In order to obtain the information on the precursors of the pyrimidine moiety of thiamine, the early production of thiamine by growing cells of thiazoleless mutant of Escherichia coli was investigated by the addition of aliphatic amino acids and citric acid cycle compounds to the growth medium from which nitrogen sources had been omitted and carbon sources reduced. The thiamine production by the growth of the mutant was presumed to depend on the synthesis of the pyrimidine portion of thiamine, since the growth could be satisfied by supplementing excess thiazole. Aspartate, out of all the amino acids tested, was effective and the combinations of aspartate and alanine or pyruvate were most effective.
The incorporation experiment of radioactive carbon from DL-aspartate-2-¹⁴C into thiamine was also carried out comparing with that of sodium formate-¹⁴C. Thiamine produced by the mutant incubated with the ¹⁴C-compounds, was isolated by Amberlite IRC-50 column chromatography, conversion to thiochrome, repeated paper chromatography and actigraphy. The incorporation of radioactive carbon of the compound was demonstrated by detection of radioactivities corresponding to the fluorescent spots of thiochrome. Aspartate as well as formate was found to be incorporated into thiamine from the specific radioactivity of the thiochrome isolated.