Degradation mechanism of difructose dianhydride III in Blautia species

Di-fructofuranose 1,2':2,3' dianhydride (DFA-III) is a cyclic fructo-disaccharide, which is produced by the condensation of two fructose molecules via the caramelization or enzymatic reaction of inulin fructotransferase. A strain of Blautia producta was known to utilize DFA-III as a carbohydrate source; however, the mechanisms remain unclear. In this study, we characterized the glycoside hydrolase (GH) family 91 DFA-III hydrolase (DFA-IIIase) from B. parvula NBRC 113351. Recombinant BpDFA-IIIase catalyzed the reversible conversion of DFA-III to inulobiose, which is further degraded to fructose by the cooperative action of DFA-IIIase and GH32 β-D-fructofuranosidase. DFA-III was utilized in several Blautia species with a gene cluster for DFA-III degradation (e.g., B. parvula NBRC 113351, B. hydrogenotrophica JCM 14656, and B. wexlerae JCM 35486), but not by B. wexlerae JCM 31267, which does not possess the gene cluster. Furthermore, B. hansenii JCM 14655, which cannot metabolize fructose, could not utilize DFA-III; however, it could degrade DFA-III to fructose in the presence of DFA-III-degrading enzymes. Fecal fermentation tests showed that Blautia species are important gut microbe for degrading DFA-III. KEY POINTS: • BpDFA-IIIase is the first characterized DFA-IIIase in intestinal non-pathogenic bacteria. • DFA-IIIase is widely conserved in Blautia species. • DFA-III is degraded to d-fructose through inulobiose by the cooperative action of DFA-IIIase and β-d-fructofuranosidase.