Exploration of Yeast Species Suitable for Preparation of Stable Isotope-Labeled Internal Standards Extracts (SILIS).

Isotope dilution mass spectrometry is a widely used method for measuring intracellular metabolite concentrations, relying on the ratio of peak areas between the target compound and its stable isotope-labeled internal standard. For metabolome analysis of microorganisms, comprehensive concentration measurements have been achieved through the preparation of stable isotope-labeled internal standard extracts (SILIS). Methods have been developed to prepare SILIS by extracting crude metabolites from fully 13C-labeled bacteria Escherichia coli and yeasts Saccharomyces cerevisiae and Pichia pastoris (Komagataella phaffii). For cost-effective preparation of SILIS, ideal characteristics of host yeasts include rapid cell growth, high biomass production, and significant metabolite accumulation. In this study, suitable yeast species for SILIS production were investigated from diverse candidates. Batch cultures of 15 yeast species from 12 genera were performed in synthetic defined medium, with cells harvested at different growth phases and metabolites extracted using the methanol/chloroform/water method. Metabolomic analysis by liquid chromatography-tandem mass spectrometry revealed the relative concentrations of 65 metabolites. The results demonstrated that S. cerevisiae and Kluyveromyces marxianus in the stationary phase were the most effective for SILIS production of central metabolic intermediates. SILIS production using S. cerevisiae and K. marxianus can be widely applied in standard laboratories because these species are safe, the media are commercially available, and the extraction methods are easily implementable.