Isotopic labelling-based analysis elucidates biosynthesis pathways in Saccharomyces cerevisiae for Melatonin, Serotonin and Hydroxytyrosol formation

•Bioactive compounds are produced during alcoholic fermentation.•Yeasts can synthetize melatonin by different pathways: from tryptophan and serotonin.•Yeasts produce Hydroxytyrosol from labelled tyrosine and also from other sources.•Isotopic labelling proved useful as strategy to elucidate biosynthetic pathways. Yeasts can synthetise bioactive compounds such as Melatonin (MEL), Serotonin (SER) and Hydroxytyrosol (HT). Deciphering the mechanisms involved in their formation can lead to exploit this fact to increase the bioactive potential of fermented beverages. Quantitative analysis using labelled compounds, 15-N2 l-tryptophan and 13-C tyrosine, allowed tracking the formation of the above-mentioned bioactive compounds during the alcoholic fermentation of synthetic must by two different Saccharomyces cerevisiae strains. Labelled and unlabelled MEL, SER and HT were undoubtedly identified and quantified by High Resolution Mass Spectrometry (HRMS). Our results prove that there are at least two pathways involved in MEL biosynthesis by yeast. One starts with tryptophan as precursor being known for the vertebrates’ pathway. Additionally, MEL is produced from SER which in turn is consistent with the plants’ biosynthesis pathway. Concerning HT, it can be formed both from labelled tyrosine and from intermediates of the Erlich pathway.