Fig. 1
Schematic riboflavin biosynthesis from vegetable oil in A. gossypii under industrial process conditions (a). Riboflavin biosynthesis with A. gossypii entails multiple compartments and different metabolic pathways. The terminal biosynthesis starts from GTP, which is formed in the purine biosynthesis, and ribulose 5-phosphate originating from the pentose phosphate (PP) pathway. The map includes qualitative data from our previous study [16]: in three parallel 13C tracer experiments, the contribution of the respective tracer to growth as well as riboflavin biosynthesis was assessed. The 13C tracers used were glycine (blue), formate (red), and serine (green). All other carbon sources (vegetable oil, yeast extract, glutamate) remained naturally labeled (grey). The small circles indicate 13C contribution of the respective tracer to proteinogenic amino acids or riboflavin. Note that the size of the circles is not quantitative. The schematic presentation of the riboflavin molecule highlights the single carbon origin also in a qualitative manner: fully 13C-labeled glycine contributes to two distinct carbon atoms, while [3-13C] serine and [13C] formate contribute their 13C label to another carbon atom. Naturally labeled medium ingredients contribute to all carbon atoms in riboflavin. The one-carbon metabolism is only drawn in the cytosol. It can be assumed, however, that there is also a one-carbon metabolism in the mitochondrion. The 13C labeling strategy for the present work (b). In two parallel approaches, fully 13C-labeled yeast extract and glutamate replaced the naturally labeled substrates in an otherwise naturally labeled medium. Mass isotopomer distributions (MIDs) of the t-butyl-dimethylsilyl derivatized amino acids from the custom-synthesized and hydrolyzed [U13C] yeast extract (c). Naturally labeled amino acids served as control. The mass isotopomer M + 0 represents the relative amount of non-labeled, M + 1 the amount of singly-labeled mass isotopomer fraction and so on. All data are corrected for natural isotopes. Amino acids are designated with their three letter code. The error of the measurement was below 1%. 3PG, 3-phosphoglycerate; CH2-THF, 5,10-methylenetetrahydrofolate; AcCoA, acetyl-CoA; CHO-THF, 10-formyltetrahydrofolate; FA, fatty acid; FOR(EXTR), (extracellular) formate; GAR, glycineamide ribonucleotide; GLUEXTR, extracellular glutamate; GLY(EXTR), (extracellular) glycine; GTP, guanosine triphosphate; OAA, oxaloacetate; PYR, pyruvate; Ru5P, ribulose 5-phosphate; SER(EXTR), (extracellular) serine; THF, tetrahydrofolate; YE, yeast extract