The Role of the PAA1 Gene on Melatonin Biosynthesis in Saccharomyces cerevisiae: A Search of New Arylalkylamine N-Acetyltransferases.

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.

The potential role of yeasts in the mitigation of health issues related to beer consumption.

Food consumption of healthier products has become an essential trend in the food sector. This is also the case in beer, a biochemical process of transformation performed by yeast cells. More and more studies proclaim the need to reduce ethanol content in alcoholic drinks, certainly the most important health issue of beer consumption. In this review we gather key health issues related to beer consumption and the last advances regarding the use of yeast to attenuate those health problems. Furthermore, we have included the latest findings about the general positive impact of yeast in health as a consequence of its ability to biotransform polyphenolic compounds present in the wort, producing healthy compounds as hydroxytyrosol or melatonin, and its ability to perform as a probiotic driver. Besides, a group of population with chronic diseases as diabetes or celiac disease could take advantage of low carbohydrate or gluten-free beers, respectively. The role of yeast in beer production has been traditionally associated to its fermentative power. But here we have found a change in this dogma in the last years toward yeasts being a main driver to enhance healthy aspects of beer. The key findings are discussed and possible future directions are proposed.

Genomic Adaptations of Saccharomyces Genus to Wine Niche.

Wine yeast have been exposed to harsh conditions for millennia, which have led to adaptive evolutionary strategies. Thus, wine yeasts from Saccharomyces genus are considered an interesting and highly valuable model to study human-drive domestication processes. The rise of whole-genome sequencing technologies together with new long reads platforms has provided new understanding about the population structure and the evolution of wine yeasts. Population genomics studies have indicated domestication fingerprints in wine yeast, including nucleotide variations, chromosomal rearrangements, horizontal gene transfer or hybridization, among others. These genetic changes contribute to genetically and phenotypically distinct strains. This review will summarize and discuss recent research on evolutionary trajectories of wine yeasts, highlighting the domestication hallmarks identified in this group of yeast.

Genome-wide effect of non-optimal temperatures under anaerobic conditions on gene expression in Saccharomyces cerevisiae.

Understanding of thermal adaptation mechanisms in yeast is crucial to develop better-adapted strains to industrial processes, providing more economical and sustainable products. We have analyzed the transcriptomic responses of three Saccharomyces cerevisiae strains, a commercial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and a commercial bioethanol strain, Ethanol Red, grown at non-optimal temperatures under anaerobic chemostat conditions. Transcriptomic analysis of the three strains revealed a huge complexity of cellular mechanisms and responses. Overall, cold exerted a stronger transcriptional response in the three strains comparing with heat conditions, with a higher number of down-regulating genes than of up-regulating genes regardless the strain analyzed. The comparison of the transcriptome at both sub- and supra-optimal temperatures showed the presence of common genes up- or down-regulated in both conditions, but also the presence of common genes up- or down-regulated in the three studied strains. More specifically, we have identified and validated three up-regulated genes at sub-optimal temperature in the three strains, OPI3, EFM6 and YOL014W. Finally, the comparison of the transcriptomic data with a previous proteomic study with the same strains revealed a good correlation between gene activity and protein abundance, mainly at low temperature. Our work provides a global insight into the specific mechanisms involved in temperature adaptation regarding both transcriptome and proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance.

Generation of intra- and interspecific Saccharomyces hybrids with improved oenological and aromatic properties.

Non-wine yeasts could enhance the aroma and organoleptic profile of wines. However, compared to wine strains, they have specific intolerances to winemaking conditions. To solve this problem, we generated intra- and interspecific hybrids using a non-GMO technique (rare-mating) in which non-wine strains of S. uvarum, S. kudriavzevii and S. cerevisiae species were crossed with a wine S. cerevisiae yeast. The hybrid that inherited the wine yeast mitochondrial showed better fermentation capacities, whereas hybrids carrying the non-wine strain mitotype reduced ethanol levels and increased glycerol, 2,3-butanediol and organic acid production. Moreover, all the hybrids produced several fruity and floral aromas compared to the wine yeast: ß-phenylethyl acetate, isobutyl acetate, γ-octalactone, ethyl cinnamate in both varietal wines. Sc × Sk crosses produced three- to sixfold higher polyfunctional mercaptans, 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexanol (3MH). We proposed that the exceptional 3MH release observed in an S. cerevisiae × S. kudriavzevii hybrid was due to the cleavage of the non-volatile glutathione precursor (Glt-3MH) to detoxify the cell from the presence of methylglyoxal, a compound related to the high glycerol yield reached by this hybrid. In conclusion, hybrid generation allows us to obtain aromatically improved yeasts concerning their wine parent. In addition, they reduced ethanol and increased organic acids yields, which counteracts climate change effect on grapes.

Modulation of aroma and chemical composition of Albariño semi-synthetic wines by non-wine Saccharomyces yeasts and bottle aging.

Saccharomyces yeasts from different origins and species fermented in a semi-synthetic must containing aroma precursor of cv. Albariño and polyfunctional mercaptans precursors. The resulting wines were subjected to accelerate anoxic aging. Afterward, aroma profiles were analyzed by distinct gas chromatography methodologies. Cryotolerant strains showed better fermentation performances with significant differences in volatile and non-volatile fermentation products than Saccharomyces cerevisiae (S. cerevisiae). We suggested that the highest levels γ-butyrolactone and diethyl succinate in Saccharomyces uvarum (S. uvarum) strains, together with their substantial succinic acid yields, could be related to greater flux through the GABA shunt. These strains also had the highest production of ß-phenylethyl acetate, geraniol, and branched-chain ethyl esters. The latter compounds were highly increased by aging, while acetates and some terpenes decreased. S. kudriavzevii strains showed a remarkable ability to release polyfunctional mercaptans, with SK1 strain yielding up to 47-fold and 8-fold more 4-methyl-4-mercaptopentan-2-one (4MMP) than S. cerevisiae and S. uvarum strains, respectively. The wild S. cerevisiae beer isolate showed a particular aroma profile due to the highest production of ethyl 4-methylvalerate (lactic and fruity notes), γ-octalactone (coconut), and furfurylthiol (roasted coffee). The latter compound is possibly produced from the pentose phosphate pathway (PPP). Since erythritol, another PPP intermediate was largely produced by this strain.

Effect of non-wine Saccharomyces yeasts and bottle aging on the release and generation of aromas in semi-synthetic Tempranillo wines.

Interest in the use of non-conventional yeasts in wine fermentation has been increased in the last years in the wine sector. The main objective of this manuscript was to explore the aromatic diversity produced by wild and non-wine strains of S. cerevisiae, S. eubayanus, S. kudriavzevii, and S. uvarum species in young and bottle-aged Tempranillo wines as well as evaluate their fermentation capacity and the yield on ethanol, glycerol, and organic acids, that can contribute to diminishing the effects of climate change on wines. S. uvarum strain U1 showed the highest ability to release or de novo produce monoterpenes, such as geraniol and citronellol, whose values were 1.5 and 3.5-fold higher than those of the wine S. cerevisiae strain. We found that compared to the normal values for red wines, ß-phenylethyl acetate was highly synthesized by U1 and E1 strains, achieving 1 mg/L. Additionally, after aging, wines of S. eubayanus strains contained the highest levels of this acetate. Malic acid was highly degraded by S. kudriavzevii yeasts, resulting in the highest yields of lactic acid (>5-fold) and ethyl lactate (>2.8-fold) in their wines. In aged wines, we observed that the modulating effects of yeast strain were very high in ß-ionone. S. uvarum strains U1 and BMV58 produced an important aging attribute, ethyl isobutyrate, which was highly enhanced during the aging. Also, the agave S. cerevisiae strain develops an essential aroma after aging, reaching the highest ethyl leucate contents. According to the results obtained, the use of wild non-wine strains of S. cerevisiae and strains of the cryotolerant species S. eubayanus, S. kudriavzevii, and S. uvarum in Tempranillo wine fermentation increase the aroma complexity. In addition, wines from S. kudriavzevii strains had twice additional glycerol, those from S. uvarum 4-fold more succinic acid, while wines from wild strains yielded 1% v/v less ethanol which may solve wine problems associated with climate change.

Metabolic engineering of Saccharomyces cerevisiae for hydroxytyrosol overproduction directly from glucose.

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.

Impact of Nitrogen Addition on Wine Fermentation by S. cerevisiae Strains with Different Nitrogen Requirements.

In modern oenology, supplementation of nitrogen sources is an important strategy to prevent sluggish or stuck fermentation. The present study thoroughly determined the effect of nitrogen addition timing and nitrogen source type on fermentation kinetics and aroma production, carried out by yeast strains with low and high nitrogen requirements. The results revealed that yeast strains with different nitrogen requirements have divergent reactions to nitrogen addition. Nitrogen addition clearly shortened the fermentation duration, especially for the high-nitrogen-demanding yeast strain. Nitrogen addition at 1/3 fermentation was the most effective in terms of fermentation activity, nitrogen assimilation, and production of acetate esters. Interestingly enough, yeast cells preferentially took up amino acids related to fermentative aroma synthesis with the addition at 2/3 fermentation. The addition of nitrogen sources also largely affected the production of important metabolites. Generally speaking, acetic acid, glycerol, and succinic acid reduced with the supplementation of nitrogen sources. The results revealed significant application importance for the winemaking industry.

Screening of Saccharomyces strains for the capacity to produce desirable fermentative compounds under the influence of different nitrogen sources in synthetic wine fermentations.

A collection of 33 Saccharomyces yeasts were used for wine fermentation with a sole nitrogen source: ammonium and four individual aroma-inducing amino acids. The fermentation performance and chemical wine composition were evaluated. The most valuable nitrogen sources were valine as a fermentation promoter on non-cerevisiae strains, phenylalanine as fruity aromas enhancer whereas the ethanol yield was lessened by leucine and isoleucine. S. cerevisiae SC03 and S. kudriavzevii SK02 strains showed to be the greatest producers of fruity ethyl esters while S. kudriavzevii strains SK06 and SK07 by shortening the fermentation duration. S. uvarum strains produced the greatest succinic acid amounts and, together with S. eubayanus, they reached the highest production of 2-phenylethanol and its acetate ester; whereas S. kudriavzevii strains were found to be positively related to high glycerol production.

Thermo-adaptive evolution to generate improved Saccharomyces cerevisiae strains for cocoa pulp fermentations.

Cocoa pulp fermentation is a consequence of the succession of indigenous yeasts, lactic acid bacteria and acetic acid bacteria that not only produce a diversity of metabolites, but also cause the production of flavour precursors. However, as such spontaneous fermentations are less reproducible and contribute to produce variability, interest in a microbial starter culture is growing that could be used to inoculate cocoa pulp fermentations. This study aimed to generate robust S. cerevisiae strains by thermo-adaptive evolution that could be used in cocoa fermentation. We evolved a cocoa strain in a sugary defined medium at high temperature to improve both fermentation and growth capacity. Moreover, adaptive evolution at high temperature (40 °C) also enabled us to unveil the molecular basis underlying the improved phenotype by analysing the whole genome sequence of the evolved strain. Adaptation to high-temperature conditions occurred at different genomic levels, and promoted aneuploidies, segmental duplication, and SNVs in the evolved strain. The lipid profile analysis of the evolved strain also evidenced changes in the membrane composition that contribute to maintain an appropriate cell membrane state at high temperature. Our work demonstrates that experimental evolution is an effective approach to generate better-adapted yeast strains at high temperature for industrial processes.

Phenotypic and genomic differences among S. cerevisiae strains in nitrogen requirements during wine fermentations.

Nitrogen requirements by S. cerevisiae during wine fermentation are highly strain-dependent. Different approaches were applied to explore the nitrogen requirements of 28 wine yeast strains. Based on the growth and fermentation behaviour displayed at different nitrogen concentrations, high and low nitrogen-demanding strains were selected and further verified by competition fermentation. Biomass production with increasing nitrogen concentrations in the exponential fermentation phase was analysed by chemostat cultures. Low nitrogen-demanding (LND) strains produced a larger amount of biomass in nitrogen-limited synthetic grape musts, whereas high nitrogen-demanding (HND) strains achieved a bigger biomass yield when the YAN concentration was above 100 mg/L. Constant rate fermentation was carried out with both strains to determine the amount of nitrogen required to maintain the highest fermentation rate. Large differences appeared in the analysis of the genomes of low and high-nitrogen demanding strains showed for heterozygosity and the amino acid substitutions between orthologous proteins, with nitrogen recycling system genes showing the widest amino acid divergences. The CRISPR/Cas9-mediated genome modification method was used to validate the involvement of GCN1 in the yeast strain nitrogen needs. However, the allele swapping of gene GCN1 from low nitrogen-demanding strains to high nitrogen-demanding strains did not significantly influence the fermentation rate.

Differential proteomic analysis by SWATH-MS unravels the most dominant mechanisms underlying yeast adaptation to non-optimal temperatures under anaerobic conditions.

Elucidation of temperature tolerance mechanisms in yeast is essential for enhancing cellular robustness of strains, providing more economically and sustainable processes. We investigated the differential responses of three distinct Saccharomyces cerevisiae strains, an industrial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and an industrial bioethanol strain, Ethanol Red, grown at sub- and supra-optimal temperatures under chemostat conditions. We employed anaerobic conditions, mimicking the industrial processes. The proteomic profile of these strains in all conditions was performed by sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS), allowing the quantification of 997 proteins, data available via ProteomeXchange (PXD016567). Our analysis demonstrated that temperature responses differ between the strains; however, we also found some common responsive proteins, revealing that the response to temperature involves general stress and specific mechanisms. Overall, sub-optimal temperature conditions involved a higher remodeling of the proteome. The proteomic data evidenced that the cold response involves strong repression of translation-related proteins as well as induction of amino acid metabolism, together with components related to protein folding and degradation while, the high temperature response mainly recruits amino acid metabolism. Our study provides a global and thorough insight into how growth temperature affects the yeast proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance.

Isotopic Tracers Unveil Distinct Fates for Nitrogen Sources during Wine Fermentation with Two Non-Saccharomyces Strains.

Non-Saccharomyces yeast strains have become increasingly prevalent in the food industry, particularly in winemaking, because of their properties of interest both in biological control and in complexifying flavour profiles in end-products. However, unleashing the full potential of these species would require solid knowledge of their physiology and metabolism, which is, however, very limited to date. In this study, a quantitative analysis using 15N-labelled NH4Cl, arginine, and glutamine, and 13C-labelled leucine and valine revealed the specificities of the nitrogen metabolism pattern of two non-Saccharomyces species, Torulaspora delbrueckii and Metschnikowia pulcherrima. In T. delbrueckii, consumed nitrogen sources were mainly directed towards the de novo synthesis of proteinogenic amino acids, at the expense of volatile compounds production. This redistribution pattern was in line with the high biomass-producer phenotype of this species. Conversely, in M. pulcherrima, which displayed weaker growth capacities, a larger proportion of consumed amino acids was catabolised for the production of higher alcohols through the Ehrlich pathway. Overall, this comprehensive overview of nitrogen redistribution in T. delbrueckii and M. pulcherrima provides valuable information for a better management of co- or sequential fermentation combining these species with Saccharomyces cerevisiae.

GTR1 Affects Nitrogen Consumption and TORC1 Activity in Saccharomyces cerevisiae Under Fermentation Conditions.

The TORC1 pathway coordinates cell growth in response to nitrogen availability present in the medium, regulating genes related to nitrogen transport and metabolism. Therefore, the adaptation of Saccharomyces cerevisiae to changes in nitrogen availability implies variations in the activity of this signaling pathway. In this sense, variations in nitrogen detection and signaling pathway are one of the main causes of differences in nitrogen assimilation during alcoholic fermentation. Previously, we demonstrated that allelic variants in the GTR1 gene underlying differences in ammonium and amino acids consumption between Wine/European (WE) and West African (WA) strains impact the expression of nitrogen transporters. The GTR1 gene encodes a GTPase that participates in the EGO complex responsible for TORC1 activation in response to amino acids availability. In this work, we assessed the role of the GTR1 gene on nitrogen consumption under fermentation conditions, using a high sugar concentration medium with nitrogen limitation and in the context of the WE and WA genetic backgrounds. The gtr1Δ mutant presented a reduced TORC1 activity and increased expression levels of nitrogen transporters, which in turn favored ammonium consumption, but decreased amino acid assimilation. Furthermore, to identify the SNPs responsible for differences in nitrogen consumption during alcoholic fermentation, we studied the polymorphisms present in the GTR1 gene. We carried out swapping experiments for the promoter and coding regions of GTR1 between the WE and WA strains. We observed that polymorphisms in the coding region of the WA GTR1 gene are relevant for TORC1 activity. Altogether, our results highlight the role of the GTR1 gene on nitrogen consumption in S. cerevisiae under fermentation conditions.

Selection and subsequent physiological characterization of industrial Saccharomyces cerevisiae strains during continuous growth at sub- and- supra optimal temperatures.

A phenotypic screening of 12 industrial yeast strains and the well-studied laboratory strain CEN.PK113-7D at cultivation temperatures between 12 °C and 40 °C revealed significant differences in maximum growth rates and temperature tolerance. From those 12, two strains, one performing best at 12 °C and the other at 40 °C, plus the laboratory strain, were selected for further physiological characterization in well-controlled bioreactors. The strains were grown in anaerobic chemostats, at a fixed specific growth rate of 0.03 h-1 and sequential batch cultures at 12 °C, 30 °C, and 39 °C. We observed significant differences in biomass and ethanol yields on glucose, biomass protein and storage carbohydrate contents, and biomass yields on ATP between strains and cultivation temperatures. Increased temperature tolerance coincided with higher energetic efficiency of cell growth, indicating that temperature intolerance is a result of energy wasting processes, such as increased turnover of cellular components (e.g. proteins) due to temperature induced damage.

Differential Contribution of the Parental Genomes to a S. cerevisiae × S. uvarum Hybrid, Inferred by Phenomic, Genomic, and Transcriptomic Analyses, at Different Industrial Stress Conditions.

In European regions of cold climate, S. uvarum can replace S. cerevisiae in wine fermentations performed at low temperatures. S. uvarum is a cryotolerant yeast that produces more glycerol, less acetic acid and exhibits a better aroma profile. However, this species exhibits a poor ethanol tolerance compared with S. cerevisiae. In the present study, we obtained by rare mating (non-GMO strategy), and a subsequent sporulation, an interspecific S. cerevisiae × S. uvarum spore-derivative hybrid that improves or maintains a combination of parental traits of interest for the wine industry, such as good fermentation performance, increased ethanol tolerance, and high glycerol and aroma productions. Genomic sequencing analysis showed that the artificial spore-derivative hybrid is an allotriploid, which is very common among natural hybrids. Its genome contains one genome copy from the S. uvarum parental genome and two heterozygous copies of the S. cerevisiae parental genome, with the exception of a monosomic S. cerevisiae chromosome III, where the sex-determining MAT locus is located. This genome constitution supports that the original hybrid from which the spore was obtained likely originated by a rare-mating event between a mating-competent S. cerevisiae diploid cell and either a diploid or a haploid S. uvarum cell of the opposite mating type. Moreover, a comparative transcriptomic analysis reveals that each spore-derivative hybrid subgenome is regulating different processes during the fermentation, in which each parental species has demonstrated to be more efficient. Therefore, interactions between the two subgenomes in the spore-derivative hybrid improve those differential species-specific adaptations to the wine fermentation environments, already present in the parental species.

Growth and lipid production of Rhodotorula glutinis R4, in comparison to other oleaginous yeasts.

Some Rhodotorula spp. have been characterized as oleaginous yeasts. Under certain culture conditions they can accumulate neutral lipids, which are mainly triglycerides (TAG). Microbial TAG that can be used as raw material for biodiesel synthesis are attractive for the biofuel industry. In this study, the ability to synthesize lipids of Rhodotorula glutinis R4, isolated in Antarctica, was compared with eight strains belonging to the genera Rhodotorula and Yarrowia with the aim of proposing a novel source of oils for biodiesel synthesis. All strains were cultured under nitrogen (N) limiting conditions and an excess of carbon (C) in the culture medium. We found that yeasts accumulated between 9-48.9 % (w/w) of lipids. Among them, R. glutinis R4 showed the highest growth (14 g L-1, µmax 0,092 h-1) and lipid production (7 g L-1; 47 % w/w). Microbial oils produced by R. glutinis R4 are similar to vegetable oils, with 61 % of oleic acid, indicating that it is adequate for biodiesel synthesis. Our results demonstrate that biodiesel derived from R. glutinis R4 complies with international fuel standards ASTM D6751 and EN 14214. Therefore, this work demonstrates that Rhodotorula glutinis R4 is a novel and valuable source of microbial oils for biodiesel synthesis.

Overproduction of hydroxytyrosol in Saccharomyces cerevisiae by heterologous overexpression of the Escherichia coli 4-hydroxyphenylacetate 3-monooxygenase.

Hydroxytyrosol (HT), which is a polyphenol with a high antioxidant power and many associated health benefits, has been found in wines. Wine yeasts are capable of producing high amounts of the higher alcohol tyrosol, which is the precursor for HT synthesis. We have improved the ability of Saccharomyces cerevisiae to produce HT by heterologously expressing the HpaBC enzyme complex of Escherichia coli, which hydroxylates tyrosol into HT. By overexpressing the hpaB and hpaC genes, we achieved HT titers of 1.15 ±â€¯0.05 mg/L and 4.6 ±â€¯0.9 mg/L in a minimal medium in which either 1 mM tyrosine or 1 mM tyrosol were respectively added. This work demonstrates that the overexpression of HpaBC in yeast is a promising tool to overproduce HT at the expense of endogenous tyrosol through central carbon catabolism flux redirection to tyrosine catabolism.

Nitrogen sources preferences of non-Saccharomyces yeasts to sustain growth and fermentation under winemaking conditions.

Wine-related non-Saccharomyces yeasts are becoming more widely used in oenological practice for their ability to confer wine a more complex satisfying aroma, but their metabolism remains unknown. Our study explored the nitrogen utilisation profile of three popular non-Saccharomyces species, Torulaspora delbrueckii, Metschnikowia pulcherrima and Metschnikowia fructicola. The nitrogen source preferences to support growth and fermentation as well as the uptake order of different nitrogen sources during wine fermentation were investigated. While T. delbrueckii and S. cerevisiae strains shared the same nitrogen source preferences, Metschnikowia sp. Displayed a lower capacity to efficiently use the preferred nitrogen compounds, but were able to assimilate a wider range of amino acids. During alcoholic fermentation, the non-Saccharomyces strains consumed different nitrogen sources in a similar order as S. cerevisiae, but not as quickly. Furthermore, when all the nitrogen sources were supplied in the same amount, their assimilation order was similarly affected for both S. cerevisiae and non-Saccharomyces strains. Under this condition, the rate of nitrogen source consumption of non-Saccharomyces strains and S. cerevisiae was comparable. Overall, this study expands our understanding about the preferences and consumption rates of individual nitrogen sources by the investigated non-Saccharomyces yeasts in a wine environment. This knowledge provides useful information for a more efficient exploitation of non-Saccharomyces strains that improves the management of the wine fermentation.