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

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 H-1 NMR spectroscopy supported by automated comparative bucket analyses, followed by quantitative H-1 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-oxoocty1)-, N-(1-oxodecanyl)-, N-(1-oxododecanyl)-, N-(1-oxornyristiny1)-, N-(1-oxopalmity1)-, and N-(1-oxooleoyl)-L-tyrosine imparted a kokumi taste enhancement above their recognition thresholds ranging between 145 and 1432 mu mol/L (model broth). Finally, carbon module labeling (CAMOLA) and carbon bond labeling (CABOLA) experiments with C-13(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.