Deciphering the melatonin metabolism in Saccharomyces cerevisiae by the bioconversion of related metabolites

Accepted Article This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jpi.12554 This article is protected by copyright. All rights reserved. PROFESSOR JOSE MANUEL GUILLAMON (Orcid ID : 0000-0001-5414-0787) Article type : Original Manuscript Deciphering the melatonin metabolism in Saccharomyces cerevisiae by the bioconversion of related metabolites Running title: Melatonin synthesis in yeasts Sara Muñiz-Calvo1, Ricardo Bisquert1, Edwin Fernández-Cruz2, María Carmen García- Parrilla2, José Manuel Guillamón1* 1Food Biotechnology Department. Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino, 7, 46980 Paterna, Valencia, Spain. 2Department of Food Science and Nutrition, Facultad de Farmacia, University of Seville, C/ Profesor García González 2, 41012 Seville, Spain * Corresponding authors: [email protected] Abstract Melatonin (Mel), originally considered a neurohormone, has been detected in beverages and food-fermented products in which yeast metabolism is highly important. This indolamine is synthesized from serotonin, with L-tryptophan being the initial substrate of both. Regarding Mel metabolism, the biosynthetic pathway in mammals consists in four-step reactions. However, six genes are implicated in the synthesis of Mel in plants, which suggest the presence of many pathways. The aim of this study is to provide new empirical data on the production of Mel and other indole-related compounds in the yeast Saccharomyces cerevisiae Accepted Article This article is protected by copyright. All rights reserved. (S. cerevisiae). To this end, we performed the addition of the pathway intermediates in S. cerevisiae cells in different growth stages (exponential and arrested cells) to follow the bioconversion and new indolic compound production from them. The different bioconverted indolic compounds tested (L-tryptophan, 5-hydroxytryptophan, tryptamine, serotonin, N- acetylserotonin, 5-metoxytryptamine and Mel) were analyzed by UHPLC-MS/MS from the extra- and intracellular contents. Our results showed that serotonin, in yeast, was prevalently formed via tryptophan decarboxylation, followed by tryptamine hydroxylation as in plants. Mel production from serotonin can be achieved by either N-acetylation, followed by O- methylation or O-methylation, in turn followed by N-acetylation. Accordingly, the classic pathway of Mel synthesis in vertebrates does not seems prevalent in yeast. Keywords: Indolic compounds, bioconversion, melatonin metabolism, yeast, substrate pulses, indole biosynthesis pathway 1. Introduction Lerner et al. in 1958 described melatonin (Mel) to be a neurohormone in the bovine pineal gland (1). It was subsequently, demonstrated that this indoleamine was also present in multiple extrapineal tissues (2,3). In the last two decades, Mel has been well accepted as a ubiquitous molecule present in many organisms belonging to different biological kingdoms (4–6). In 1999, Sprenger et al. (7) conducted the first study about Mel synthesis in yeast. In this work, the authors reported that baker´s yeast was able to synthesize Mel from L-tryptophan (L-Trp), serotonin (Ser), N-acetylserotonin (NAS) and 5-methoxytryptamine (5MT) as potential precursors. Since then, very few studies have been carried out on this topic since most research has focused on demonstrating the presence of Mel or biosynthesis by yeasts in a fermentative context (8–12). Moreover, the biosynthetic pathway of Mel and other ICs is Accepted Article This article is protected by copyright. All rights reserved. completely unknown in yeasts. The only gene described and characterized as being involved in Mel production is PAA1, a polyamine acetyltransferase identified as the homolog of aralkylamine N-acetyltransferase (AANAT) (13), while the remaining genes and enzymes of the route are unknown to date. Another issue to find out in the coming future is the physiological sense of Mel synthesis in yeasts. As for its properties, it is clear that Mel has a multiple effect on different organisms, such as the regulation of circadian rhythms and seasonal reproduction, immune system modulator, inhibitor of tumor growth and protection against UV light, among others (14–18). Mel’s antioxidant action is well established, and it can act as a radical scavenger, by regulating antioxidant enzymes or contributing to the homeostasis of mitochondria (19–22). Recently, three independent works have also proved the role of Mel as an antioxidant and as UV protector in S. cerevisiae (23–25). Mel is also hypothesized to act as a growth signal molecule, (26) but the role of produced Mel is not very well known. This work aims to provide new empirical data to unveil Mel production in S. cerevisiae. To this end, we carried out several pulses with the intermediates of the pathway (L-Trp, 5- hydroxytryptophan (5-OH-Trp), Ser, NAS, tryptamine and 5-MT) in cells in different growth stages. We evaluated the bioconversion of several indolic compounds (ICs) related with Mel in exponentially growing yeast cells using yeast minimal media (SD) in the batch-mode and synthetic must (SM) in a continuous culture. We also performed the experiment in arrested cells in salt medium (SLT) by following, but extending, the study conducted by Sprenger et al. (7). The intracellular content of cells and extracellular media, were analyzed by ultrahighperformance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Accepted Article This article is protected by copyright. All rights reserved. 2. Material and Methods 2.1. Chemicals and reagents All the ICs used were: L-Trp, 5-OH-Trp, tryptamine, Ser, 5-MT, NAS, Mel purchased from Sigma-Aldrich (Madrid, Spain). Stock solutions were prepared and filtered through a 0.22 µm nylon filter. The calibration curve for HPLC-MS/MS was freshly prepared every time by diluting the solutions of 1 mg/mL of each IC in MilliQ-water to a final concentrations of 0.1, 1, 10, 100 and 1000 ng/ml. Stock solutions were stored at -20 ºC for no longer than 1 month. HPLC grade ethanol and acetonitrile were obtained from Panreac (Panreac AppliChem, Spain). 2.2.Yeast strains and inoculum preparation The S. cerevisiae yeast strain used in this study was wine commercial strain QA23, provided by Lallemand S.A. (Canada) as active dry yeasts (ADY). For ADY QA23, the inoculum was prepared by rehydrating cells in sterile distilled water during 30 min at 37 ºC, and it was inoculated in the same media used for the indicated experiment. 2.3.Batch cultivations For batch cultivations, two different growth modes were used. The first consisted of arresting cells in the depletion/restoration experiments described by Sprenger et al. (7) with some modifications. Yeast cells were suspended at cell densities of ∼108 cells/mL in SLT (50 mM Na2HPO4, 0.5% NaCl, adjusted to pH 5.8 using citric acid) and incubated in Erlenmeyer flasks with orbital agitation (150 rpm) at 28 °C for 4 h. After the incubation period in SLT, ICs were added at a final concentration of 1 mM. Sampling was done 15 min after the indolic pulse. For harvesting, 10 mL were centrifuged at 4000 rpm for 3 min at 4 ºC. The obtained pellet (∼109 cells) was washed twice with sterile distilled water. Pellets were frozen with Accepted Article This article is protected by copyright. All rights reserved. liquid nitrogen and stored at -80 ºC and supernatants were stored at -20 ºC until further extractions. The second batch-mode was performed in SD (0.17% (w/v) yeast nitrogen base, 0.5% (w/v) ammonium sulfate, 2% (w/v) glucose). Strain QA23, from the overnight preculture in SD, was inoculated in 20 ml SD at an optical density of 600 nm (OD600) of 0.1 and incubated at 28 ºC until the OD600 reached ∼0.8. When cells were in the exponential growth stage, ICs at the 1mM final concentration were added. A volume of ∼ 10 units of OD600 was sampled at 15 min and centrifuged at 4000 rpm for 3 min at 4 ºC. After centrifugation, the cell suspension was washed twice with sterile distilled water. Finally the pellet was frozen with liquid nitrogen and stored at -80 ºC until intracellular extraction and the supernatant was stored at -20 ºC until further extraction. 2.4. Chemostat cultivation The synthetic fermentation medium used for the continuous culture was a modified SM described by Bely et al. (27) with some modifications, such as 200 g/L of reducing sugars (100 g/L glucose and 100 g/L fructose), no anaerobic factors, and the total assimilable nitrogen source (YAN) 100 mg N/L came in the ammonium form (NH4Cl). Chemostat cultivation was performed in a 500 mL mini bioreactor (My Control miniBio, Applikon biotechnology, The Netherlands) with a working volume of 300 mL. The dilution rate (D) was 0.15 h-1, the stirrer employed worked at 300 rpm and temperature was kept at 28 ºC. The medium pH remained at 3.3 using a pH sensor, and was controlled by an alkali pump by adding 1N NaOH. Before starting the continuous culture, strain QA23 was inoculated at ∼2x106 cells/mL and grown in the batch mode to achieve enough biomass. The continuous culture was connected when the batch culture entered the stationary phase. The IC addition pulse in the culture was for a 0.5mM final concentration, and was performed only after all the Accepted Article This article is protected by copyright. All rights reserved. continuous cultures had been running for at least five residence times and the biomass values were constant. Once addition was done, a volume of ∼ 35 units of OD600 was sampled at 15 min and centrifuged at 4000 rpm for 3 min at 4 ºC. After centrifugation, the cell suspension was washed twice with sterile distilled water, and the pellet was frozen with liquid nitrogen and stored at -80 ºC until intracellular extractions. Finally, the supernatant was stored at -20 ºC until further extractions. 2.5. Sample preparation For the determination of the intra and extracellular ICs, culture supernatants and the intracellular content were extracted and analyzed by UHPLC-MS/MS. The intracellular metabolite content was obtained by adding 1 mL of cold mixture ethanol/water 50% (v/v) to the cell pellets and 0.3 mL of glass beads. Cells were ruptured by a Tehtnica MillMix 20 homogenizer (Tehtnica, Slovenia) with three shaking cycles at 30 s−1 for 30 s at 4 °C. Lysed cells were centrifuged at 5000 × g and 4 °C for 10 min and supernatant was filtered by a 0.22-µm nylon filter, collected and placed inside a new tube and stored at -20 °C until further use. For the extracellular extraction, an equal amount of absolute ethanol was added to the supernatant. The mixture was filtered by a 0.22-µm nylon filter (Phenomenex, USA) and stored at -20 ºC until analysis. 2.6. Indolic compound analysis by UHPLC-MS/MS The indolic studied metabolites were quantified by UHPLC-MS/MS as described previously (28). The MRM transitions used for quantification were m/z 205 → 118.03 and m/z 205 → 145.85 for L-Trp, m/z 177 → 132.21 and 177 → 160.03 for Ser, m/z 221 → 162.19 and 221 → 203.99 for 5-OH-Trp, m/z 219 → 159.96 and m/z 219 → 202.00 for NAS, m/z 233 → Accepted Article This article is protected by copyright. All rights reserved. 174.10 and 233 → 216.10 for Mel, m/z 161> 144 for tryptamine and m/z 191.2> 174 for 5- MT. 3. Results and Discussion It is known that Mel is produced by yeasts during alcoholic fermentation and this production depends on the medium, mainly the L-Trp and sugar concentrations, and also the growth phase (9,11,29–31), among others conditions. However, the biosynthetic pathway of Mel and other ICs is completely unknown in yeasts. Conversely, this metabolic route has been extensively studied and well established in other organisms. Although differences in the intermediates and branches of the route have been found in different organisms (32), there is a high degree of conservation in the enzymatic reactions which leads to the synthesis of Mel. To unveil the putative biosynthetic route and the order of the enzymatic steps in yeasts, we studied the production of ICs, both intra- and extracellularly, after pulses of different intermediates of the route in different growth systems: exponential growth conditions in SD and continuous culture with SM. Chemostat cultures help to accurately control many variables, such as the specific growth rate, temperature and pH, among others. This steady state is only disrupted by the pulses of different intermediates, to produce an over-flow of its metabolic conversion. 3.1. Pulses of melatonin intermediates to yeast cells: a general overview L-Trp, 5-OH-Trp, Ser, NAS, tryptamine and 5-MT were pulsed to the cells grown in SD (the batch culture) or SM (the continuous culture), and to the cells incubated in a non-proliferative medium SLT (arrested-cells). After 15 min of the pulses, the intracellular and extracellular metabolites were analyzed by UHPLC/HRMS (Table S1). As many data were collected, and to provide a better understanding, we represented the increases in the different ICs on a heatmap (Fig. 1). Accepted Article This article is protected by copyright. All rights reserved. Although different growth systems provide considerable similarities in the different indole bioconversions after each pulse, we also detected differences in the production of the indolic metabolites among growth media, which could be explained by either a higher permeability of cells to the pulsed compounds or a different bioconversion rate in the various growth states. Most of the produced metabolites were detected either intracellularly or extracellularly, but a direct correlation was not always found between the presence in the medium and in the cellular compartment. This could also be due to the fact that, under specific conditions, certain compounds, are released to the medium or are rapidly converted into other metabolites (33). Previous publications have also reported the rapid turnover of Mel, as well as other 5-methoxylated related indoles, as a result of different mechanisms, such as photooxidation (34), hydroxylation by free radicals (35) or metabolization by different enzymes (36), which can also contribute to these differences observed among the various growth systems and cellular states. The conversion of L-Trp into Ser involves two reactions: hydroxylation and decarboxylation. However, the order of these reactions described for distinct organisms differs. On the one hand, L-Trp is hydroxylated to 5-OH-Trp, by the tryptophan hydroxylase (TPH) enzyme, and is then decarboxylated, by aromatic L-amino acid decarboxylase (AADC), which gives rise to Ser. On the other hand, and alternative way, AADC can decarboxylate L-Trp into tryptamine, and then tryptamine is hydroxylated by tryptamine 5-hydroxylase (T5H), to result in Ser. The second part of the biosynthetic route consists in the conversion of Ser into Mel. Once again, this conversion can also occur in two different ways and the enzymes described as being involved are: AANAT or acetylserotonin O-methyltransferase (ASMT)/caffeic acid O- methyltransferase (COMT). If Ser is acetylated by AANAT, it produces NAS, which can then be O-methylated by ASMT/COMT to produce Mel. However, Ser can first be O-methylated and then the resulting 5-MT is acetylated by AANAT to generate Mel. Accepted Article This article is protected by copyright. All rights reserved. 3.1.1. Hydroxylation activity As explained above, there are two hydroxylation activities involved in the synthesis of Ser. The hydroxylation of L-Trp to 5-OH-Trp is carried out by the TPH enzyme. This reaction is known as the first step in the classic Mel biosynthetic pathway described for animals. Conversely, in plants, hydroxylation is a second step in the biosynthetic pathway, and occurs by T5H converting tryptamine into Ser. In order to determine which hydroxylation reaction preferentially occurs in S. cerevisiae, we used L-Trp and tryptamine as substrates in feeding experiments. When L-Trp was added as a precursor, we were not able to detect 5-OH-Trp either intra or extracellularly for any of the growth systems (Fig. 1). Nonetheless, when we supplemented the media with tryptamine, we mostly detected Ser both extra- and intracellularly in all the growth systems (Table S1 and Fig. 2A). We highlight the vast amount of intracellular Ser observed in SLT (more than 300 ng/mL), which was by far the highest concentration detected for any analyzed conditions. In light of these results, the conversion of tryptamine into Ser seemed the preferential hydroxylation reaction, whereas L- Trp hydroxylation must be a very scarce or a null enzymatic reaction in S. cerevisiae. Aromatic amino acid hydroxylases (AAAH) are non-heme iron family of enzymes in which TPH is encompassed together with phenylalananine and tyrosine hydroxylase (PAH and TH) (37). Moreover, AAAH are pterin-dependent enzymes that require the presence of special cofactors, such as tetrahydrobiopterin or tetrahydromonapterin (38), except for a TPH from dinoflagellate Gonyaulax polyedra, which doesn’t require tetrahydropterin cofactors (39). 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Figure legends Fig. 1 Effect of the addition of different potential melatonin precursors (rows) on the increase of its related products (columns) extra- and intracellularly in Saccharomyces cerevisiae. Differences in concentrations were normalized and the increases of the compound used in each pulse are not considered (grey boxes). Defined synthetic (SD), salt medium (SLT) and synthetic must (SM). Fig. 2 Metabolite production in yeast after addition of exogenous tryptamine (A), 5- hydroxytryptophan (B), serotonin (C) and 5-methoxytryptamine (D) in the extra- and intracellular production (dotted and stripped bars respectively). Different growth systems are defined synthetic (SD), salt medium (SLT) and synthetic must (SM). The error bars represent SD. Fig. 3 Melatonin catabolites produced extracellularly after 1 mM exogenous melatonin addition to arrested yeast cells in salt medium. N-acetylserotonin (NAS), 5- methoxytryptamine (5MT). Error bars represent SD. Fig. 4 The hypothesized melatonin biosynthetic pathway in yeast. L-Trp (tryptophan), Tryp (tryptamine), 5OH-Trp (5-hydroxytryptophan), Ser (serotonin), NAS (N-acetylserotonin), 5MT (5 methoxytryptamine). Enzymatic steps are shown in numbers: 1 (tryptophan decarboxylation), 2 (tryptamine hydroxylation), 3 (5-hydroxytryptophan decarboxylation), 4 (5-hydroxytryptophan carboxylation), 5 (serotonin N-acetyltation), 6 (N-acetylserotonin O- methylation), 7 (N-acetylserotonin O-demethylation), 8 (serotonin O-methylation), 9 (5- methoxytriptamine N-acetylation), 10 (melatonin deacetylation). Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved. Accepted Article This article is protected by copyright. All rights reserved.