The Hydrophilic C-terminus of Yeast Plasma-membrane Na+/H+ Antiporters Impacts Their Ability to Transport K.

Yeast plasma-membrane Na+/H+ antiporters (Nha/Sod) ensure the optimal intracellular level of alkali-metal cations and protons in cells. They are predicted to consist of 13 transmembrane segments (TMSs) and a large hydrophilic C-terminal cytoplasmic part with seven conserved domains. The substrate specificity, specifically the ability to recognize and transport K+ cations in addition to Na+ and Li+, differs among homologs. In this work, we reveal that the composition of the C-terminus impacts the ability of antiporters to transport particular cations. In the osmotolerant yeast Zygosaccharomyces rouxii, the Sod2-22 antiporter only efficiently exports Na+ and Li+, but not K+. The introduction of a negative charge or removal of a positive charge in one of the C-terminal conserved regions (C3) enabled ZrSod2-22 to transport K+. The same mutations rescued the low level of activity and purely Li+ specificity of ZrSod2-22 with the A179T mutation in TMS6, suggesting a possible interaction between this TMS and the C-terminus. The truncation or replacement of the C-terminal part of ZrSod2-22 with the C-terminus of a K+-transporting Nha/Sod antiporter (Saccharomyces cerevisiae Nha1 or Z. rouxii Nha1) also resulted in an antiporter with the capacity to export K+. In addition, in ScNha1, the replacement of three positively charged arginine residues 539-541 in the C3 region with alanine caused its inability to provide cells with tolerance to Li+. All our results demonstrate that the physiological functions of yeast Nha/Sod antiporters, either in salt tolerance or in K+ homeostasis, depend on the composition of their C-terminal parts.

Sustainable bio-manufacturing of D-arabitol through combinatorial engineering of Zygosaccharomyces rouxii, bioprocess optimization and downstream separation.

Biosynthesis of D-arabitol, a high value-added platform chemical, from renewable carbon sources provides a sustainable and eco-friendly alternative to the chemical industry. Here, a robust brewing yeast, Zygosaccharomyces rouxii, capable of naturally producing D-arabitol was rewired through genome sequencing-based metabolic engineering. The recombinant Z. rouxii obtained by reinforcing the native D-xylulose pathway, improving reductive power of the rate-limiting step, and inhibiting the shunt pathway, produced 73.61% higher D-arabitol than the parent strain. Subsequently, optimization of the fermentation medium composition for the engineered strain provided 137.36 g/L D-arabitol, with a productivity of 0.64 g/L/h in a fed-batch experiment. Finally, the downstream separation of D-arabitol from the complex fermentation broth using an ethanol precipitation method provided a purity of 96.53%. This study highlights the importance of D-xylulose pathway modification in D-arabitol biosynthesis, and pave a complete and efficient way for the sustainable manufacturing of this value-added compound from biosynthesis to preparation.

Serine Improves Lactic Acid Stress Tolerance and Ethanol Production in Zygosaccharomyces bailii in Baijiu Fermentation.

Lactic acid is the primary inhibitor of the growth and ethanol production of yeasts in Baijiu fermentation. Certain amino acids have been found to be related to stress tolerance in yeasts. This study explored the effect of lactic acid stress on the ethanol-producing yeast Zygosaccharomyces bailii and evaluated the ability of serine to increase the lactic acid tolerance of Z. bailii in vitro. Serine significantly improved Z. bailii viability by 16.5% and ethanol production by 226.6% under lactic acid stress. Under lactic acid stress, serine supplementation led to an increase of 41.9% in cell wall integrity, 31.9% in cell membrane integrity, 296.6% in intracellular adenosine triphosphate (ATP), and 18.4% in the mitochondrial membrane potential. Finally, field emission scanning electron microscopy (FESEM) indicated that serine supplementation maintained the cell shape and reduced cell leakage. This study revealed a novel lactic acid tolerance mechanism of core functional yeasts during Jiang-flavor Baijiu fermentation.

Exploring the correlation of metabolites changes and microbial succession in solid-state fermentation of Sichuan Sun-dried vinegar.

BACKGROUND: The traditional Sichuan Sun-dried vinegar (SSV) with unique flavor and taste is believed to be generated by the solid-state fermentation craft. However, how microorganisms and their metabolites change along with fermentation has not yet been explored. RESULTS: In this study, our results demonstrated that the middle and late stages of SSV fermentation were the periods showing the largest accumulation of organic acids and amino acids. Furthermore, in the bacterial community, the highest average relative abundance was Lactobacillus (ranging from 37.55 to 92.50%) in all fermentation stages, while Acetobacters ranked second position (ranging from 20.15 to 0.55%). The number of culturable lactic acid bacteria is also increased during fermentation process (ranging from 3.93 to 8.31 CFU/g). In fungal community, Alternaria (29.42%), Issatchenkia (37.56%) and Zygosaccharomyces (69.24%) were most abundant in different fermentation stages, respectively. Interestingly, Zygosaccharomyces, Schwanniomyces and Issatchenkia were first noticed as the dominant yeast genera in vinegar fermentation process. Additionally, spearman correlation coefficients exhibited that Lactobacillus, Zygosaccharomyces and Schwanniomyces were significant correlation with most metabolites during the fermentation, implying that these microorganisms might make a significant contribution to the flavor formation of SSV. CONCLUSION: The unique flavor of SSV is mainly produced by the core microorganisms (Lactobacillus, Zygosaccharomyces and Schwanniomyces) during fermentation. This study will provide detailed information related to the structure of microorganism and correlation between changes in metabolites and microbial succession in SSV. And it will be very helpful for proposing a potential approach to monitor the traditional fermentation process.

Transcriptomics Reveals the Effect of Strain Interactions on the Growth of A. Oryzae and Z. Rouxii.

The microbial community structure in traditional fermented foods is quite complex, making the relationship between strains unclear. In this regard, the co-culture system can simulate microbial interactions during food fermentation and reveal the morphological changes, metabolic processes, and gene expression of microbial communities. The present study sought to investigate the effects of microbial interactions on the growth of Aspergillus oryzae and Zygosaccharomyces rouxii through omics. After co-cultivation, the pH value and dry weight were consistent with the pure culture of Z. rouxii. Additionally, the consumption of reducing sugar decreased, and the enzymatic activity increased compared with the pure culture of fungus. The analysis of volatile organic compounds (VOCs) and transcriptomics showed that co-culture significantly promoted the effect on Z. rouxii. A total of 6 different VOCs and 2202 differentially expressed genes were identified in the pure and co-culture of Z. rouxii. The differentially expressed genes were mainly related to the endonucleolytic cleavage of rRNA, ribosome biogenesis in eukaryotes, and RNA polymerase metabolic pathways. The study results will provide insights into the effect of microbial interactions on the growth of A. oryzae and Z. rouxii.

High-level production of d-arabitol by Zygosaccharomyces rouxii from glucose: Metabolic engineering and process optimization.

d-Arabitol is a top value-added compound with wide applications in the food, pharmaceutical and biochemical industries. Nevertheless, sustainable biosynthesis of d-arabitol is limited by lack of efficient strains and suitable fermentation process. Herein, metabolic engineering and process optimization were performed in Zygosaccharomyces rouxii to overcoming these limitations. Adopting systems metabolic engineering include enhancement of innate biosynthetic pathway, supply of precursor substrate d-ribulose-5P and cofactors regeneration, a novel recombinant strain ZR-5A with good performance was obtained, which boosted d-arabitol production up to 29.01 g/L, 59.31 % higher than the parent strain. Further with the optimum medium composition and fed-batch fermentation, the strain ZR-5A finally produced 149.10 g/L d-arabitol with the productivity of 1.04 g/L/h, which was the highest titer ever reported by Z.rouxii system. This is the first report on the use of metabolic engineering to construct Z. rouxii chassis for the sustainable production of d-arabitol.

Molecular Tools for Leveraging the Potential of the Acid-Tolerant Yeast Zygosaccharomyces bailii as Cell Factory.

Microorganisms offer a tremendous potential as cell factories, and they are indeed been used by humans since the previous centuries for biotransformations. Among them, yeasts combine the advantage of a unicellular state with a eukaryotic organization. Moreover, in the era of biorefineries, their biodiversity can offer solutions to specific process constraints. Zygosaccharomyces bailii, an ascomycete budding yeast, is widely known for its peculiar tolerance to different stresses, among which are organic acids. Moreover, the recent reclassification of the species, including diverse hybrids, is further expanding both fundamental and applied interests. It is therefore reasonable that despite the possibility to apply with this yeast some of the molecular tools and protocols routinely used to manipulate Saccharomyces cerevisiae, adjustments and optimizations are necessary. Here we describe in detail the methods for determining chromosome number, size, and aneuploidy, transformation, classical target gene disruption or gene integration, and designing of episomal expression plasmids helpful for engineering the yeast Z. bailii .

Effects of microbial community structure and its co-occurrence on the dynamic changes of physicochemical properties and free amino acids in the Cantonese soy sauce fermentation process.

The soy sauce produced by Cantonese fermentation has a unique flavor, among which brine fermentation plays an important role. In this fermentation process, 61 volatile compounds, including 19 esters, 10 aldehydes, 9 alcohols, 5 phenols, and 18 others, were identified by headspace solid-phase microextraction-gas chromatography-mass spectrometry. Seventeen kinds of free amino acids were detected by high-performance liquid chromatography. Results showed that Touyou, which comprised 1.5 g/100 g total nitrogen, 1.0 g/100 mL amino acid nitrogen, 3.66 g/100 g reducing sugar, 1.44 g/100 mL total acid, 17.04 g/100 mL salt content, and 27.3% umami free amino acids, had excellent quality. High-throughput sequencing was used to identify microorganisms. The top 3 of bacteria were Weissella, Staphylococcus, and Lactobacillus, and the top 3 fungi were Aspergillus, Zygosaccharomyces, and Candida. The co-occurrence network analysis of microorganisms showed that the top-ranked microorganisms were Plectosphaerella, Aureobasidium, unidentified_Mortierellales_sp, Glutinomyces, Faecalibacterium, and Cladophialophora. Then, eight microorganisms (VIP[pred] > 1) were obtained by two-way orthogonal partial least squares model, namely, Staphylococcus, Candida, Weissella, Aspergillus, Zygosaccharomyces, Lactobacillus, Monilinia, and Clavispora. Correlation analysis showed that these microorganisms were strongly related to flavor metabolites. This study explored the dynamics of traditional Cantonese fermentation, which has positive implications for optimizing this traditional fermentation process.

Metabolomics analysis of salt tolerance of Zygosaccharomyces rouxii and guided exogenous fatty acid addition for improved salt tolerance.

BACKGROUND: Zygosaccharomyces rouxii plays an irreplaceable role in the manufacture of traditional fermented foods, which are produced in a high-salt environment. However, there is little research on strategies for improving salt tolerance of Z. rouxii. RESULTS: In this study, metabolomics was used to reveal the changes in intracellular metabolites under salt stress, and the results show that most of the carbohydrate contents decreased, the contents of xanthohumol and glycerol increased (fold change 4.07 and 5.35, respectively), while the contents of galactinol, xylitol and d-threitol decreased (fold change -9.43, -5.83 and -3.59, respectively). In addition, the content of four amino acids and six organic acids decreased, while that of the ten nucleotides increased. Notably, except for stearic acid (C18:0), all fatty acid contents increased. Guided by the metabolomics results, the effect of addition of seven exogenous fatty acids (C12:0, C14:0, C16:0, C18:0, C16:1, C18:1, and C18:2) on the salt tolerance of Z. rouxii was analyzed, and the results suggested that four exogenous fatty acids (C12:0, C16:0, C16:1, and C18:1) can increase the biomass yield and maximum growth rate. Physiological analyses demonstrated that exogenous fatty acids could regulate the distribution of fatty acids in the cell membrane, increase the degree of unsaturation, improve membrane fluidity, and maintain cell integrity, morphology and surface roughness. CONCLUSION: These results are applicable to revealing the metabolic mechanisms of Z. rouxii under salt stress and screening potential protective agents to improve stress resistance by adding exogenous fatty acids. © 2022 Society of Chemical Industry.

Regulation of mating and mating-type-specific genes in Zygosaccharomyces sp. yeast.

Zygosaccharomyces sp. is an industrially important yeast for the production traditional fermented foods in Japan. At present, however, there is no easy method for mating Zygosaccharomyces sp. strains in the laboratory; furthermore, little is known about the expression of mating-type-specific genes in this yeast. Here, mating was observed when Zygosaccharomyces sp. was subjected to nitrogen-starvation conditions. The expression of mating-type-specific genes, Zygo STE6 and Zygo MFα1, was induced under nitrogen-starvation conditions, as confirmed by lacZ reporter assay. This expression was mating-type-specific: Zygo STE6 was expressed specifically for mating-type a, whereas and Zygo MFα1 was expressed specifically for mating-type α. Yeast strains Zygosaccharomyces rouxii DL2 and DA2, derived from type strain Z. rouxii CBS732, did not show mating even under nitrogen-starvation conditions. Gene sequencing revealed that the Zygo STE12 in Z. rouxii CBS732 has a frameshift mutation. Under nitrogen starvation, mating was observed in both DL2 and DA2 transformed with the wild-type Zygo STE12. The expression of Zygo STE6 in Z. rouxii DL2 transformed with wild-type Zygo STE12 under nitrogen-starvation conditions was confirmed by lacZ reporter assay. Collectively, these results revealed that, under nitrogen-starvation conditions, Zygosaccharomyces sp. can mate and mating-type-specific genes are expressed. Furthermore, Zygo Ste12 is essential for both mating and the expression of mating-type-specific genes in Zygosaccharomyces sp.

Nuclear magnetic resonance investigation of water transport through the plasma membrane of various yeast species.

A specific technique of nuclear magnetic resonance (NMR) spectroscopy, filter-exchange spectroscopy (FEXSY), was employed to investigate water transport through the plasma membrane in intact yeast cells. This technique allows water transport to be monitored directly, thus avoiding the necessity to subject the cells to any rapid change in the external conditions, e.g. osmotic shock. We established a sample preparation protocol, a data analysis procedure and verified the applicability of FEXSY experiments. We recorded the exchange rates in the temperature range 10-40°C for Saccharomyces cerevisiae. The resulting activation energy of 29 kJ mol-1 supports the hypothesis that water exchange is facilitated by water channels-aquaporins. Furthermore, we measured for the first time water exchange rates in three other phylogenetically unrelated yeast species (Schizosaccharomyces pombe, Candida albicans and Zygosaccharomyces rouxii) and observed remarkably different water exchange rates between these species. Findings of our work contribute to a better understanding of as fundamental a cell process as the control of water transport through the plasma membrane.

A set of plasmids carrying antibiotic resistance markers and Cre recombinase for genetic engineering of nonconventional yeast Zygosaccharomyces rouxii.

The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T . By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX-loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATαP expression locus with the loxP-kanMX-loxP cassette.

Characterizing the metabolites and the microbial communities of the soy sauce mash affected by temperature and hydrostatic pressure.

The effect of hydrostatic pressure (HSP) and constant temperature fermentation (CTF) on the microbial community of soy sauce mash and metabolites (volatile and non-volatile) in raw soy sauce were investigated by multiphase methods. Soy sauce inoculated with yeasts (YG) or a mixture of yeasts and bacteria (MYG) were used in the present research. The results suggested that the effect of HSP resulted in decreasing of fungal community diversity, while CTF caused changes of the evenness of fungi species, which were also confirmed by alpha/beta diversity and clustering analysis. The volatile compounds analysis indicated that the total volatiles decreased by 47.53% in the raw soy sauce fortified by MYG under HSP, while that of organic acids and free amino acids were increased. The effect of CTF on volatile compounds depended on the fortified pattern. The community diversity and metabolite features between fortified and conventional soy sauce were also different.

Heterologous expression and functional analysis of the F-box protein Ucc1 from other yeast species in Saccharomyces cerevisiae.

The ubiquitin-proteasome system plays an important role in metabolic regulation. In a previous study, we reported that, in Saccharomyces cerevisiae, when glucose is available, the SCFUcc1 ubiquitin ligase complex targets citrate synthase 2 (Cit2) for proteasomal degradation, thereby suppressing the glyoxylate cycle, an anabolic pathway that replenishes the TCA cycle with succinate for the activation of gluconeogenesis. However, the roles of Ucc1 in other yeast species remain unclear. Here, we cloned orthologs of the F-box protein Ucc1 from Zygosaccharomyces bailii, an aggressive food spoilage microorganism that is the most acetic acid-tolerant yeast species, and Candida glabrata, an emerging fungal pathogen. These orthologs were expressed in S. cerevisiae, and their activities were tested genetically and biochemically. The results showed that Z. bailii Ucc1 rescued the ucc1Δ phenotype, suggesting the existence of a similar mechanism regulating the glyoxylate cycle in Z. bailii. By contrast, C. glabrata Ucc1 did not complement the ucc1Δ phenotype or exhibit a dominant negative effect on Ucc1. These results suggest the importance of analysing the regulatory mechanisms of glyoxylate cycle in a broad range of yeast species.

Zygosaccharomyces bailii and Z. rouxii induced ethanol formation in apple juice supplemented with different natural preservatives: A response surface methodology approach.

In this study, ethanol produced by osmophilic yeasts, Zygosaccharomyces bailii and Z. rouxii, in apple juice preserved with mint essential oil (MEO), carvacrol and natamycin instead of synthetic preservatives was modeled. Some processing parameters such as sodium benzoate (SB, 0-0.1%) used as a positive control, storage temperature (4-20 °C) and storage time (1-41 days) were selected in the study. Box-Behnken design in response surface methodology was used to evaluate the effects of processing parameters on ethanol levels of apple juice and three models were created for three preservatives for each yeast. Preservative type affected the ethanol formation in apple juice for both yeasts studied. Increase of preservative concentration decreased the ethanol formation during the storage period. The best effective preservative was determined as MEO and Z. bailii was found to be quite resistant yeast against to the preserving agents for three models as compared to Z. rouxii. Ethanol level increased with the increase of both storage temperature and time for both yeasts. The results showed that apple juice could be preserved by these three preservatives, but the MEO was the most effective agent for apple juice during the storage.

Effect of raw material and starters on the metabolite constituents and microbial community diversity of fermented soy sauce.

BACKGROUND: The quality of soy sauce is strongly affected by microorganisms and raw materials (defatted soybean or whole soybean). The present study investigated the effect of two types of fortified pattern, including inoculation with starters (Tetragenococcus halophilus combined with Zygosaccharomyces rouxii and Candida versatilis), and adding culture medium (saccharified rice flour solution), on the metabolite profiles and microbial community of soy sauce produced from defatted soybean (DP) and whole soybean (HD). Relationships between microbes and volatiles, and their interactions, were shown. RESULTS: The dominant metabolites differed in the soy sauce samples except for isoflavones. Alcohols and phenols were higher in DP moromi. Two classes of dominant esters, long-chain fatty acid esters (LFAE) and unsaturated-short-chain fatty acid esters (USFAE), were higher in HD moromi than DP. Weissella, Leuconostoc, and Aspergillus were the dominant microbes. Leuconostoc, and Aspergillus increased, and Weissella decreased in moromi inoculated with starters compared with a control. Similar changes to Leuconostoc were observed in moromi added culture medium. CONCLUSIONS: The microbes were responsible for the formation of volatiles. The intergeneric interactions with microbes were affected by fortified pattern. The effect of starters or culture medium on microbial community and metabolites of soy sauce depended on the raw material. © 2019 Society of Chemical Industry.

Effect of co-culture with Tetragenococcus halophilus on the physiological characterization and transcription profiling of Zygosaccharomyces rouxii.

Zygosaccharomyces rouxii and Tetragenococcus halophilus are widely existed and play vital roles during the manufacture of fermented foods such as soy sauce. The aim of this study was to elucidate the effect of T. halophilus CGMCC 3792 on the physiological characterizations and transcription profiling of Z. rouxii CGMCC 3791. Salt tolerance analysis revealed that co-culture with T. halophilus enhanced the salt tolerance of Z. rouxii during salt stress. Analysis of the volatile compounds revealed that co-culture reduced the level of 1-butanol, improved the level of octanoic acid which all were produced by T. halophilus and reduced the level of phenylethyl alcohol produced by Z. rouxii. The presence of Z. rouxii decreased the contents of 3,4-dimethylbenzaldehyde and acetic acid produced by T. halophilus. In addition, co-culture improved the content of benzyl alcohol significantly. Analysis of membrane fatty acid showed that co-culture improved the content of palmitic (C16:0) and stearic (C18:0) acids in cells of Z. rouxii, and reduced the contents of myristic (C14:0), palmitoleic acid (C16:1) and oleic acid (C18:1). In order to further explore the interactions between the two strains, RNA-seq technology was used to investigate the effect of co-culture with T. halophilus on the transcription profiling of Z. rouxii. By comparing cells incubated in co-culture group with cells incubated in single-culture group, a total of 967 genes were considered as differentially expressed genes (DEGs). Among the DEGs, 72 genes were up-regulated, while 895 genes were down-regulated. These DEGs took party in various activities in cells of Z. rouxii, and the result showed co-culture with T. halophilus had a positive effect on proteolysis, the attachment of a cell to another cell, extracellular protein accumulation, energy metabolism, and a negative effect on oxidative phosphorylation, small molecular substances metabolism, DNA replication and repair, and transcription in cells of Z. rouxii. Results presented in this study may contribute to further understand the interactions between Zygosaccharomyces rouxii and Tetragenococcus halophilus.

Soy sauce fermentation: Microorganisms, aroma formation, and process modification.

Soy sauce is an increasingly popular oriental fermented condiment produced through a two-step fermentation process called koji (solid-state fermentation) and moromi (brine fermentation). Complex microbial interactions play an essential role in its flavor development during the fermentation. Tetragenococcus halophilus and Zygosaccharomyces rouxii are predominant among the microbes involved in the moromi stage. Despite their importance for producing a wide range of volatile compounds, antagonism can occur due to different growth condition requirements. Furthermore, microbial interactions in moromi fermentation are affected by current efforts to reduce salt in soy sauce, in order to tackle slow fermentation due to low metabolic activity of microbes and increased health risk related to high sodium intake. Attempts to enhance and accelerate flavor formation in the presence of high salt concentration include the inoculation with mixed starter cultures, genetic modification, cell, and enzyme immobilization. Although salt reduction can accelerate the microbial growth, the flavor quality of soy sauce is compromised. Several approaches have been applied to compensate such loss in quality, including the use of salt substitutes, combination of indigenous cultures, pretreatment of raw material and starter cultures encapsulation. This review discusses the role of microorganisms in soy sauce production in relation to flavor formation and changes in production practices.

Physiological Genomics of the Highly Weak-Acid-Tolerant Food Spoilage Yeasts of Zygosaccharomyces bailii sensu lato.

Zygosaccharomyces bailii and two closely related species, Z. parabailii and Z. pseudobailii ("Z. bailii species complex", "Z. bailii sensu lato" or simply "Z. bailii (s.l.)"), are frequently implicated in the spoilage of acidified preserved foods and beverages due to their tolerance to very high concentrations of weak acids used as food preservatives. The recent sequencing and annotation of these species' genomes have clarified their genomic organization and phylogenetic relationship, which includes events of interspecies hybridization. Mechanistic insights into their adaptation and tolerance to weak acids (e.g., acetic and lactic acids) are also being revealed. Moreover, the potential of Z. bailii (s.l.) to be used in industrial biotechnological processes as interesting cell factories for the production of organic acids, reduction of the ethanol content, increase of alcoholic beverages aroma complexity, as well as of genetic source for increasing weak acid resistance in yeast, is currently being considered. This chapter includes taxonomical, ecological, physiological, and biochemical aspects of Z. bailii (s.l.). The focus is on the exploitation of physiological genomics approaches that are providing the indispensable holistic knowledge to guide the effective design of strategies to overcome food spoilage or the rational exploitation of these yeasts as promising cell factories.

Fermentative capabilities of native yeast strains grown on juices from different Agave species used for tequila and mezcal production.

The Asparagaceae family is endemic from America, being the Agave genus the most important. The Agave species possess economic relevance and are use as raw material to produce several distilled alcoholic beverages, as bacanora, tequila, and mezcal. The fermentation process has been carry out either spontaneously or by adding a selected yeast strain. The latter is generally responsible for the production of ethanol and volatile compounds. This study comprised five Agave species (A. angustifolia, A. cupreata, A. durangensis, A. salmiana, and A. tequilana) and eight endogenous yeast strains: five of them were non-Saccharomyces (Torulaspora delbrueckii, Zygosaccharomyces bisporus, Candida ethanolica, and two Kluyveromyces marxianus) and three Saccharomyces cerevisiae strains. The results showed that the S. cerevisiae strains were not able to grow on A. durangensis and A. salmiana juices. The Kluyveromyces marxianus strains grew and fermented all the agave juices and displayed high ethanol production (48-52 g L-1) and volatile compounds. The ethanol production was higher on A. angustifolia juice (1.1-2.8-fold), whereas the volatile compound was dependent on both yeast strain and the Agave species. The use of endogenous non-Saccharomyces yeast strains is feasible, as they may outperform S. cerevisiae regarding the production of fermented beverages from agave plants with a high content of ethanol and aromatic compounds. Graphical abstract.