Isolation, genomic characterization, and safety assessment of an O -desmethylangolensin-producing Clostridium beijerinckii strain from Chinese Stinky Tofu

ABSTRACT The health benefits of dietary soy isoflavones are largely mediated by specific microbial metabolites, such as O -desmethylangolensin ( O -DMA). However, the diversity and application potential of O -DMA-producing strains remain poorly explored, primarily due to the limited availability of isolated strains, narrow ecological sources, and a lack of practical applications. In this study, an O -DMA-producing bacterium, designated strain FRJF5, was isolated from Chinese stinky tofu under anaerobic conditions and was identified as Clostridium beijerinckii . The biosynthesized O -DMA exhibited an enantiomeric excess (e.e.) of 78.6%. Based on phylogenetic and average nucleotide identity analyses against 235 public C. beijerinckii genomes, the clustering of FRJF5 with strains from diverse habitats—including industrial fermentation settings, animal feces, and soil—highlights the broad ecological diversity within this species. Functional gene mining and intra-species comparative genomics revealed a unique flavonoid metabolism gene cluster in FRJF5. Using apigenin as a representative flavonoid, we confirmed the successful conversion to 3-(4-hydroxyphenyl)-propionic acid. Moreover, the strain was predicted and verified to possess a substantial butyrate-producing capacity. Genomic screening for virulence or antibiotic resistance genes, combined with phenotypic tests (hemolysis, antibiotic susceptibility, and mouse gavage), revealed a favorable safety profile for strain FRJF5. Finally, intervention experiments in a mouse model of colitis supported its potential in alleviating the disease. Collectively, this study identifies C. beijerinckii FRJF5 as a strain capable of simultaneously producing O -DMA and butyrate, highlighting its potential for future applications in functional foods. IMPORTANCE Soy isoflavones require gut bacterial conversion into bioactive metabolites—such as the anti-inflammatory compound O -desmethylangolensin ( O -DMA)—to exert health benefits. Yet O -DMA-producing strains remain scarce, largely confined to fecal sources, and poorly characterized. Here, we isolated Clostridium beijerinckii FRJF5 from Chinese stinky tofu, an unexplored ecological niche. This strain not only produces enantiomerically enriched O -DMA but also co-produces butyrate, a metabolite known to strengthen gut barrier function. Genomic mining uncovered a unique flavonoid metabolism gene cluster responsible for this dual activity. Combined with favorable safety profiles, FRJF5 emerges as a strong candidate for functional food applications. This work expands the known diversity of O -DMA producers and bridges traditional fermented foods with next-generation probiotic development.