Oxidation of biological molecules with age and induced oxidative stress in different growth phases of Saccharomyces cerevisiae.

One of the mechanistic approaches for explaining ageing is the oxidative stress theory of ageing. Saccharomyces cerevisiae has been used as a model to study ageing due to many factors. We have attempted to investigate if the differential ability to withstand oxidative stress can be correlated with their lifespans. In all the four strains studied (AP22, 699, 8C, and SP4), there was no age-associated increases in lipid peroxidation levels measured as thiobarbituric acid reactive substances (TBARS). Under induced oxidative stress conditions, there was an increased TBARS level in both the ages assessed with a quantum-fold increase in the stationary phase cells of AP22. In contrast, the late stationary phase cells of 8C exhibited the least susceptibility to induced oxidative stress. The level of TBARS in both exponential and late stationary phase cells of 699 was overall more than that in the other three strains. Protein carbonylation increased with age in 8C and 699. Induced stress increased carbonylation in the exponential cells of SP4 and 699 and the stationary phase cells of all four strains. Protein carbonylation data indicate that the AP22 cells exhibit decreased protein carbonylation vis-à-vis the other strains. Induced stress data showed that while the exponential cells of 699 are susceptible, the late stationary phase cells of 699 are most resistant. Western blotting analysis using anti-HNE antibodies showed two proteins of molecular mass ~ 56 and ~ 84 kDa that were selectively modified with age in all the strains. Under induced stress conditions, an additional protein of ~ 69 kDa was oxidized. Our investigation shows that the difference in lifespan between the four strains of S. cerevisiae may be regulated by oxidative stress. Knowledge of the identity of the oxidized proteins will significantly facilitate a better understanding of the effect of oxidative stress conditions on the cells of S. cerevisiae.

Caracterização do papel de Nop53 na montagem da subunidade ribossomal maior em Saccharomyces cerevisiae / Characterization of the role of Nop53 in the assembly of the large ribosomal subunit in Saccharomyces cerevisiae

Os ribossomos são complexos ribonucleoproteicos conservados formados por duas subunidades assimétricas (40S e 60S em eucariotos) responsáveis pela tradução da informação genética e catálise da síntese proteica. A montagem destes complexos em eucariotos é mais bem descrita em S. cerevisiae, constituindo um processo celular energeticamente dispendioso e com múltiplas etapas. Ela tem origem no nucléolo com a transcrição do pré-rRNA 35S e requer o recrutamento hierárquico e transiente de cerca de 200 fatores de montagem para garantir a formação correta dos centros funcionais aptos à tradução. Neste processo, que se estende no núcleo e citoplasma, 79 proteínas ribossomais associam-se gradativamente à medida que o prérRNA é dobrado, modificado e processado. O processamento do pré-rRNA 35S consiste na remoção progressiva de espaçadores internos (ITS1 e ITS2) e externos (5ETS e 3ETS), que separam e flanqueiam os rRNAs maduros componentes de ambas subunidades ribossomais. A clivagem do ITS1 separa as vias de maturação do pré-60S e do pré-40S. O ITS2, que, em associação a fatores de montagem, forma uma estrutura denominada ITS2-foot, é o último espaçador do pré-60S a ser removido. A composição do ITS2-foot permanece inalterada no nucléolo até a transição entre o estado E nucleolar e a formação da partícula Nog2 nuclear. Nesta etapa, a liberação do fator Erb1 permite o recrutamento do fator de montagem conservado e essencial Nop53. Na base do ITS2-foot, Nop53 recruta o exossomo via RNA helicase Mtr4 para a clivagem 3-5 exonucleolítica de parte do ITS2 levando à desmontagem do ITS2-foot. O fato de Nop53 atuar como ponte entre dois grandes complexos e apresentar uma estrutura flexível e estendida nos levou a aprofundar a caracterização de seu papel durante a maturação do pré60S. Neste trabalho, usando análise proteômica quantitativa label-free baseada em espectrometria de massas, caracterizou-se o interactoma de Nop53, e avaliou-se o impacto da depleção de Nop53 no interactoma da subunidade catalítica do exossomo Rrp6 e na composição de pré-ribossomos representativos de quase todas as etapas de maturação do pré-60S. Em paralelo, foram caracterizados mutantes truncados de Nop53 e avaliada por pull-down a interação de Nop53 com componentes do exossomo. Os resultados obtidos mostraram que Nop53 é capaz de interagir com o cofator do exossomo Mpp6, sugerindo pontos adicionais de interação durante o recrutamento do exossomo ao pré-60S. A análise do interactoma de Rrp6 mostrou uma associação precoce do exossomo aos intermediários pré-ribossomais nucleolares mais iniciais, anteriores aos previamente descritos. Mudanças na composição dos intermediários pré-60S revelaram que a depleção de Nop53 afeta a transição entre o estado E e a partícula Nog2, afetando eventos tardios de maturação como o recrutamento de Yvh1. Comparando-se o efeito da depleção de Nop53 com o de mutantes nop53 desprovidos da região de recrutamento do exossomo, obtivemos evidências bioquímicas do papel estrutural de Nop53 na base do ITS2- foot. Em conjunto, estas observações, à luz de estruturas de intermediários pré-ribossomais recentemente descritas, nos permitiram concluir que o recrutamento de Nop53 ao pré-60S contribui para a estabilização de eventos de remodelamento do rRNA que antecedem a formação da partícula Nog2

Ribosomes are conserved ribonucleoprotein complexes formed by two asymmetric subunits (the 40S and the 60S in eukaryotes) responsible for translating the genetic information and catalyzing protein synthesis. The assembly of these complexes in eukaryotes is best described in S. cerevisiae. It is an energetically demanding, multi-step cellular process, that starts in the nucleolus with the transcription of the 35S pre-rRNA. It requires the hierarchical and transient recruitment of about 200 assembly factors to ensure the correct formation of the functional centers suitable for translation. In this process, which extends into the nucleus and cytoplasm, 79 ribosomal proteins gradually associate as the pre-rRNA is folded, modified, and processed. The 35S pre-rRNA processing happens with the progressive removal of internal (ITS1 and ITS2) and external (5'ETS and 3'ETS) transcribed spacers, which separate and flank the mature rRNA components of both ribosomal subunits. The cleavage at the ITS1 separates the pre-60S and pre40S maturation pathways. The ITS2, which in association with assembly factors constitutes a structure called ITS2-foot, is the last pre-60S spacer to be removed. The composition of the ITS2- foot remains unchanged in the nucleolus until the transition between the nucleolar state E and the nuclear Nog2 particle. At this stage, the release of Erb1 allows the recruitment of the conserved and essential assembly factor Nop53. At the base of the ITS2-foot, Nop53 recruits the exosome via the RNA helicase Mtr4 for the ITS2 3'-5' exonucleolytic cleavage leading to the ITS2-foot disassembly. The fact that Nop53 acts as a bridge between these two large complexes and exhibits a flexible and extended structure led us to further characterize its role in the pre-60S maturation. In this work, using mass spectrometry-based label-free quantitative proteomics, we characterized the interactome of Nop53, as well as the impact of the depletion of Nop53 on the interactome of the exosome catalytic subunit Rrp6 and on the composition of pre-ribosomes representative of almost all pre-60S maturation stages. In parallel, we characterized nop53 truncated mutants and evaluated the interaction of Nop53 with exosome components by pulldown assays. The results showed that Nop53 can interact with the exosome cofactor Mpp6, suggesting the contribution of additional points of interaction during the exosome recruitment to the pre-60S. The analysis of the Rrp6 interactome revealed an early association of the exosome with pre-ribosomal intermediates at very early nucleolar stages, before those previously described. Changes in the composition of pre-60S intermediates revealed that Nop53 depletion affects the transition between the state E and the Nog2 particle, affecting late pre-60S maturation events, such as the Yvh1 recruitment. Comparing the effect of Nop53 depletion with that of nop53 mutants lacking the exosome interacting region, we obtained biochemical evidence of the structural role of Nop53 at the base of the ITS2-foot. Altogether, and in light of recently described structures of pre-ribosomal intermediates, these observations allowed us to conclude that the recruitment of Nop53 to the pre-60S contributes to the stabilization of rRNA remodeling events that precede the formation of the Nog2 particle

A modified fluctuation assay reveals a natural mutator phenotype that drives mutation spectrum variation within Saccharomyces cerevisiae.

Although studies of Saccharomyces cerevisiae have provided many insights into mutagenesis and DNA repair, most of this work has focused on a few laboratory strains. Much less is known about the phenotypic effects of natural variation within S. cerevisiae's DNA repair pathways. Here, we use natural polymorphisms to detect historical mutation spectrum differences among several wild and domesticated S. cerevisiae strains. To determine whether these differences are likely caused by genetic mutation rate modifiers, we use a modified fluctuation assay with a CAN1 reporter to measure de novo mutation rates and spectra in 16 of the analyzed strains. We measure a 10-fold range of mutation rates and identify two strains with distinctive mutation spectra. These strains, known as AEQ and AAR, come from the panel's 'Mosaic beer' clade and share an enrichment for C > A mutations that is also observed in rare variation segregating throughout the genomes of several Mosaic beer and Mixed origin strains. Both AEQ and AAR are haploid derivatives of the diploid natural isolate CBS 1782, whose rare polymorphisms are enriched for C > A as well, suggesting that the underlying mutator allele is likely active in nature. We use a plasmid complementation test to show that AAR and AEQ share a mutator allele in the DNA repair gene OGG1, which excises 8-oxoguanine lesions that can cause C > A mutations if left unrepaired.

Production of neoagarooligosaccharides by probiotic yeast Saccharomyces cerevisiae var. boulardii engineered as a microbial cell factory.

BACKGROUND: Saccharomyces cerevisiae var. boulardii is a representative probiotic yeast that has been widely used in the food and pharmaceutical industries. However, S. boulardii has not been studied as a microbial cell factory for producing useful substances. Agarose, a major component of red macroalgae, can be depolymerized into neoagarooligosaccharides (NAOSs) by an endo-type ß-agarase. NAOSs, including neoagarotetraose (NeoDP4), are known to be health-benefiting substances owing to their prebiotic effect. Thus, NAOS production in the gut is required. In this study, the probiotic yeast S. boulardii was engineered to produce NAOSs by expressing an endo-type ß-agarase, BpGH16A, derived from a human gut bacterium Bacteroides plebeius. RESULTS: In total, four different signal peptides were compared in S. boulardii for protein (BpGH16A) secretion for the first time. The SED1 signal peptide derived from Saccharomyces cerevisiae was selected as optimal for extracellular production of NeoDP4 from agarose. Expression of BpGH16A was performed in two ways using the plasmid vector system and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. The production of NeoDP4 by engineered S. boulardii was verified and quantified. NeoDP4 was produced by S. boulardii engineered using the plasmid vector system and CRISPR-Cas9 at 1.86 and 0.80 g/L in a 72-h fermentation, respectively. CONCLUSIONS: This is the first report on NAOS production using the probiotic yeast S. boulardii. Our results suggest that S. boulardii can be considered a microbial cell factory to produce health-beneficial substances in the human gut.

Inhibitory Activity and Mechanism Investigation of Hypericin as a Novel α-Glucosidase Inhibitor.

α-glucosidase is a major enzyme that is involved in starch digestion and type 2 diabetes mellitus. In this study, the inhibition of hypericin by α-glucosidase and its mechanism were firstly investigated using enzyme kinetics analysis, real-time interaction analysis between hypericin and α-glucosidase by surface plasmon resonance (SPR), and molecular docking simulation. The results showed that hypericin was a high potential reversible and competitive α-glucosidase inhibitor, with a maximum half inhibitory concentration (IC50) of 4.66 ± 0.27 mg/L. The binding affinities of hypericin with α-glucosidase were assessed using an SPR detection system, which indicated that these were strong and fast, with balances dissociation constant (KD) values of 6.56 × 10-5 M and exhibited a slow dissociation reaction. Analysis by molecular docking further revealed that hydrophobic forces are generated by interactions between hypericin and amino acid residues Arg-315 and Tyr-316. In addition, hydrogen bonding occurred between hypericin and α-glucosidase amino acid residues Lys-156, Ser-157, Gly-160, Ser-240, His-280, Asp-242, and Asp-307. The structure and micro-environment of α-glucosidase enzymes were altered, which led to a decrease in α-glucosidase activity. This research identified that hypericin, an anthracene ketone compound, could be a novel α-glucosidase inhibitor and further applied to the development of potential anti-diabetic drugs.

Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion.

Monoclonal antibodies, antibody fragments and fusion proteins derived thereof have revolutionized the practice of medicine. Major challenges faced by the biopharmaceutical industry are however high production costs, long processing times and low productivities associated with their production in mammalian cell lines. The yeast Saccharomyces cerevisiae, a well-characterized eukaryotic cell factory possessing the capacity of post-translational modifications, has been industrially exploited as a secretion host for production of a range of products, including pharmaceuticals. However, due to the incompatible surface glycosylation, few antibody molecules have been functionally expressed in S. cerevisiae. Here, three non-glycosylated antibody fragments from human and the Camelidae family were chosen for expression in a S. cerevisiae strain (HA) previously evolved for high α-amylase secretion. These included the Fab fragment Ranibizumab (Ran), the scFv peptide Pexelizumab (Pex), and a nanobody consisting of a single V-type domain (Nan). Both secretion and biological activities of the antibody fragments were confirmed. In addition, the secretion level of each protein was compared in the wild type (LA) and two evolved strains (HA and MA) with different secretory capacities. We found that the secretion of Ran and Nan was positively correlated with the strains' secretory capacity, while Pex was most efficiently secreted in the parental strain. To investigate the mechanisms for different secretion abilities in these selected yeast strains for the different antibody fragments, RNA-seq analysis was performed. The results showed that several bioprocesses were significantly enriched for differentially expressed genes when comparing the enriched terms between HA.Nan vs. LA.Nan and HA.Pex vs. LA.Pex, including amino acid metabolism, protein synthesis, cell cycle and others, which indicates that there are unique physiological needs for each antibody fragment secretion.

Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants.

BACKGROUND: Tailoring gene expression to balance metabolic fluxes is critical for the overproduction of metabolites in yeast hosts, and its implementation requires coordinated regulation at both transcriptional and translational levels. Although synthetic minimal yeast promoters have shown many advantages compared to natural promoters, their transcriptional strength is still limited, which restricts their applications in pathway engineering. RESULTS: In this work, we sought to expand the application scope of synthetic minimal yeast promoters by enhancing the corresponding translation levels using specific Kozak sequence variants. Firstly, we chose the reported UASF-E-C-Core1 minimal promoter as a library template and determined its Kozak motif (K0). Next, we randomly mutated the K0 to generate a chimeric promoter library, which was able to drive green fluorescent protein (GFP) expression with translational strengths spanning a 500-fold range. A total of 14 chimeric promoters showed at least two-fold differences in GFP expression strength compared to the K0 control. The best one named K528 even showed 8.5- and 3.3-fold increases in fluorescence intensity compared with UASF-E-C-Core1 and the strong native constitutive promoter PTDH3, respectively. Subsequently, we chose three representative strong chimeric promoters (K540, K536, and K528) from this library to regulate pathway gene expression. In conjunction with the tHMG1 gene for squalene production, the K528 variant produced the best squalene titer of 32.1 mg/L in shake flasks, which represents a more than 10-fold increase compared to the parental K0 control (3.1 mg/L). CONCLUSIONS: All these results demonstrate that this chimeric promoter library developed in this study is an effective tool for pathway engineering in yeast.

Significance of two intracellular triacylglycerol lipases of Aspergillus oryzae in lipid mobilization: A perspective in industrial implication for microbial lipid production.

Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.

Potential application of yeasts from Ecuadorian chichas in controlled beer and chicha production.

The potential of yeasts isolated from traditional chichas as starter cultures, either for controlled production of the native beverage or for industrial beer production, has been investigated. Three S. cerevisiae strains and one T. delbrueckii strain isolated from four different Ecuadorian chichas were compared to ale and lager beer strains with respect to fermentation performance, sugar utilisation, phenolic off-flavour production, flocculation and growth at low temperature. Fermentations were performed in 15 °P all-malt wort and in a model chicha substrate at 12 °C and 20 °C. Tall-tube fermentations (1.5 L) were also performed with both substrates to assess yeast performance and beer quality. Among the strains tested, only one Ecuadorian S. cerevisiae strain was able to ferment the wort sugars maltose and maltotriose. Fermentations with all Ecuadorian strains were poor in wort at 12 °C relative to 20 °C, but were similar in model chicha substrate at both temperatures. The aromatic profile was different between species and strains. These results indicate the potential of yeasts derived from traditional Andean fermented beverages for commercial applications. One of the chicha strains demonstrated traits typical of domesticated brewery strains and could be suitable for ale fermentation, while the other strains may have potential for low-alcohol beer or chicha production.

Aerobic growth physiology of Saccharomyces cerevisiae on sucrose is strain-dependent.

Present knowledge on the quantitative aerobic physiology of the yeast Saccharomyces cerevisiae during growth on sucrose as sole carbon and energy source is limited to either adapted cells or to the model laboratory strain CEN.PK113-7D. To broaden our understanding of this matter and open novel opportunities for sucrose-based biotechnological processes, we characterized three strains, with distinct backgrounds, during aerobic batch bioreactor cultivations. Our results reveal that sucrose metabolism in S. cerevisiae is a strain-specific trait. Each strain displayed distinct extracellular hexose concentrations and invertase activity profiles. Especially, the inferior maximum specific growth rate (0.21 h-1) of the CEN.PK113-7D strain, with respect to that of strains UFMG-CM-Y259 (0.37 h-1) and JP1 (0.32 h-1), could be associated to its low invertase activity (0.04-0.09 U/mgDM). Moreover, comparative experiments with glucose or fructose alone, or in combination, suggest mixed mechanisms of sucrose utilization by the industrial strain JP1, and points out the remarkable ability of the wild isolate UFMG-CM-259 to grow faster on sucrose than on glucose in a well-controlled cultivation system. This work hints to a series of metabolic traits that can be exploited to increase sucrose catabolic rates and bioprocess efficiency.

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.

Large-scale sequencing and comparative analysis of oenological Saccharomyces cerevisiae strains supported by nanopore refinement of key genomes.

Saccharomyces cerevisiae has long been part of human activities related to the production of food and wine. The industrial demand for fermented beverages with well-defined and stable characteristics boosted the isolation and selection of strains conferring a distinctive aroma profile to the final product. To uncover variants characterizing oenological strains, the sequencing of 65 new S. cerevisiae isolates, and the comparison with other 503 publicly available genomes were performed. A hybrid approach based on short Illumina and long Oxford Nanopore reads allowed the in-depth investigation of eleven genomes and the identification of putative laterally transferred regions and structural variants. A comparative analysis between clusters of strains belonging to different datasets allowed the identification of novel relevant genetic features including single nucleotide polymorphisms, insertions and structural variants. Detection of oenological single nucleotide variants shed light on the existence of different levels of modulation for the mevalonate pathway relevant for the biosynthesis of aromatic compounds.

Improving isobutanol tolerance and titers through EMS mutagenesis in Saccharomyces cerevisiae.

Improving yeast tolerance toward isobutanol is a critical issue enabling high-titer industrial production. Here, we used EMS mutagenesis to screen Saccharomyces cerevisiae with greater tolerance toward isobutanol. By this method, we obtained EMS39 with high-viability in medium containing 16 g/L isobutanol. Then, we metabolically engineered isobutanol synthesis in EMS39. About 2µ plasmids carrying PGK1p-ILV2, PGK1p-ILV3 and TDH3p-cox4-ARO10 were used to over-express ILV2, ILV3 and ARO10 genes, respectively, in EMS39 and wild type W303-1A. And the resulting strains were designated as EMS39-20 and W303-1A-20. Our results showed that EMS39-20 increased isobutanol titers by 49.9% compared to W303-1A-20. Whole genome resequencing analysis of EMS39 showed that more than 59 genes had mutations in their open reading frames or regulatory regions. These 59 genes are enriched mainly into cell growth, basal transcription factors, cell integrity signaling, translation initiation and elongation, ribosome assembly and function, oxidative stress response, etc. Additionally, transcriptomic analysis of EMS39-20 was carried out. Finally, reverse engineering tests showed that overexpression of CWP2 and SRP4039 could improve tolerance of S.cerevisiae toward isobutanol. In conclusion, EMS mutagenesis could be used to increase yeast tolerance toward isobutanol. Our study supplied new insights into mechanisms of tolerance toward isobutanol and enhancing isobutanol production in S. cerevisiae.

Distribution and function of dominant yeast species in the fermentation of strong-flavor baijiu.

The role of the yeast community in Chinese strong-flavor baijiu fermentation was investigated by culture-independent and culture-dependent methods based on 26S rDNA sequence analysis. 92 yeast species belonging to 54 genera were found by Illumina sequencing during the fermentation of strong-flavor baijiu "Wuliangye" and 306 strains belonging to 28 species, which covered all species with more than 1% of relative abundance, were isolated and identified. Kazachstania exigua, Geotrichum silvicola, Pichia kudriavzevii, Saccharomyces cerevisiae, Zyosaccharomyces bailii, and K. humilis were found to be the first six dominant species, and their proportion varied with different fermentation stages. K. exigua was the most important yeast species responsible for the production of ethanol with the assistance of P. kudriavzevii and Z. bailii in the vigorous stage and P. kudriavzevii and G. silvicola in the continuous fermentation stage. Higher alcohols were mainly produced by K. exigua, P. kudriavzevii, S. cerevisiae, and Z. bailii in the vigorous fermentation stage, which was stimulated by more oxygen in the grains of the upper strata. K. exigua, P. kudrizevii, S. cerevisiae, Z. bailii, G. silvicola, and Trichosporon ovoides promoted the formation of ethyl alcohol. The results revealed the key roles of K. exigua, G. silvicola, and P. kudriavzevii in the fermentation of strong-flavor baijiu. The functions of these species should be confirmed by a further study based on the fermentation characteristics of isolated yeast strains gathered in this study. Distribution and function of dominant yeast species in the fermentation of strong flavor baijiu.

Evaluating accessibility, usability and interoperability of genome-scale metabolic models for diverse yeasts species.

Metabolic network reconstructions have become an important tool for probing cellular metabolism in the field of systems biology. They are used as tools for quantitative prediction but also as scaffolds for further knowledge contextualization. The yeast Saccharomyces cerevisiae was one of the first organisms for which a genome-scale metabolic model (GEM) was reconstructed, in 2003, and since then 45 metabolic models have been developed for a wide variety of relevant yeasts species. A systematic evaluation of these models revealed that-despite this long modeling history-the sequential process of tracing model files, setting them up for basic simulation purposes and comparing them across species and even different versions, is still not a generalizable task. These findings call the yeast modeling community to comply to standard practices on model development and sharing in order to make GEMs accessible and useful for a wider public.

Genomic Evidence of an Ancient East Asian Divergence Event in Wild Saccharomyces cerevisiae.

Comparative genome analyses have suggested East Asia to be the cradle of the domesticated microbe Brewer's yeast (Saccharomyces cerevisiae), used in the food and biotechnology industry worldwide. Here, we provide seven new, high-quality long-read genomes of nondomesticated yeast strains isolated from primeval forests and other natural environments in China and Taiwan. In a comprehensive analysis of our new genome assemblies, along with other long-read Saccharomycetes genomes available, we show that the newly sequenced East Asian strains are among the closest living relatives of the ancestors of the global diversity of Brewer's yeast, confirming predictions made from short-read genomic data. Three of these strains (termed the East Asian Clade IX Complex here) share a recent ancestry and evolutionary history suggesting an early divergence from other S. cerevisiae strains before the larger radiation of the species, and prior to its domestication. Our genomic analyses reveal that the wild East Asian strains contain elevated levels of structural variations. The new genomic resources provided here contribute to our understanding of the natural diversity of S. cerevisiae, expand the intraspecific genetic variation found in this heavily domesticated microbe, and provide a foundation for understanding its origin and global colonization history.

Ethanol production process driving changes on industrial strains.

Ethanol production has key differences between the two largest producing countries of this biofuel, Brazil and the USA, such as feedstock source, sugar concentration and ethanol titers in industrial fermentation. Therefore, it is highly probable that these specificities have led to genome adaptation of the Saccharomyces cerevisiae strains employed in each process to tolerate different environments. In order to identify particular adaptations, in this work, we have compared the genomes of industrial yeast strains widely used to produce ethanol from sugarcane, corn and sweet sorghum, and also two laboratory strains as reference. The genes were predicted and then 4524 single-copy orthologous were selected to build the phylogenetic tree. We found that the geographic location and industrial process were shown as the main evolutionary drivers: for sugarcane fermentation, positive selection was identified for metal homeostasis and stress response genes, whereas genes involved in membrane modeling have been connected with corn fermentation. In addition, the corn specialized strain Ethanol Red showed an increased number of copies of MAL31, a gene encoding a maltose transporter. In summary, our work can help to guide new strain chassis selection for engineering strategies, to produce more robust strains for biofuel production and other industrial applications.

Biodiversity and oenological attitude of Saccharomyces cerevisiae strains isolated in the Montalcino district: biodiversity of S. cerevisiae strains of Montalcino wines.

The biodiversity of Saccharomyces cerevisiae was studied in the Montalcino area (Italy). Two wineries were involved in the study, which compared the genotypic and oenological characteristics of the S. cerevisiae strains isolated in spontaneous fermentations. After isolation yeasts were identified by 26S rRNA gene sequence analysis, and S. cerevisiae strains were characterized through interdelta sequence analysis (ISA). Oenological tests were performed in synthetic grape must by varying the magnitude of the main wine-imiting factors. The evolution of alcoholic fermentation was monitored by measuring sugar consumption and flow cytometry. The results revealed the prevalence of S. cerevisiae from the third day of fermentation and the presence of a wide range of S. cerevisiae strains having ISA profiles characteristic of each winery. From an oenological point of view, the features of such strains, in terms of resistance to wine-limiting factors, seemed to be linked to the main oenological variables applied in the production process of each winery. Extreme fermentation temperatures and copper residues are the variables that mostly depress the yeast population, in terms of fermentation rate and cell viability. Flow cytometry revealed the different impact of limiting factors on the viability of yeast by the quantification of the ratio between live/dead yeast cells of each strain, suggesting different mechanisms of inhibition, for instance stuck of cell growth or cell killing, in response to the different stress factors.

Bioconversion of ferulic acid into aroma compounds by newly isolated yeast strains of the Latin American biodiversity.

Nine yeast strains isolated from Latin American biodiversity were screened for ferulic acid (FA) consumption and conversion into aroma compounds such as vanillin, vanillic acid (VA), and 4-vinylguaiacol (VG). Selected strains (Rhodotorula mucilaginosa UFMG-CM-Y3647, UFMG-CM-Y2190, UFMG-CM-Y665) were evaluated in flask experiments to investigate the influence of the pH media on bioconversion and a two-step process was conducted to maximize the metabolites production. The effect of pH was found to be significantly important for FA bioconversion, as acidic conditions (pH < 6.0) improved VA accumulation, with highest production of 1.14 ± 0.02 and 1.25 ± 0.03 g/L shown by UFMG-CM-Y3647 and UFMG-CM-Y2190, respectively. The two-step process favored 4-VG production for most strains, being UFMG-CM-Y2190 the best producer, its cultures reaching 1.63 ± 0.09 g/L after 55 hr, showing a productivity of 29.59 ± 1.55 mg/(L·hr), as glucose affected the metabolites pool and redirected yeast metabolism. R mucilaginosa UFMG-CM-Y3647 was selected for scaled-up cultivations in a 2-L bioreactor, where pH-controlled pH 5.5 and aeration of 2.5 vvm was found to be the best condition to improve VA productivity, attaining final concentrations of 1.20 ± 0.02 g/L-1 (78% molar yield) and a productivity of 40.82 ± 0.57 mg/(L·hr).

The impact of different Saccharomyces cerevisiae strains on microbial composition and quality of Chinese rice wine fermentations.

Chinese rice wine (CRW) is a popular fermented product in China, with complicated microbial composition and flavour compounds. To reveal the effects of different strains of Saccharomyces cerevisiae (N85 and XZ11) on the microbial composition in the process of brewing, metagenomic sequencing approaches were carried out to explore the dynamic changes of bacteria and fungi. Furthermore, the volatile compounds and organic acids in CRW were identified by headspace solid phase microextraction (HS-SPME)/gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) at the end of the brewing. Our results indicated that different S. cerevisiae strains could influence microbial compositions and especially affected the growth of Lactobacillus brevis and Pantoea ananatis. The changes in the microbial community structure contributed to the remarkable difference in the content of lactic acid, esters, alcohols, and aldehydes. Moreover, functional network analysis provided insights into the biological correlations between microbial species and metabolic pathways, that is, Lactobacillus genus had negative effects on metabolic activities. This study expands the idea of improving the quality of CRW by controlling the microbiome.