Identification and cloning of the first O -demethylase gene of isoflavones from Bifidobacterium breve INIA P734

Biochanin A and formononetin are methylated isoflavones, abundant in legumes, which can be converted in genistein and daidzein by means of an O -demethylation reaction. Our objective was to transform these methylated isoflavones into higher biological activity and bioavailability compounds, such as daidzein and genistein. Prior screening, using 10 lactic acid bacteria (LAB) and 3 bifidobacteria strains, revealed that only 7 lactobacilli strains were able to transform biochanin A into genistein, while none of the strains demethylated formononetin. As the dmt 734 gene from Bifidobacterium breve INIA P734 was putatively annotated as O- demethylase, it was cloned in a vector expressing antibiotic resistance (pNZ:TuR.dmt734) as well as in a food grade vector (pLEB590.dmt734), which were subsequently transformed in LAB and bifidobacteria O- demethylase lacking strains. Those recombinant strains harbouring pNZ:TuR.dmt734, or pLEB590.dmt734, managed to transform biochanin A into genistein, but did not transform formononetin into daidzein. These results confirm the demethylation specificity of isoflavones and the biotechnological interest of B. breve INIA P734 O -demethylase, as well as the food grade strains harbouring O -demethylase activity, which would be of value for the development of fermented vegetable-based foods enriched in genistein.