Differential Off-line LC-NMR (DOLC-NMR) Metabolomics To Monitor Tyrosine-Induced Metabolome Alterations in <i>Saccharomyces cerevisiae</i>

A novel differential off-line LC-NMR approach (DOLC-NMR) was developed to capture and quantify nutrient-induced metabolome alterations in Saccharomyces cerevisiae . Off-line coupling of HPLC separation and 1 H NMR spectroscopy supported by automated comparative bucket analyses, followed by quantitative 1 H NMR using ERETIC 2 (electronic reference to access in vivo concentrations), has been successfully used to quantitatively record changes in the metabolome of S. cerevisiae upon intervention with the aromatic amino acid l -tyrosine. Among the 33 metabolites identified, glyceryl succinate, tyrosol acetate, tyrosol lactate, tyrosol succinate, and N -acyl-tyrosine derivatives such as N -(1-oxooctyl)-tyrosine are reported for the first time as yeast metabolites. Depending on the chain length, N -(1-oxooctyl)-, N -(1-oxodecanyl)-, N -(1-oxododecanyl)-, N -(1-oxomyristinyl)-, N -(1-oxopalmityl)-, and N -(1-oxooleoyl)- l -tyrosine imparted a kokumi taste enhancement above their recognition thresholds ranging between 145 and 1432 μmol/L (model broth). Finally, carbon module labeling (CAMOLA) and carbon bond labeling (CABOLA) experiments with 13 C 6 -glucose as the carbon source confirmed the biosynthetic pathway leading to the key metabolites; for example, the aliphatic side chain of N -(1-oxooctyl)-tyrosine could be shown to be generated via de novo fatty acid biosynthesis from four C 2 -carbon modules (acetyl-CoA) originating from glucose.