In Vitro Interaction Between Yeast Extracellular Vesicles and Human Monocyte-Derived Dendritic Cells.

Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host's immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host's immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro-generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.

Golden Gate Cloning for Efficient Biosynthesis of Lycopene in Synthetic Yeast.

Golden Gate Assembly (GGA) represents a versatile method for assembling multiple DNA fragments into a single molecule, which is widely used in rapid construction of complex expression cassettes for metabolic engineering. Here we describe the GGA method for facile construction and optimization of lycopene biosynthesis pathway by the combinatorial assembly of different transcriptional units (TUs). Furthermore, we report the method for characterizing and improving lycopene production in the synthetic yeast chassis.

Yeast Two-Hybrid Assay for Investigating Antiviral Innate Immunity.

Yeast two-hybrid (YTH) technology is a powerful tool for studying protein interactions and has been widely used in various fields of molecular biology, including the study of antiviral innate immunity. This chapter presents detailed information and experimental procedures for identifying virus-host protein interactions involved in immune regulation using yeast two-hybrid technology.

Protein Kinase R (PKR) as a Novel dsRNA Sensor in Antiviral Innate Immunity.

Protein kinase R (PKR), a key double-stranded RNA (dsRNA)-activated sensor, is pivotal for cellular responses to diverse stimuli. This protocol delineates a comprehensive methodological framework employing single luciferase assays, yeast assays, immunoblot assays, and quantitative PCR (qPCR) to discern and validate PKR activities and their downstream impacts on NF-κB-activating signaling pathways. These methodologies furnish a systematic approach to unraveling the role of PKR as a dsRNA sensor and effector in antiviral innate immunity, enabling in-depth analyses of dsRNA sensor activities.

Impact of fermentation by Saccharomyces Cerevisiae on the macronutrient and in vitro digestion characteristics of Chinese noodles.

This study examined the impact of live bread yeast (Saccharomyces cerevisiae) on the nutritional characteristics of Asian dried noodles. Micronutrient analysis of fermented noodles revealed a 6.9% increase in the overall amino acid content, a 37.1% increase in the vitamin B content and a 63.0% decrease in the phytic acid level. Molecular weight analysis of starch and protein contents revealed moderate decrease in the fermented noodles. The in vitro digestion of fermented noodles showed a slightly faster initial acidification, four-fold decrease in the initial shear viscosity (from 8.85 to 1.94 Pa·s). The initial large food particle count (>2 mm diameter) was 19.5% lower in the fermented noodles. The fermented noodles contained slightly higher free sugar content (73.5 mg g-1 noodle) during the gastric digestion phase. The overall nutrition and digestion results indicate nutritional improvement and digestion-easing attributes in the fermented noodles.

Novel Antarctic Endo-Polygalacturonase for Pectin Extraction and Vegetal Tissue Maceration at Mild Temperatures.

The aim of the present work was to partially purify and characterize an Antarctic polygalacturonase and to determine the enzyme's potential in pectin extraction and vegetal maceration at 20 °C. Polygalacturonase was purified by chromatography to obtain an enzymatic preparation of specific activity 30.3 U.mg-1. Optimal conditions for the polygalacturonase activity were 45 °C and pH 5.0-6.0, and the activation energy for the reaction was 41.8 kJ.mol-1. Of the enzyme activity, 100% was retained after 3 h at 40 °C. The enzyme was remarkably stable for an hour over a wide range of pH (2.0-12.0). Polygalacturonase activity was slightly reduced in the presence of Ca+2, Fe+3, K+, Mn+2, and Zn+2, whereas Hg+2 reduced the activity by 60%, suggesting a thiol-dependent catalysis. The apparent molecular weight of the enzyme was 33 kDa. The kinetic constants evaluated against polygalacturonic acid were 0.17 mg.ml-1 (Km), 480 s-1 (Kcat), and 7.9 µmol.mg-1.min-1 (Vmax). The enzyme was active against different pectic substrates. Thin-layer chromatography revealed an endo-mechanism of action. Polygalacturonase digested lime pomace to aid the extraction of high-methoxylated pectin at 20 °C and increased the vegetal maceration of Capsicum annuum by 24% over the control values.

Bioengineered yeast for preventing age-related diseases.

The aging process entails a multifaceted decline in the capacity to restore homeostasis in response to stress. A prevalent characteristic of many age-related diseases is the presence of low-grade chronic inflammation, a risk factor contributing significantly to morbidity and mortality in the elderly population. Specific lifestyle interventions, such as regular physical activity, targeted diet, and supplementation, can delay the accumulation of chronic age-associated conditions by mitigating inflammation processes. Bioengineered yeast-producing compounds with distinctive bioactivities, including anti-inflammatory properties, have the potential to provide rich dietary alternatives for the prevention of age-related diseases. This review highlights recent achievements in engineering effective yeast platforms, namely Saccharomyces cerevisiae and Yarrowia lipolytica, that hold promise in retarding the onset of aging and age-related ailments.

Yeast cell biocarrier for the encapsulation of ascorbic acid: effect of plasmolysis process, suspension media and ascorbic acid levels on the physicochemical, morphological and bioactive properties of microcapsules.

BACKGROUND: Ascorbic acid is a water-soluble vitamin and shows weak stability against external factors such as heat, oxygen, light etc. Due to its lower stability, encapsulation is an effective process for the preservation of its activity. Although there are a wide variety of encapsulation methods, the technique of encapsulation with yeast cells has been followed with increasing interest in recent years. In this study, encapsulation possibilities of ascorbic acid by yeast cells were investigated. In this context, Saccharomycess cerevisiae yeast cells in plasmolyzed and non-plasmolyzed forms were used in two different suspension media (water and ethanol) and effect of ascorbic acid concentrations (10, 20 and 50 g per 10 g yeast) were studied. A total of 12 different yeast microcapsule samples were produced and some physicochemical, bioactive and structural characterizations were performed. RESULTS: The ascorbic acid level of yeast microcapsule samples was determined as 206.4-713.9 and 202.8-726.1 mg g-1 for plasmolyzed and non-plasmolyzed yeast cell types, respectively. ABTS radical scavenging activity increased from 27.23 to 233.04 µg TE g-1 by increased ascorbic acid levels. Ascorbic acid capsules were used in soft candy processing against free ascorbic acid and it was found that 47.9% ascorbic acid loss was detected for control sample at the 24-day storage while the ascorbic acid loss was approximately 25% for yeast microcapsules. CONCLUSION: It was concluded that yeast cells are capable of preserving ascorbic acid stability during storage and yeast cells can be used effectively and safely for the manufacturing of the ascorbic acid microcapsules. © 2024 Society of Chemical Industry.

Yeast culture enhances long-term fermentation of corn straw by ruminal microbes for volatile fatty acid production: Performance and mechanism.

Ruminal microbes can efficiently ferment biomass waste to produce volatile fatty acids (VFAs). However, keeping long-term efficient VFA production efficiency has become a bottleneck. In this study, yeast culture (YC) was used to enhance the VFA production in long-term fermentation. Results showed that YC group improved the volatile solid removal and VFA concentration to 47.8% and 7.82 g/L, respectively, 18.6% and 16.1% higher than the control, mainly enhancing the acetic, propionic, and butyric acid production. YC addition reduced the bacterial diversity, changed the bacterial composition, and improved interactions among bacteria. The regulation mechanism of YC was to increase the abundance and activity of hydrolytic and acidogenic bacteria such as Prevotella and Treponema, improve bacterial interactions, and further promote expression of functional genes. Ultimately, a long-term efficient ruminal fermentation of corn straw into VFAs was achieved.

Cytotoxicity, genotoxicity and mutagenicity of mixed ternary mononuclear Mg complex based on valproic acid with 1,10-phenanthroline in Saccharomyces cerevisiae and V79 cells.

Valproic acid (VA) is a widely used drug for the treatment of diseases affecting the central nervous system. Due to its epigenetic modulatory potential, it has been studied for possible therapeutic application in anticancer therapies. However, the VA exhibits different side effects in its application. Thus, synthetic coordination complexes with valproate can generate promising candidates for new active drugs with reduced toxicity. In this sense, we investigated the genotoxic and mutagenic potential of the sodium valproate and of the mixed ternary mononuclear Mg complex based on VA with 1,10-phenanthroline (Phen) ligand - [Mg (Valp)2Phen], in Saccharomyces cerevisiae and V79 cells. The MTT and clonal survival assays in V79 cells indicated that the Mg complex has higher cytotoxicity than sodium valproate. A similar cytotoxicity profile is observed in yeast. This fact is possibly due to the intercalation capacity of [Mg(Valp)2Phen], inducing DNA strand breaks, as observed in the comet assay and micronucleus test. In this sense, members of the NER, HR, NHEJ and TLS repair pathways are required for the repair of DNA lesions induced by [Mg(Valp)2Phen]. Interestingly, BER proteins apparently increase the cytotoxic potential of the drug. Furthermore, the [Mg(Valp)2Phen] showed higher cytotoxicity in V79 cells and yeast when compared to sodium valproate indicating applicability as a cytotoxic agent.

Investigation of impact of siderophore and process variables on production of iron enriched Saccharomyces boulardii by Plackett-Burman design.

The primary cause of anemia worldwide is due to poor diet and iron deficiency. Iron (Fe) enriched yeast can be the most effective way to manage anemia because of the capability for biotransformation of mineral to organic and bioavailable iron. To overcome the low richness of yeast, the use of siderophore as cellular iron carriers is a new approach. In this research, for the first time the potential of siderophore in increasing the Fe enrichment of Saccharomyces boulardii (S. boulardii), which is important because of its probiotic properties and resistance to different stresses, has been investigated to produce of potential iron supplements. For this purpose, siderophore was produced by Pseudomonas aeruginosa (P. aeruginosa). Siderophore impact, along with ten other independent process variables, has been studied on the efficiency of iron biotransformation by the Plackett-Burman design (PBD). The results showed that the highest biotransformation yield was 17.77 mg Fe/g dry cell weight (DCW) in the highest biomass weight of 9 g/l. Iron concentration is the most important variable, with contributions of 46% and 70.79% for biomass weight and biotransformation, respectively, followed by fermentation time, agitation speed, and KH2PO4 concentration. But increasing the level of siderophore and zinc led to a significant negative effect. siderophore inefficiency may be attributed to the absence of membrane receptors for pyoverdine (Pvd) and pyochelin (Pch) siderophores. Also, the steric hindrance of the cell wall mannan, the stickiness and sediment ability of the yeast, can create limitations in the absorption of elements. Such yeast can be used as a potential source of iron even for vegetarians and vegans in the form of medicinal and fortified food products to improve the treatment of anemia. It is recommended that further research be focused on increasing the iron enrichment of yeast by overcoming the structural barrier of the cell wall, investigating factors affecting membrane permeability and iron transport potential of other types of siderophores.

Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress.

Protein ubiquitination is essential to govern cells' ability to cope with harmful environments by regulating many aspects of protein dynamics from synthesis to degradation. As important as the ubiquitination process, the reversal of ubiquitin chains mediated by deubiquitinating enzymes (DUBs) is critical for proper recovery from stress and re-establishment of proteostasis. Although it is known that ribosomes are decorated with K63-linked polyubiquitin (K63-ub) chains that control protein synthesis under stress, the mechanisms by which these ubiquitin chains are reversed and regulate proteostasis during stress recovery remain elusive. Here, we showed in budding yeast that the DUB Ubp2 is redox-regulated during oxidative stress in a reversible manner, which determines the levels of K63-ub chains present on ribosomes. We also demonstrate that Ubp2 can cleave single ubiquitin moieties out of chain and its activity is modulated by a series of repeated domains and the formation of disulfide bonds. By combining cellular, biochemical, and proteomics analyses, we showed that Ubp2 is crucial for restoring translation after stress cessation, indicating an important role in determining the cellular response to oxidative stress. Our work demonstrates a novel role for Ubp2, revealing that a range of signaling pathways can be controlled by redox regulation of DUB activity in eukaryotes, which in turn will define cellular states of health and diseases.

Investigation of cold-resistance mechanisms in cryophylactic yeast Metschnikowia pulcherrima based on comparative transcriptome analysis.

Introduction: Low temperature inhibits the growth of most microorganisms. However, some microbes can grow well in a low temperature, even a freezing temperature. Methods: In this study, the mechanisms conferring cold resistance in the cryophylactic yeast Metschnikowia (M.) pulcherrima MS612, an isolate of the epidermis of ice grapes, were investigated based on comparative transcriptome analysis. Results: A total of 6018 genes and 374 differentially expressed genes (> 2-fold, p < 0.05) were identified using RNA-Seq. The differentially expressed genes were mainly involved in carbohydrate and energy metabolism, transport mechanisms, antifreeze protection, lipid synthesis, and signal transduction. M. pulcherrima MS612 maintained normal growth at low temperature (5°C) by enhancing energy metabolism, sterol synthesis, metal ion homeostasis, amino acid and MDR transport, while increased synthesis of glycerol and proline transport to improve its resistance to the freezing temperature (-5°C). Furthermore, cAMP-PKA and ERAD signaling pathways contribute to resist the low temperature and the freezing temperature, respectively. Conclusion: This study provides new insights into cold resistance in cryophylactic microorganisms for maneuvering various metabolism to resist different cold environment.

Current upstream and downstream process strategies for sustainable yeast lipid production.

An increasing global population demands more lipids for food and chemicals, but the unsustainable growth of plant-derived lipid production and an unreliable supply of certain lipids due to environmental changes, require new solutions. One promising solution is the use of lipids derived from microbial biomass, particularly oleaginous yeasts. This critical review begins with a description of the most promising yeast lipid replacement targets: palm oil substitute, cocoa butter equivalent, polyunsaturated fatty acid source, and animal fat analogue, emphasizing sustainability aspects. Subsequently, the review focuses on the most recent advances in upstream methodologies, particularly fermentation strategies that promote circularity, such as waste valorisation, co-cultivation and co-product biosynthesis. Downstream processing methods for minimising energy consumption and waste generation, including bioflocculation energy-efficient and environmentally friendly cell lysis and extraction, and integrated co-product recovery methods, are discussed. Finally, the current challenges are outlined. Integrating these strategies advances sustainable yeast lipid production for high-value applications.

Sow reproductive and progeny growth performance when fed Pichia guilliermondii yeast postbiotic: systematic review and meta-analysis.

Previous research suggested that feeding sows with a product containing an inactivated strain specific Pichia guilliermondii yeast postbiotic (PG; Citristim, ADM Animal Nutrition, Quincy, IL) has the potential to support fecundity, and progeny performance at birth, weaning, and after weaning. To summarize these effects, a systematic review followed by a meta-analysis was carried out to determine the effects of feeding sows with PG during gestation and lactation on reproductive the performance of sows and the growth of progeny after weaning. All experiments included were randomized trials reporting side-by-side comparisons of an appropriate control (CON) and the CON with the inclusion of PG. The effects of PG inclusion in sow diets were evaluated using the raw mean difference and effect size calculations. Analysis included seven trials for sow reproductive and litter performance until weaning, and eight trials for progeny performance after weaning. The risk of publication bias was assessed by funnel plots. In the case of publication bias, the Trim and Fill method was used. Heterogeneity was assessed using I 2 statistics. Sows fed PG during gestation and lactation had more piglets born alive (BA), BA + stillborn, and BA + stillborn + mummies (P < 0.001). The individual birth weight of the piglets was not affected by the supplementation (P = 0.835). As a result, litter weight at birth was greater in sows-fed PG (P < 0.001). Piglets born from PG-fed sows tended to be weaned 0.34 d younger than those from CON-fed sows (P = 0.060). Twenty-one-day adjusted pig weight at weaning tended to be lighter by 0.122 kg in the PG sow group (P = 0.069); however, litter weight at weaning adjusted to 21 d remained similar across groups (P = 0.516). The number of piglets weaned and mortality-adjusted number of piglets weaned per sow were greater in PG than in CON sows (P < 0.023). A carryover effect was observed for progeny of PG-fed sows after weaning. Piglets born from PG-supplemented sows had greater weight gain (P = 0.030) and tended to have a better survival rate (P = 0.055) until the end of the nursery phase. These results indicate that feeding PG to sows during gestation and lactation consistently and significantly improves not only the performance of sows at farrowing but also performance of the progeny after weaning.

Effects of yeast cultures on meat quality, flavor composition and rumen microbiota in lambs.

Since the banning of antibiotics, the use of feed additives to improve meat quality to satisfy people's pursuit of high quality has become a research hotspot. Yeast culture (YC) is rich in proteins, mannan oligosaccharides, peptides, and yeast cell metabolites, etc., and its use as a feed additive has a positive impact on improving meat quality. So the study aimed to provide a theoretical basis for YC improving mutton flavor and quality by detecting and analyzing the effects of YC on muscle physicochemical properties, amino acids, fatty acids, flavor composition, expression of related genes, and rumen microbiota of lambs. A total of 20 crossbred F1 weaned lambs (Australian white sheep♂ × Hu sheep♀; average 23.38 ± 1.17 kg) were randomly assigned to 2 groups, the control group (CON) and the 1.0% YC supplemented group (YC) (n = 10), and were reared in separate pens. The experiment had a pre-feeding period of 10 d and a treatment period of 60 d. After the experiment, 6 lambs in each group were randomly selected for slaughtering. The results showed that dietary YC supplementation increased rumen total VFA and acetate concentrations (p < 0.05), and muscle carcass fat (GR), a∗ value, intramuscular fat (IMF), lysine (Lys), arginine (Arg), nonessential amino acid (NEAA), oleic acid (C18:1n9c), and eicosanoic acid (C20:1) contents were significantly increased (p < 0.05), while cooking loss and γ-linolenic acid (C18: 3n6) were decreased (p < 0.05). Furthermore, we found that dietary YC improved the types of flavor compounds, and the key flavor substances such as hexanal, nonanal, styrene, benzaldehyde, p-xylene, and 1-octen-3-ol contents were changed (p < 0.05). Additionally, the expression of fat metabolism related genes PPARγ, FASN, and FABP4 were increased. Adding 1% YC to lamb diets increased profits by 47.70 CNY per sheep after 60 d of fattening. All of which indicated that YC could improve meat quality, especially flavor, which may be related to the regulation of the relative abundance of rumen microorganisms Bacteroidota, Prevotella_7, Succiniclasticum and Lachnospiraceae_NK3A20_group.

An undergraduate research experience in CRISPR-Cas9 mediated eukaryotic genome editing to teach fundamental biochemistry techniques.

CRISPR-Cas9 technology is an established, powerful tool for genome editing through the ability to target specific DNA sequences of interest for introduction of desired genetic modifications. CRISPR-Cas9 is utilized for a variety of purposes, ranging from a research molecular biology tool to treatment for human diseases. Due to its prominence across a variety of applications, it is critical that undergraduates in the life sciences are educated on CRISPR-Cas9 technology. To this end, we created an intensive eight-week long course-based undergraduate research experience (CURE) designed for students to understand CRISPR-Cas9 genome editing and perform it in Saccharomyces cerevisiae. Students enrolled in the CURE participate in 2, 3-h sessions a week and are engaged in the entire process of CRISPR-Cas9 genome editing, from preparation of genome editing reagents to characterization of mutant yeast strains. During the process, students master fundamental techniques in the life sciences, including sterile technique, Polymerase Chain Reaction (PCR), primer design, sequencing requirements, and data analysis. The course is developed with flexibility in the schedule for repetition of techniques in the event of a failed experiment, providing an authentic research experience for the students. Additionally, we have developed the course to be easily modified for the editing of any yeast gene, offering the potential to expand the course in research-driven classroom or laboratory settings.

Microtubule reorganization and quiescence: an intertwined relationship.

Quiescence is operationally defined as a reversible proliferation arrest. This cellular state is central for both organism development and homeostasis, its dysregulation causing many pathologies. The quiescent state encompasses very diverse cellular situations depending on the cell type and its environment. Further, quiescent cell properties evolve with time, a process that is thought to be at the origin of aging in multicellular organisms. Microtubules are found in all eukaryotes, and are essential for cell proliferation as they support chromosome segregation and intracellular trafficking. Upon proliferation cessation and quiescence establishment, the microtubule cytoskeleton was shown to undergo significant remodeling. The purpose of this review is to examine the literature in search of evidence to determine whether the observed microtubule reorganizations are merely a consequence of quiescence establishment or if they somehow participate in this cell fate decision.

Evidence for a hydrogen sulfide-sensing E3 ligase in yeast.

In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor that activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCFMet30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the trans-sulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30's WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCFMet30 dynamically senses the flow of sulfur metabolites through the trans-sulfuration pathway to regulate the synthesis of these special amino acids.

Evolutionary modes of wtf meiotic driver genes in Schizosaccharomyces pombe.

Killer meiotic drivers (KMDs) are a class of selfish genetic elements that bias inheritance in their favor by destroying meiotic progeny that do not carry them. How KMDs evolve is not well understood. In the fission yeast Schizosaccharomyces pombe, the largest gene family, known as the wtf genes, is a KMD family that causes intraspecific hybrid sterility. Here, we investigate how wtf genes evolve using long-read-based genome assemblies of 31 distinct S. pombe natural isolates, which encompass the known genetic diversity of S. pombe. Our analysis, involving nearly 1,000 wtf genes in these isolates, yields a comprehensive portrayal of the intraspecific diversity of wtf genes. Leveraging single-nucleotide polymorphisms in adjacent unique sequences, we pinpoint wtf-gene-containing loci that have recently undergone gene conversion events and infer their pre-gene-conversion state. These events include the revival of wtf pseudogenes, lending support to the notion that gene conversion plays a role in preserving this gene family from extinction. Moreover, our investigation reveals that solo long terminal repeats (LTRs) of retrotransposons, frequently found near wtf genes, can act as recombination arms, influencing the upstream regulatory sequences of wtf genes. Additionally, our exploration of the outer boundaries of wtf genes uncovers a previously unrecognized type of directly oriented repeats flanking wtf genes. These repeats may have facilitated the early expansion of the wtf gene family in S. pombe. Our findings enhance the understanding of the mechanisms influencing the evolution of this KMD gene family.