## 3 Results and Discussion
### Biotin and Thiamine Impact on the Wine Metabolome
#### 3.4.2 Impact of Thiamine on the Wine Metabolome
However, this further annotation process has led to the identification of the pathways in which those biomarkers are involved, and therefore, associated with the exometabolome changes that were observed here (Supplementary Table S3, Figure S1). In contrast to the low number of pathways affected by the initial biotin, there was, here, a thorough influence of high thiamine on numerous metabolic pathways, since 46 named pathways were matched through KEGG [27] for its associated biomarkers, while a far lesser number of metabolic pathways were influenced by low thiamine. Interestingly, high thiamine seems to exert a strong influence on both amino acid and carbon metabolisms, since they displayed the highest number of total annotated biomarkers (53 and 51, respectively), distributed in numerous, more specified pathways. Thiamine, as such, appears to be a relevant actor in amino acid synthesis, matching with 10 biomarkers, as well as being strongly involved in phenylalanine, valine, leucine, isoleucine, tyrosine, and lysine metabolisms, more specifically. The overall effect of the vitamin on amino acid metabolism might reflect, overall, its role as a cofactor involved in the Ehrlich pathway, with TPP being essential to the decarboxylation step; however, it might also reflect, indirectly, the relevance of pyridoxine in the transamination of the Ehrlich pathway as well, which can proceed to the conversion of an amino acid towards its associated \(\alpha\)-ketoacid, and inversely [42; 61]. Since thiamine biosynthesis relies on pyridoxine in case of thiamine deficiencies, in a mechanism that takes priority over all other pyridoxine-dependent reactions [34], it is not impossible that this effect on amino acid metabolism is not a significant effect. Figure 5: Metabolomic foot-printing of _S. cerevisiae_ grown in high or low thiamine conditions based on LC-MS-MS untargeted analysis data. Van Krevelen diagrams based on the H/C and O/C atomic ratios for the significantly more intense features (extracted through ANOVA; \(p<0.01\)), histogram proportion of their elemental compositions, and pie chart distribution of the predicted families for these biomarkers in high thiamine conditions (**left**, 378 markers) and low thiamine conditions (**right**, 79 markers). The point size indicates the relative intensity of the extracted masses. acid synthesis also reflects the indirect influence of pyridoxine. Plus, such an influence of thiamine on both phenylalanine and tyrosine, more specifically, appears consistent with the role held by the vitamin in the pentose phosphate pathway through TPP-dependent transketolases Tkll/2 [42], from which prephenate is derived, itself a precursor in the biosynthesis of both aromatic amino acids [62]. Similarly, the differences observed regarding both valine and leucine metabolism might reflect the thiamine-dependent character of acetlactate synthase Ilv2 [42], which is involved in the synthesis of both branched-chain amino acids [63]. All in all, such an influence held by thiamine on the amino acid pathways in the _S. cerevisiae_ exometabolome appears consistent with our previous observations regarding the wine volatile compounds, which highlighted a similar effect. The strong influence exerted by high thiamine on carbon metabolism, similarly, affects a large number of pathways involved in the CCM, and highlights, notably, a remarkable effect on keto acids, such as 2-oxoxcarboxylic acids, C5-branched dibasic acids, and pyruvate, which might reflect the TPP-dependency of the Ehrlich decarboxylation reaction of \(\alpha\)-keto acids towards their associated aldehydes [42; 61], as well as the conversion of pyruvate towards S-acetlactate through TPP-dependent Ilv2 [42]. The observed influence of thiamine on both the propanoate metabolism and the TCA cycle biomarkers, similarly, might be a reflection of the cofactor role played by TPP in the conversion of \(\alpha\)-ketoglutarate to succinyl-CoA by Kgd1 [42], which is further derived towards propanoyl-CoA [64].