Plants produce many specific secondary metabolites as a response to environmental stress, especially biological stress. These compounds show strong biological activities and high stability against degradation by microbes and animals. Berberine, a benzylisoquinoline alkaloid, is found in many plant species and has strong antimicrobial activity, and is often included in traditional herbal medicines. We previously investigated how berberine is degraded in nature and we isolated two berberine-utilizing bacteria. In this study, we characterized the gene encoding the enzyme that degrades the 2,3-methylenedioxy ring of berberine; this ring is important for its activity and stability. Further characterization of several other berberine-utilizing bacteria and the genes encoding key demethylenation enzymes revealed that these enzymes are tetrahydrofolate dependent and similar to demethylation enzymes such as GcvT. Because the degradation of O-methyl groups or the methylenedioxy ring in phenolic compounds such as lignin, lignan and many other natural products, including berberine, is the key step for the catabolism of these compounds, our discovery reveals the common origin of the catabolism of these stable chemicals in bacteria.Plants produce many secondary metabolites to respond to environmental stress, especially biological stress. These compounds have strong biological activities and high stability against degradation by microbes and animals. Berberine (BBR) is a benzylisoquinoline alkaloid that is produced by various higher plants, such as Coptis japonica, Berberis fremontii and Mahonia aquifolium 1 , and shows strong antimicrobial and chemical defense activities 2 . BBR was also used as an antidiarrheal in ancient times, and interest in its use toward improving intestinal microflora is increasing 3, 4 . Although few studies have characterized the structure-activity relationship of protoberberine alkaloids, chemicals with a methylenedioxy ring, such as BBR, tend to demonstrate more activity than those with dimethoxy or hydroxy groups, such as palmatine 5,6 .The metabolism of BBR has been mainly investigated in mammals. CYP2D6, CYP1A2 and CYP3A4 are responsible for metabolizing BBR to demethyleneberberine (D-BBR) in human liver microsomes 7 . Interestingly, BBR was not metabolized by the intestinal bacteria of humans and rats in anaerobic conditions 8,9 . Liquid chromatography/time-of-flight mass spectrometry analysis of bile, plasma and urine in rats revealed the presence of BBR metabolites, including thalifendine, berberrubine, D-BBR, jatrorrhizine, palmatine, columbamine, 3,9-demethyl-palmatine, hydroxylated BBR and hydroxylated D-BBR; BBR was converted into sulfate and glucuronic acid conjugates for excretion 10 .There is currently a new opportunity to study the environmental fate of BBR: two bacteria, Sphingobium sp. strain BD3100 and Rhodococcus sp. strain BD7100, that utilize BBR as their sole carbon source were isolated from the sludge of a berberine-producing factory 11 . The formation of demethyleneberberine ...