Pyruvate was shown to be the precursor of diacetyl and acetoin in Streptococcus diacetilactis, but dialyzed cell-free extracts of S. diacetilactis and Leuconostoc citrovorum that had been treated with anion-exchange resin to remove coenzyme A (CoA) formed only acetoin from pyruvate in the presence of thiamine pyrophosphate (TPP) and Mg++ or Mn++ ions. The ability to produce diacetyl was restored by the addition of acetyl-CoA. Acetyl-phosphate did not replace the acetyl-CoA. Neither diacetyl nor acetoin was formed when the otherwise complete reaction system was modified by using boiled extract or by omitting the extract, pyruvate, TPP, or the metal ions. Free acetaldehyde was not involved in the biosynthesis of diacetyl or acetoin from pyruvate, dialyzed cell-free extracts of the bacteria produced only acetoin (besides CO2) from α-acetolactate, and acetoin was not involved in the biosynthesis of diacetyl. Only one of the optical isomers present in racemic α-acetolactate was attacked by the extracts, and there was no appreciable spontaneous decarboxylation of the α-acetolactate at the pH (4.5) used in experiments.
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