Non-conventional Yeast Species Research Articles

Unravelling yeast cellulase potential: A computational approach to structural study, cellulolytic activity, and docking

The discovery of highly efficient catalysts capable of enhancing the biomass conversion represents a significant challenge within the biofuel production sector. Cellulolytic enzymes originating from microorganisms are promising catalysts for bioethanol conversion. In this study, we conducted a comparative analysis of cellulase, especially Endo β-1,4 glucanase enzymes, from five different non-conventional yeast species. These enzymes were evaluated in the presence of microcrystalline cellulose as the substrate, and their structural, physicochemical properties and molecular docking were studied using in silico screening techniques. During multiple sequence alignment, we identified conserved catalytic amino residues in yeast cellulases, highlighting the pivotal role of glutamic acid in cellulose hydrolysis. Using InterProScan, we determined that the obtained sequence belonged to the GH5 and GH45 families and domains. The key amino acid residues involved in the interaction were glutamic acid, serine, alanine, arginine, asparagine, and histidine. Notably, the docking score for Candida fabianii was significantly lower (-12.95kcal/mol) than that of the other enzymes, indicating a strong affinity for cellulose. In conclusion, our results underscore the importance of a key amino acid residue in Endo β-1,4 glucanase from Candida fabianii, which may enhance the selectivity of endoglucanase toward cellulose.

Iterative gene integration mediated by 26S rDNA and non-homologous end joining for the efficient production of lycopene in Yarrowia lipolytica

Because of its potent antioxidant effects, lycopene has been used in various industries including, but not limited to, food, medical, and cosmetic industries. Yarrowia lipolytica, a non-conventional yeast species, is a promising chassis due to its natural mevalonate (MVA) pathway, abundant precursor acetyl coenzyme A content, and oleaginous properties. Several gene editing tools have been developed for Y. lipolytica along with engineering strategies for tetraterpenoid production. In this study, we engineered Y. lipolytica following multi-level strategies for efficient lycopene accumulation. We first evaluated the performance of the key lycopene biosynthetic genes crtE, crtB, and crtI, expressed via ribosomal DNA (rDNA) mediated multicopy random integration in the HMG1- and GGS1-overexpressing background strain. Further improvement in lycopene production was achieved by overexpressing the key genes for MVA synthesis via non-homologous end joining (NHEJ) mediated multi-round iterative transformation. Efficient strategies in the MVA and lipid synthesis pathways were combined to improve lycopene production with a yield of 430.5 mg/L. This strain produced 121 mg/g dry cell weight of lycopene in a 5-L fed-batch fermentation system. Our findings demonstrated iterative gene integration mediated by 26S rDNA and NHEJ for the efficient production of lycopene in Y. lipolytica. These strategies can be applied to induce Y. lipolytica to produce other tetraterpenoids.Graphical

The Effect of Plasma-Treated Water on Microbial Growth and Biosynthesis of Gamma-Decalactones by Yarrowia lipolytica Yeast.

In recent years, the production of plasma-treated water (PTW) by low-temperature low-pressure glow plasma (LPGP) has been increasingly gaining in popularity. LPGP-treated water changes its physical and physiochemical properties compared to standard distilled water. In this study, a non-conventional lipolytic yeast species Yarrowia lipolytica was cultivated in culture media based on Nantes plasma water with heightened singlet oxygen content (Nantes PW) or in water treated with low-temperature, low-pressure glow plasma while in contact with air (PWTA) or nitrogen (PWTN). The research aimed to assess the influence of culture conditions on castor oil biotransformation to gamma-decalactone (GDL) and other secondary metabolites in media based on nanowater. The Nantes plasma water-based medium attained the highest concentration of gamma-decalactone (4.81 ± 0.51 g/L at 144 h of culture), maximum biomass concentration and biomass yield from the substrate. The amplified activity of lipases in the nanowater-based medium, in comparison to the control medium, is encouraging from the perspective of GDL biosynthesis, relying on the biotransformation of ricinoleic acid, which is the primary component of castor oil. Although lipid hydrolysis was enhanced, this step seemed not crucial for GDL concentration. Interestingly, the study validates the significance of oxygen in β-oxidation enzymes and its role in the bioconversion of ricinoleic acid to GDL and other lactones. Specifically, media with higher oxygen content (WPTA) and Nantes plasma water resulted in remarkably high concentrations of four lactones: gamma-decalactone, 3-hydroxy-gamma-decalactone, dec-2-en-4-olide and dec-3-en-4-olide.

High quality de novo genome assembly of the non-conventional yeast Kazachstania bulderi describes a potential low pH production host for biorefineries

Kazachstania bulderi is a non-conventional yeast species able to grow efficiently on glucose and δ-gluconolactone at low pH. These unique traits make K. bulderi an ideal candidate for use in sustainable biotechnology processes including low pH fermentations and the production of green chemicals including organic acids. To accelerate strain development with this species, detailed information of its genetics is needed. Here, by employing long read sequencing we report a high-quality phased genome assembly for three strains of K. bulderi species, including the type strain. The sequences were assembled into 12 chromosomes with a total length of 14 Mb, and the genome was fully annotated at structural and functional levels, including allelic and structural variants, ribosomal array and mating type locus. This high-quality reference genome provides a resource to advance our fundamental knowledge of biotechnologically relevant non-conventional yeasts and to support the development of genetic tools for manipulating such strains towards their use as production hosts in biotechnological processes.

Saccharomyces cerevisiae responds similarly to co-culture or to a fraction enriched in Metschnikowia pulcherrima extracellular vesicles.

The recent introduction of non-conventional yeast species as companion wine starters has prompted a growing interest in microbial interactions during wine fermentation. There is evidence of interactions through interference and exploitation competition, as well as interactions depending on physical contact. Furthermore, the results of some transcriptomic analyses suggest interspecific communication, but the molecules or biological structures involved in recognition are not well understood. In this work, we explored extracellular vesicles (EVs) as possible mediators of interspecific communication between wine yeasts. The transcriptomic response of Saccharomyces cerevisiae after 3h of contact with a fraction enriched in EVs of Metschnikowia pulcherrima was compared with that induced by active M.pulcherrima cells. Interestingly, there is a high level of overlap between the transcriptomic profiles of yeast cells challenged by either M.pulcherrima whole cells or the EV-enriched fraction. The results indicate an upregulation of yeast metabolism in response to competing species (in line with previous results). This finding points to the presence of a signal, in the EV-enriched fraction, that can be perceived by the yeast cells as a cue for the presence of competitors, even in the absence of metabolically active cells of the other species.

Contributions of Adaptive Laboratory Evolution towards the Enhancement of the Biotechnological Potential of Non-Conventional Yeast Species.

Changes in biological properties over several generations, induced by controlling short-term evolutionary processes in the laboratory through selective pressure, and whole-genome re-sequencing, help determine the genetic basis of microorganism's adaptive laboratory evolution (ALE). Due to the versatility of this technique and the imminent urgency for alternatives to petroleum-based strategies, ALE has been actively conducted for several yeasts, primarily using the conventional species Saccharomyces cerevisiae, but also non-conventional yeasts. As a hot topic at the moment since genetically modified organisms are a debatable subject and a global consensus on their employment has not yet been attained, a panoply of new studies employing ALE approaches have emerged and many different applications have been exploited in this context. In the present review, we gathered, for the first time, relevant studies showing the ALE of non-conventional yeast species towards their biotechnological improvement, cataloging them according to the aim of the study, and comparing them considering the species used, the outcome of the experiment, and the employed methodology. This review sheds light on the applicability of ALE as a powerful tool to enhance species features and improve their performance in biotechnology, with emphasis on the non-conventional yeast species, as an alternative or in combination with genome editing approaches.

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.

Genome editing toolboxes are essential for the exploration and exploitation of non-conventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The non-conventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1,000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses non-homologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.

Heterologous expression reveals unique properties of Trk K+ importers from nonconventional biotechnologically relevant yeast species together with their potential to support Saccharomyces cerevisiae growth.

In the model yeast Saccharomyces cerevisiae, Trk1 is the main K+ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K+ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K+ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb+ . Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K+ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li+ ions.

Antimicrobial and prebiotic activity of mannoproteins isolated from conventional and nonconventional yeast species—the study on selected microorganisms

Yeast mannoproteins are proposed as a paraprobiotics with antimicrobial and prebiotic properties. They can be used as biopreservatives in food and in diseases therapies. The knowledge about the specificity and/or capability of their influence on the growth of different microorganism is limited. The study determined the effect of mannoprotein preparations of Saccharomyces cerevisiae (S. cerevisiae) ATCC 7090 and nonconventional yeast origin [Metschnikowia reukaufii (M. reukaufii) WLP 4650 and Wickerhamomyces anomalus (W. anomalus) CCY 38-1-13] on the growth of selected bacteria of the genera: Lactobacilllus, Limosilatobacillus, Limosilatobacillus, Bifidobacterium, Staphylococcus, Enterococcus, Pseudomonas, Escherichia, Proteus and Salmonella. The degree of stimulation or growth inhibition of tested bacteria depended on the type and dose of the mannoprotein and the bacterial strain. The addition of the tested preparations in the entire range of applied concentrations had a positive effect especially on the growth of Lactobacillus arabinosus ATCC 8014 and Bifidobacterium animalis subsp. lactis B12. Mannoproteins isolated from S. cerevisiae limited the growth of the Escherichia coli (E. coli) ATCC 25922, Pseudomonas aureoginosa (P. aureoginosa) ATCC 27853, Proteus mirabilis ATCC 35659 and Salmonella Enteritidis ATCC 13076 to the greatest extent, while preparations of M. reukaufii and W. anomalus origin most effectively limited the growth of Staphylococcus aureus strains, E. coli and P. aureoginosa. The growth of Enterococcus faecalis was stimulated by the presence of all studied preparations in most of the concentrations used. Further research will determine how the purification process of studied mannoproteins or oligosaccharide fractions, its structure and composition influence on the growth of selected bacteria and what is the mechanism of its activity.

A unique, newly discovered four-member protein family involved in extracellular fatty acid binding in Yarrowia lipolytica

BackgroundYarrowia lipolytica, a nonconventional oleaginous yeast species, has attracted attention due to its high lipid degradation and accumulation capacities. Y. lipolytica is used as a chassis for the production of usual and unusual lipids and lipid derivatives. While the genes involved in the intracellular transport and activation of fatty acids in different cellular compartments have been characterized, no genes involved in fatty acid transport from the extracellular medium into the cell have been identified thus far. In this study, we identified secreted proteins involved in extracellular fatty acid binding.ResultsRecent analysis of the Y. lipolytica secretome led to the identification of a multigene family that encodes four secreted proteins, preliminarily named UP1 to UP4. These proteins were efficiently overexpressed individually in wild-type and multideletant strain (Q4: Δup1Δup2Δup3Δup4) backgrounds. Phenotypic analysis demonstrated the involvement of these proteins in the binding of extracellular fatty acids. Additionally, gene deletion and overexpression prevented and promoted sensitivity to octanoic acid (C8) toxicity, respectively. The results suggested binding is dependent on aliphatic chain length and fatty acid concentration. 3D structure modeling supports the proteins’ role in fatty acid assimilation at the molecular level.ConclusionsWe discovered a family of extracellular-fatty-acid-binding proteins in Y. lipolytica and have proposed to name its members eFbp1 to eFbp4. The exact mode of eFbps action remains to be deciphered individually and synergistically; nevertheless, it is expected that the proteins will have applications in lipid biotechnology, such as improving fatty acid production and/or bioconversion.

Antifungal activity of non-conventional yeasts against Botrytis cinerea and non-Botrytis grape bunch rot fungi.

Grapes harbour a plethora of non-conventional yeast species. Over the past two decades, several of the species have been extensively characterised and their contribution to wine quality is better understood. Beyond fermentation, some of the species have been investigated for their potential as alternative biological tools to reduce grape and wine spoilage. However, such studies remain limited to a few genera. This work aimed to evaluate the antagonistic activity of grape must-derived non-conventional yeasts against Botrytis cinerea and non-Botrytis bunch-rotting moulds and to further elucidate mechanisms conferring antifungal activity. A total of 31 yeast strains representing 21 species were screened on different agar media using a dual culture technique and liquid mixed cultures, respectively. Pichia kudriavzevii was the most potent with a minimum inhibitory concentration of 102 cells/mL against B. cinerea but it had a narrow activity spectrum. Twelve of the yeast strains displayed broad antagonistic activity, inhibiting three strains of B. cinerea (B05. 10, IWBT FF1 and IWBT FF2), a strain of Aspergillus niger and Alternaria alternata. Production of chitinases and glucanases in the presence of B. cinerea was a common feature in most of the antagonists. Volatile and non-volatile compounds produced by antagonistic yeast strains in the presence of B. cinerea were analysed and identified using gas and liquid chromatography mass spectrometry, respectively. The volatile compounds identified belonged mainly to higher alcohols, esters, organosulfur compounds and monoterpenes while the non-volatile compounds were cyclic peptides and diketopiperazine. To our knowledge, this is the first report to demonstrate inhibitory effect of the non-volatile compounds produced by various yeast species.

Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species.

As redesigning organisms using engineering principles is one of the purposes of synthetic biology (SynBio), the standardization of experimental methods and DNA parts is becoming increasingly a necessity. The synthetic biology community focusing on the engineering of Saccharomyces cerevisiae has been in the foreground in this area, conceiving several well-characterized SynBio toolkits widely adopted by the community. In this review, the molecular methods and toolkits developed for S. cerevisiae are discussed in terms of their contributions to the required standardization efforts. In addition, the toolkits designed for emerging nonconventional yeast species including Yarrowia lipolytica, Komagataella phaffii, and Kluyveromyces marxianus are also reviewed. Without a doubt, the characterized DNA parts combined with the standardized assembly strategies highlighted in these toolkits have greatly contributed to the rapid development of many metabolic engineering and diagnostics applications among others. Despite the growing capacity in deploying synthetic biology for common yeast genome engineering works, the yeast community has a long journey to go to exploit it in more sophisticated and delicate applications like bioautomation.

The cell wall and the response and tolerance to stresses of biotechnological relevance in yeasts.

In industrial settings and processes, yeasts may face multiple adverse environmental conditions. These include exposure to non-optimal temperatures or pH, osmotic stress, and deleterious concentrations of diverse inhibitory compounds. These toxic chemicals may result from the desired accumulation of added-value bio-products, yeast metabolism, or be present or derive from the pre-treatment of feedstocks, as in lignocellulosic biomass hydrolysates. Adaptation and tolerance to industrially relevant stress factors involve highly complex and coordinated molecular mechanisms occurring in the yeast cell with repercussions on the performance and economy of bioprocesses, or on the microbiological stability and conservation of foods, beverages, and other goods. To sense, survive, and adapt to different stresses, yeasts rely on a network of signaling pathways to modulate the global transcriptional response and elicit coordinated changes in the cell. These pathways cooperate and tightly regulate the composition, organization and biophysical properties of the cell wall. The intricacy of the underlying regulatory networks reflects the major role of the cell wall as the first line of defense against a wide range of environmental stresses. However, the involvement of cell wall in the adaptation and tolerance of yeasts to multiple stresses of biotechnological relevance has not received the deserved attention. This article provides an overview of the molecular mechanisms involved in fine-tuning cell wall physicochemical properties during the stress response of Saccharomyces cerevisiae and their implication in stress tolerance. The available information for non-conventional yeast species is also included. These non-Saccharomyces species have recently been on the focus of very active research to better explore or control their biotechnological potential envisaging the transition to a sustainable circular bioeconomy.

Engineering heterologous enzyme secretion in Yarrowia lipolytica

BackgroundEukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme.ResultsBy engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and alpha-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion.ConclusionsOverall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.

Exploring Yeast Diversity to Produce Lipid-Based Biofuels from Agro-Forestry and Industrial Organic Residues.

Exploration of yeast diversity for the sustainable production of biofuels, in particular biodiesel, is gaining momentum in recent years. However, sustainable, and economically viable bioprocesses require yeast strains exhibiting: (i) high tolerance to multiple bioprocess-related stresses, including the various chemical inhibitors present in hydrolysates from lignocellulosic biomass and residues; (ii) the ability to efficiently consume all the major carbon sources present; (iii) the capacity to produce lipids with adequate composition in high yields. More than 160 non-conventional (non-Saccharomyces) yeast species are described as oleaginous, but only a smaller group are relatively well characterised, including Lipomyces starkeyi, Yarrowia lipolytica, Rhodotorula toruloides, Rhodotorula glutinis, Cutaneotrichosporon oleaginosus and Cutaneotrichosporon cutaneum. This article provides an overview of lipid production by oleaginous yeasts focusing on yeast diversity, metabolism, and other microbiological issues related to the toxicity and tolerance to multiple challenging stresses limiting bioprocess performance. This is essential knowledge to better understand and guide the rational improvement of yeast performance either by genetic manipulation or by exploring yeast physiology and optimal process conditions. Examples gathered from the literature showing the potential of different oleaginous yeasts/process conditions to produce oils for biodiesel from agro-forestry and industrial organic residues are provided.

A CRISPR/Cas9 method facilitates efficient oligo-mediated gene editing in Debaryomyces hansenii.

Halophilic and osmotolerant yeast Debaryomyces hansenii has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques do not allow the full potential of D. hansenii as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism poses further challenges when precise gene targeting is required. In this study, we present a novel plasmid-based CRISPRCUG/Cas9 method for easy and efficient gene editing of the prototrophic strains of D. hansenii. Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple single-guide RNAs (sgRNAs) that provide the possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed NHEJ-deficient D. hansenii that enable our CRISPRCUG/Cas9 tools to support the highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single-stranded DNA oligonucleotides are sufficient for direct repair of DNA breaks induced by sgRNA-Cas9, resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in D. hansenii. In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.

A novel AST2 mutation generated upon whole-genome transformation of Saccharomyces cerevisiae confers high tolerance to 5-Hydroxymethylfurfural (HMF) and other inhibitors.

Development of cell factories for conversion of lignocellulosic biomass hydrolysates into biofuels or bio-based chemicals faces major challenges, including the presence of inhibitory chemicals derived from biomass hydrolysis or pretreatment. Extensive screening of 2526 Saccharomyces cerevisiae strains and 17 non-conventional yeast species identified a Candida glabrata strain as the most 5-hydroxymethylfurfural (HMF) tolerant. Whole-genome (WG) transformation of the second-generation industrial S. cerevisiae strain MD4 with genomic DNA from C. glabrata, but not from non-tolerant strains, allowed selection of stable transformants in the presence of HMF. Transformant GVM0 showed the highest HMF tolerance for growth on plates and in small-scale fermentations. Comparison of the WG sequence of MD4 and GVM1, a diploid segregant of GVM0 with similarly high HMF tolerance, surprisingly revealed only nine non-synonymous SNPs, of which none were present in the C. glabrata genome. Reciprocal hemizygosity analysis in diploid strain GVM1 revealed AST2N406I as the only causative mutation. This novel SNP improved tolerance to HMF, furfural and other inhibitors, when introduced in different yeast genetic backgrounds and both in synthetic media and lignocellulose hydrolysates. It stimulated disappearance of HMF and furfural from the medium and enhanced in vitro furfural NADH-dependent reducing activity. The corresponding mutation present in AST1 (i.e. AST1D405I) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2N406I and in presence of high inhibitor concentrations. Our work provides a powerful genetic tool to improve yeast inhibitor tolerance in lignocellulosic biomass hydrolysates and other inhibitor-rich industrial media, and it has revealed for the first time a clear function for Ast2 and Ast1 in inhibitor tolerance.

A Multiphase Multiobjective Dynamic Genome-Scale Model Shows Different Redox Balancing among Yeast Species of the Saccharomyces Genus in Fermentation.

ABSTRACTYeasts constitute over 1,500 species with great potential for biotechnology. Still, the yeast Saccharomyces cerevisiae dominates industrial applications, and many alternative physiological capabilities of lesser-known yeasts are not being fully exploited. While comparative genomics receives substantial attention, little is known about yeasts’ metabolic specificity in batch cultures. Here, we propose a multiphase multiobjective dynamic genome-scale model of yeast batch cultures that describes the uptake of carbon and nitrogen sources and the production of primary and secondary metabolites. The model integrates a specific metabolic reconstruction, based on the consensus Yeast8, and a kinetic model describing the time-varying culture environment. In addition, we proposed a multiphase multiobjective flux balance analysis to compute the dynamics of intracellular fluxes. We then compared the metabolism of S. cerevisiae and Saccharomyces uvarum strains in a rich medium fermentation. The model successfully explained the experimental data and brought novel insights into how cryotolerant strains achieve redox balance. The proposed model (along with the corresponding code) provides a comprehensive picture of the main steps occurring inside the cell during batch cultures and offers a systematic approach to prospect or metabolically engineering novel yeast cell factories.IMPORTANCE Nonconventional yeast species hold the promise to provide novel metabolic routes to produce industrially relevant compounds and tolerate specific stressors, such as cold temperatures. This work validated the first multiphase multiobjective genome-scale dynamic model to describe carbon and nitrogen metabolism throughout batch fermentation. To test and illustrate its performance, we considered the comparative metabolism of three yeast strains of the Saccharomyces genus in rich medium fermentation. The study revealed that cryotolerant Saccharomyces species might use the γ-aminobutyric acid (GABA) shunt and the production of reducing equivalents as alternative routes to achieve redox balance, a novel biological insight worth being explored further. The proposed model (along with the provided code) can be applied to a wide range of batch processes started with different yeast species and media, offering a systematic and rational approach to prospect nonconventional yeast species metabolism and engineering novel cell factories.

Levaduras no convencionales como herramientas de innovación y diferenciación en la producción de cerveza

Yeasts play a crucial role in brewing. During fermentation, besides ethanol and carbon dioxide, yeasts produce a considerable number of organic compounds, which are essential for beer flavor. In particular, Saccharomyces cerevisiae and Saccharomyces pastorianus are traditionally used in the production of ale and lager beers, respectively. Nowadays, the continuous growth of the craft beer market motivates the production of differential and innovative beers; leading specialists and brewers focus on non-conventional yeasts as tools for new product development. In this work, we describe the potential application of non-conventional yeast species such as those of the genera Brettanomyces, Torulaspora, Lachancea, Wickerhamomyces, Pichia and Mrakia in the craft brewing industry, as well as non-traditional brewing yeasts of the Saccharomyces genus. Furthermore, the fermentation conditions of these non-conventional yeasts are discussed, along with their abilities to assimilate and metabolize diverse wort components providing differential characteristics to the final product. In summary, we present a comprehensive review of the state-of-the-art of non-conventional yeasts, which is highly relevant for their application in the production of novel craft beers including flavored beers, non-alcoholic beers, low-calorie beers and functional beers.

Mutagenesis, screening and isolation of Brettanomyces bruxellensis mutants with reduced 4-ethylphenol production.

The use of non-conventional yeast species to obtain interesting flavors and aromas has become a new trend in the fermented beverages industry. Among such species, Brettanomyces bruxellensis (B. bruxellensis) has been reported as capable of producing desirable or at least singular aromas in fermented beverages like beer and wine. However, this yeast can also produce an aromatic defect by producing high concentrations of phenolic compounds like, 4-ethylguaiacol and particularly 4-ethylphenol (4-EP). In the present study, we designed a mutant screening method to isolate B. bruxellensis mutants with reduced 4-EP production. More than 1000 mutants were screened with our olfactory screening method, and after further sensory and chemical analysis we were able to select a B. bruxellensis mutant strain with a significant reduction of 4-EP production (more than threefold) and less phenolic perception. Notably, the selected strain also showed higher diversity and concentration of ethyl esters, the most important group of odor active compounds produced by yeasts. Based on these results, we consider that our selected mutant strain is a good candidate to be tested as a non-conventional yeast starter (pure or in co-inoculation) to obtain wines and beers with novel aromatic properties.