ORIGIN OF CARBON-ATOMS OF BIOTIN - C-13-NMR STUDIES ON BIOTIN BIOSYNTHESIS IN ESCHERICHIA-COLI

The origin of the carbon atoms of pimeloyl-CoA, the earliest known pre cursor in the pathway of de novo biotin biosynthesis in Escherichia co li, was investigated by C-13-NMR spectroscopy. In fermentation of the biotin-overproducing DRK332/pXBA312 strain of Escherichia coli (a repr essor mutant carrying a biotin operon fragment in the plasmid), a high dose of L-alanine (8 g/l) stimulated dethiobiotin and biotin accumula tion. Although L-alanine is a known precursor of 7-keto-8-aminopelargo nic acid in biotin biosynthesis, the C-13-NMR spectrum of dethiobiotin showed that the C-3 of L-[3-C-13]alanine was incorporated into not on ly the methyl carbon (C-9) but also alternate carbons (C-2, C-4, C-6) of the side chain, and these latter positions are the same as those la beled with D-[1-C-13]glucose. These data indicate that L-alanine can a ct as an alternative carbon source, suggesting that acetyl-CoA is a po ssible precursor for pimeloyl-CoA synthesis. In accordance with this h ypothesis, the C-1 of sodium (1-C-13)acetate and the C-2 of sodium (2- C-13)acetate were incorporated into alternate carbons in the side chai n of dethiobiotin, i.e., (C-1, C-3, C-5, C-7) and (C-1, C-2, C-4, C-6) , respectively. These results suggested firstly that in E. coli pimelo yl-CoA is biosynthesized from L-alanine and/or acetate via acetyl-CoA, but not via pimelic acid, which has been suggested as a biotin precur sor in other species, and secondly that the carboxyl group of biotin o riginates from carbon dioxide produced through the tricarboxylic acid cycle.