Akkermansia muciniphila has evolved to specialize in the degradation and utilization of host mucus, which it may use as the sole source of carbon and nitrogen. Mucus degradation and fermentation by A. muciniphila are known to result in the liberation of oligosaccharides and subsequent production of acetate, which becomes directly available to microorganisms in the vicinity of the intestinal mucosa. Coculturing experiments of A. muciniphila with non-mucus-degrading butyrate-producing bacteria Anaerostipes caccae, Eubacterium hallii, and Faecalibacterium prausnitzii resulted in syntrophic growth and production of butyrate. In addition, we demonstrate that the production of pseudovitamin B12 by E. hallii results in production of propionate by A. muciniphila, which suggests that this syntrophy is indeed bidirectional. These data are proof of concept for syntrophic and other symbiotic microbe-microbe interactions at the intestinal mucosal interface. The observed metabolic interactions between A. muciniphila and butyrogenic bacterial taxa support the existence of colonic vitamin and butyrate production pathways that are dependent on host glycan production and independent of dietary carbohydrates. We infer that the intestinal symbiont A. muciniphila can indirectly stimulate intestinal butyrate levels in the vicinity of the intestinal epithelial cells with potential health benefits to the host.

A sensitive and selective method is needed to analyse in situ produced vitamin B12 in plant-based materials, potential new dietary sources of vitamin B12. A UHPLC/UV method was developed and validated for the determination of human active vitamin B12 in cell extracts of Propionibacterium freudenreichii subsp. shermanii and after immunoaffinity purification in extracts of cereal matrices fermented by P. freudenreichii. An Acquity HSS T3 C18 column resulted in a baseline separation, a calibration curve of excellent linearity and a low limit of detection (0.075 ng/5 μL injection). As confirmed by UHPLC-MS, the active vitamin B12 could be separated from pseudovitamin B12. The recovery of vitamin B12 from purified spiked cereal matrices was good (>90%; RSD<5%). A nutritionally relevant amount of active vitamin B12 was produced by P. freudenreichii in cereal malt matrices (up to 1.9 μg/100 g) in 24h at 28 °C.

SummaryIntestinimonas is a newly described bacterial genus with representative strains present in the intestinal tract of human and other animals. Despite unique metabolic features including the production of butyrate from both sugars and amino acids, there is to date no data on their diversity, ecology, and physiology. Using a comprehensive phylogenetic approach, Intestinimomas was found to include at least three species that colonize primarily the human and mouse intestine. We focused on the most common and cultivable species of the genus, Intestinimonas butyriciproducens, and performed detailed genomic and physiological comparison of strains SRB521 T and AF211, isolated from the mouse and human gut respectively. The complete 3.3-Mb genomic sequences of both strains were highly similar with 98.8% average nucleotide identity, testifying to their assignment to one single species. However, thorough analysis revealed significant genomic rearrangements, variations in phage-derived sequences, and the presence of new CRISPR sequences in both strains. Moreover, strain AF211 appeared to be more efficient than strain SRB521 T in the conversion of the sugars arabinose and galactose. In conclusion, this study provides genomic and physiological insight into Intestinimonas butyriciproducens, a prevalent butyrate-producing species, differentiating strains that originate from the mouse and human gut.

Propionibacterium freudenreichii is the only generally recognized safe (GRAS) bacterium known to synthesize active vitamin B12 and offers previously untapped potential for naturally fortifying foods with vitamin B12. Biosynthesis of the lower ligand 5,6-dimethylbenizimidazole (DMBI) is often a key limiting factor in the production of active vitamin B12 in Propionibacteria. Here, we studied the effect of the natural food-grade precursors of DMBI [riboflavin (RF) and nicotinamide (NAM)] on vitamin B12 production by 27 P. freudenreichii and 3 Propionibacterium acidipropionici strains in whey-based medium. We employed sensitive and selective UHPLC and LC-MS/MS to confirm and quantify the synthesized vitamin B12. In 12 P. freudenreichii strains, co-supplementation with RF (40 µM) and NAM (27 mM) increased the volumetric yield of vitamin B12 up to 4-fold compared to the control cultures. For the majority of these strains, the production level with RF and NAM exceeded the yield obtained with DMBI supplementation (100 µM). The significant positive correlation between RF consumption and vitamin B12 production suggests that RF is proportionally directed towards vitamin B12 biosynthesis. This study shows that the availability of RF and NAM enhances the production of active vitamin B12 by P. freudenreichii in a strain-dependent manner.

BackgroundPropionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12. Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B12 by microbial fermentation. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Genomic data indicate that P. freudenreichii possesses a fusion gene, bluB/cobT2, coding for a predicted phosphoribosyltransferase/nitroreductase, which is presumably involved in production of vitamin B12. Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B12 production properties.ResultsHere, we investigated the activity of heterologously expressed and purified fusion enzyme BluB/CobT2. Our results show that BluB/CoBT2 is responsible for the biosynthesis of the DMBI base and its activation into α-ribazole phosphate, preparing it for attachment as the lower ligand of cobalamin. The fusion enzyme was found to be efficient in metabolite channeling and the enzymes’ inability to react with adenine, a lower ligand present in the pseudovitamin, revealed a mechanism favoring the production of the active form of the vitamin. P. freudenreichii did not produce cobalamin under strictly anaerobic conditions, confirming the requirement of oxygen for DMBI synthesis. In vivo experiments also revealed a clear preference for incorporating DMBI over adenine into cobamide under both microaerobic and anaerobic conditions.ConclusionsThe herein described BluB/CobT2 is responsible for the production and activation of DMBI. Fusing those two activities results in high pressure towards production of the true vitamin B12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway of P. freudenreichii. The GRAS organism status and the preference for synthesizing active vitamin form make P. freudenreichii a unique candidate for the in situ production of vitamin B12 within food products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0363-9) contains supplementary material, which is available to authorized users.