarase and porphyranase genes encoded in their genomes. Most of these enzymes identified and characterized so far originate from marine bacteria. Recently, Bacteroides plebeius, a human gut bacterium isolated from seaweed-eating Japanese individuals, was revealed to contain a polysaccharide utilization locus (PUL) targeting the porphyran and agarose of red seaweeds. For example, B. plebeius contains an endo-type ß-agarase, BpGH16A, belonging to glycoside hydrolase family 16. BpGH16A cleaves the ß-1,4-glycosidic linkages of agarose and produces neoagarooligosccharides from agarose. Since it is crucial to study the characteristics of BpGH16A to understand the depolymerization pathway of red seaweed polysaccharides by B. plebeius in the human gut and to industrially apply the enzyme for the depolymerization of agar, we characterized BpGH16A for the first time. According to our results, BpGH16A is an extracellular endo-type ß-agarase with an optimal temperature of 40 °C and an optimal pH of 7.0, which correspond to the temperature and pH of the human colon. BpGH16A depolymerizes agarose into neoagarotetraose (as the main product) and neoagarobiose (as the minor product). Thus, BpGH16A is suggested to be an important enzyme that initiates the depolymerization of red seaweed agarose or agar in the human gut by B. plebeius. KEY POINTS: • Bacteroides plebeius is a human gut bacterium isolated from seaweed-eating humans. • BpGH16A is an extracellular endo-type ß-agarase with optimal conditions of 40 °C and pH 7.0. • BpGH16A depolymerizes agarose into neoagarotetraose and neoagarobiose.