We found that Lactobacillus reuteri CRL1098, a lactic acid bacterium isolated from sourdough, is able to produce cobalamin. The sugar-glycerol cofermentation in vitamin B 12 -free medium showed that this strain was able to reduce glycerol through a well-known cobalamin-dependent reaction with the formation of 1,3-propanediol as a final product. The cell extract of L. reuteri corrected the coenzyme B 12 requirement of Lactobacillus delbrueckii subsp. lactis ATCC 7830 and allowed the growth of Salmonella enterica serovar Typhimurium (metE cbiB) and Escherichia coli (metE) in minimal medium. Preliminary genetic studies of cobalamin biosynthesis genes from L. reuteri allowed the identification of cob genes which encode the CobA, CbiJ, and CbiK enzymes involved in the cobalamin pathway. The cobamide produced by L. reuteri, isolated in its cyanide form by using reverse-phase high-pressure liquid chromatography, showed a UV-visible spectrum identical to that of standard cyanocobalamin (vitamin B 12 ).

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We have reported previously on the ability of Lactobacillus reuteri to produce a compound with vitamin B 12 activity. Here we report on the chemical characterisation of this corrinoid-like molecule. High performance liquid chromatography coupled to an ultraviolet diode array detector, mass spectrometry and nuclear magnetic resonance spectroscopy has enabled us to identify the compound as Coa-[a-(7-adenyl)]-Cob-cyanocobamide or pseudovitamin B 12 . This molecule differs from cobalamin in the a-ligand, where it has adenine instead of 5,6-dimethylbenzimidazole bound in a a-glycosidic linkage to C-1 of ribose. L. reuteri is the first lactic acid bacterium in which the production of a cobalamin-like molecule has been identified and the first microorganism reported to produce exclusively pseudo-B 12 .

To clone, express and characterize uroporphyrinogen III synthase/methyltransferase gene (cobA/hemD) from Lactobacillus reuteri. Some strains of Lb. reuteri produce cobalamin (vitamin B(12)). Cobalamin biosynthesis relies on the sequential action of more than 25 enzymes in a complex metabolic pathway. We have cloned, expressed and characterized the gene in Lb. reuteri that codes for the S-adenosy L: -methionine uroprophyrinogen III methyltransferase/synthase (CobA/HemD), a key bifunctional enzyme in the biosynthesis of cobalamin and other tetrapyrrols.