Biochemical and molecular basis involved in the synthesis of melatonin and other derivatives of aromatic amino acids in saccharomyces cerevisiae

• Autores: Sara Muñiz Calvo
• Directores de la Tesis: José Manuel Guillamón Navarro (dir. tes.)
• Lectura: En la Universitat de València ( España ) en 2021
• Idioma: español
• Tribunal Calificador de la Tesis: Paloma Manzanares Mir (presid.), M. Jesús Torija Martínez (secret.), Pau Ferrer Alegre (voc.)
• Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
• Enlaces
  • Tesis en acceso abierto en: TESEO
• Resumen

  • Recently, the metabolism of aromatic amino acids in yeasts has been linked to the synthesis of bioactive molecules (melatonin, serotonin, tyrosol, hydroxytyrosol, etc.) that could be relevant from different aspects related to both yeast regulation and human health. The activities of these compounds as a potent antioxidant and other aspects beneficial to consumer health makes it really interesting to study its synthesis. However, few information is known about the synthesis of these bioactive molecules by yeast because it is a very recent topic of study.

    Therefore, the working hypothesis of the present thesis is: Through a better understanding of the aromatic amino acid metabolism routes in S. cerevisiae yeast, we can increase the content of bioactive molecules in products of alcoholic fermentation. Thus, yeast metabolism can increase the content of bioactive molecules in fermented beverages and foods, which could have an impact on the health of the consumer and which will undoubtedly increase the added value of these beverages, better positioning them in an increasingly competitive and global market. In this context, the main objective of this thesis work was to study the molecular and physiological mechanisms involved in the production of bioactive compounds derived from the aromatic amino acid metabolism, mainly tryptophan derived compounds such as melatonin and serotonin, in S. cerevisiae.

    In this thesis we adapt and set up a simple, rapid, and low-cost technique for detecting the presence of melatonin in S. cerevisiae intracellular samples based on voltammetry. Also, we evaluated the possible effect of melatonin on BY4743 laboratory strain in the presence and absence of H2O2 and UV radiation. Our results demonstrate that intracellular melatonin was an efficient antioxidant and UV protector molecule in S. cerevisiae. Regarding melatonin biosynthetic pathway, it is still far unknown which activities or genes are involved in this pathway in yeast. To unveil the melatonin biosynthetic pathway in S. cerevisiae we evaluated the products generated from different substrates of the route (L-tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, tryptamine, and 5‐methoxytryptamine), in the wine yeast strain QA23 at different stages of growth by HPLC-MS/MS. Besides in order to identify the genes that respond to the synthesis of melatonin in yeasts, we performed a BLAST analysis using the protein sequences of animals and plants. The genes selected as putative orthologs, as well as a positive gene as a control (from animals or plants), were overexpressed both in S. cerevisiae and E. coli. Importantly, our results suggested that the only gene that has been proposed as homolog of the arylalkylamine N-acetyltransferase of vertebrates in yeast, PAA1, may not be the exclusive enzyme in the acetylation of arylalkylamines such as serotonin, tryptamine and 5-methoxytryptamine in S. cerevisiae.

    Finally, we constructed a S. cerevisiae strain modified by multiple integration into the genome of the E. coli hydroxylase HpaBC complex in order to overproduce hydroxytyrosol. Subsequently, several changes in yeast metabolism (including overexpression of ARO genes, suchas ARO3, ARO4, ARO7 and ARO10 as well as the specific mutations on some of these genes) were made to increase the flow of the route to the synthesis of tyrosol, the higher alcohol derived from tyrosine catabolism. The combination of ARO4K229L overexpression together with HpaBC integration showed the greatest effect on hydroxytyrosol production achieving 374.5 mg/L in shake flask experiments which exceeded the production by more than 230,000 times that of the control strain.