Summary: Hydroxytyrosol (HT) is one of the most powerful dietary antioxidants with numerous applications in different areas, including cosmetics, nutraceuticals and food. In the present work, heterologous hydroxylase complex HpaBC from Escherichia coli was integrated into the Saccharomyces cerevisiae genome in multiple copies. HT productivity was increased by redirecting the metabolic flux towards tyrosol synthesis to avoid exogenous tyrosol or tyrosine supplementation. After evaluating the potential of our selected strain as an HT producer from glucose, we adjusted the medium composition for HT production. The combination of the selected modifications in our engineered strain, combined with culture conditions optimization, resulted in a titre of approximately 375 mg l ⁻¹ of HT obtained from shake‐flask fermentation using a minimal synthetic‐defined medium with 160 g l ⁻¹ glucose as the sole carbon source. To the best of our knowledge, this is the highest HT concentration produced by an engineered S. cerevisiae strain.

Recently, the presence of melatonin in fermented beverages has been correlated with yeast metabolism during alcoholic fermentation. Melatonin, originally considered a unique product of the pineal gland of vertebrates, has been also identified in a wide range of invertebrates, plants, bacteria, and fungi in the last two decades. These findings bring the challenge of studying the function of melatonin in yeasts and the mechanisms underlying its synthesis. However, the necessary information to improve the selection and production of this interesting molecule in fermented beverages is to disclose the genes involved in the metabolic pathway. So far, only one gene has been proposed as involved in melatonin production in Saccharomyces cerevisiae, PAA1, a polyamine acetyltransferase, a homolog of the vertebrate’s aralkylamine N-acetyltransferase (AANAT). In this study, we assessed the in vivo function of PAA1 by evaluating the bioconversion of the different possible substrates, such as 5-methoxytryptamine, tryptamine, and serotonin, using different protein expression platforms. Moreover, we expanded the search for new N-acetyltransferase candidates by combining a global transcriptome analysis and the use of powerful bioinformatic tools to predict similar domains to AANAT in S. cerevisiae. The AANAT activity of the candidate genes was validated by their overexpression in E. coli because, curiously, this system evidenced higher differences than the overexpression in their own host S. cerevisiae. Our results confirm that PAA1 possesses the ability to acetylate different aralkylamines, but AANAT activity does not seem to be the main acetylation activity. Moreover, we also prove that Paa1p is not the only enzyme with this AANAT activity. Our search of new genes detected HPA2 as a new arylalkylamine N-acetyltransferase in S. cerevisiae. This is the first report that clearly proves the involvement of this enzyme in AANAT activity.

Melatonin synthesis by yeast has been described on several occasions, mainly in a fermentative context. However, the genetic determinants involved in its synthesis remain undefined. Understanding melatonin synthesis in yeast is important because it can provide insights into the broader mechanisms of indolamine production, which has implications for both basic biological research and industrial applications. Although two genes with N ‐acetyltransferase (NAT) activity ( PAA1 and HPA2 ) have been identified in Saccharomyces cerevisiae , these genes do not seem to be major contributors to the production of melatonin and other indolamines in yeast in vivo. In this study, we identified the uncharacterized gene YDR391C as the gene encoding a protein with NAT activity, herein named IAT4 . By comparing different substrates using the purified Iat4, we found that the K _m values were 353, 356, and 930 µM towards 5‐methoxytryptamine, tryptamine, and serotonin, respectively. The substrate affinity of Iat4 towards serotonin was approximately five times higher than that reported for the previous homolog of the melatonin enzyme arylalkylamine N ‐acetyltransferase ( PAA1 ), suggesting that IAT4 could play a more significant role in melatonin biosynthesis. This enhanced affinity could lead to more efficient production of N ‐acetylserotonin, potentially improving yields in biotechnological applications. Finally, we demonstrate the conversion of serotonin into microbially‐produced N ‐acetylserotonin by overexpressing IAT4 in a serotonin‐overproducing yeast strain at a titer of 14.5 mg/L. These findings represent the first steps towards the development of yeast strains optimized for the biological production of N ‐acetylserotonin and related compounds, which might aid in studying the regulatory mechanisms and functions related to melatonin biosynthesis in S. cerevisiae and other yeast species.

The EU Green Deal prioritises the transformation of the chemical industry to a more environmentally sustainable model. This involves using microorganisms, such as Saccharomyces cerevisiae , to produce molecules more sustainably through biotechnological approaches. In this study, we demonstrate an example of serotonin production using S. cerevisiae as a cell factory, along with its optimisation and upscaling. To achieve this, we introduced two heterologous genes, the combination of tryptophan decarboxylase from Clostridium sporogenes ( Cs TDC) and tryptamine 5‐hydroxylase from Oryza sativa ( Os T5H), to complete the serotonin biosynthetic pathway using L‐tryptophan (L‐TRP) as a precursor. By modifying ARO4 to a feedback‐resistant version ( ARO4 *), the flux of the shikimate pathway was significantly increased and serotonin production was achieved at levels up to 120 mg/L directly from the glucose source. After a medium optimisation, a final concentration of 80 g/L glucose and 300 mg/L of nitrogen resulted in better conditions for increasing serotonin titres. Using this medium in a 1 L bioreactor fermentation resulted in approximately 250 mg/L of serotonin. A targeted metabolomic study of the bioreactor growth medium identified potential bottlenecks in the serotonin‐overproducing strain and future targets for increasing its titre. We have constructed a strain of S. cerevisiae that represents the first steps towards feasible industrial production of serotonin using a sustainable and environmentally friendly approach, paving the way for the development of similar biotechnological strategies in the future.