Search Results (66)

CRISPR technology, which is derived from the bacterial adaptive immune system, has transformed traditional genetic engineering techniques, made strain engineering significantly easier, and become a very versatile genome editing system that allows for precise, programmable modifications to a wide range of microbial genomes. The economies of fermentation-based manufacturing are changing because of its quick acceptance in both academic and industry labs. CRISPR processes have been used to modify industrially significant bacteria, including the lactic acid producers, Clostridium spp., Escherichia coli, and Corynebacterium glutamicum, in order to increase the yields of bioethanol, butanol, succinic acid, acetone, and polyhydroxyalkanoate precursors. CRISPR-mediated promoter engineering and single-step multiplex editing have improved inhibitor tolerance, raised ethanol titers, and allowed for the de novo synthesis of terpenoids, flavonoids, and recombinant vaccines in yeasts, especially Saccharomyces cerevisiae and emerging non-conventional species. While enzyme and biopharmaceutical manufacturing use CRISPR for quick strain optimization and glyco-engineering, food and beverage fermentations benefit from starter-culture customization for aroma, texture, and probiotic functionality. Off-target effects, cytotoxicity linked to Cas9, inefficient delivery in specific microorganisms, and regulatory ambiguities in commercial fermentation settings are some of the main challenges. This review provides an industry-specific summary of CRISPR–Cas9 applications in microbial fermentation and highlights technical developments, persisting challenges, and industrial advancements. Full article

Aquaculture is the fastest-growing food production sector. However, it faces significant challenges, including demand from a growing global population, which is estimated to reach 10.4 billion by the year 2100, disease outbreaks, environmental impacts, and the overuse of antibiotics. To address these issues, sustainable alternatives such as the use of microorganisms (probiotics, bacteriophages, and genetically modified microorganisms) have gained attention. This review examines the effects of these microorganisms on fish aquaculture, focusing on their potential to improve growth, health, and disease resistance while reducing environmental impacts. Probiotics, particularly lactic acid bacteria and yeasts, have been shown to enhance immune responses, digestive enzyme activity, and nutrient absorption in fish. Bacteriophages offer a promising alternative to antibiotics for controlling bacterial pathogens, with studies demonstrating their efficacy in reducing mortality rates in infected fish. Additionally, genetically modified microorganisms (GMMs) have been explored for their ability to produce beneficial compounds, such as enzymes and antimicrobial peptides, which can improve fish health and reduce the need for chemical treatments. Despite their potential, challenges such as regulatory hurdles, public acceptance, and environmental risks must be addressed. This review highlights the importance of further research to optimize the use of microorganisms in aquaculture and underscores their role in promoting sustainable practices. By integrating these biological tools, the aquaculture industry can move towards a more sustainable and environmentally friendly future. Full article

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are incurable neurodegenerative disorders sharing pathological and genetic features, including mutations in the FUS gene. FUS is an RNA-binding protein that mislocalizes to the cytoplasm and aggregates in ALS/FTD. In a yeast model, FUS proteinopathy is connected to changes in the epigenome, including reductions in the levels of H3S10ph, H3K14ac, and H3K56ac. Exploiting the same model, we reveal novel connections between FUS aggregation and epigenetic dysregulation. We show that the histone-modifying enzymes Ipl1 and Rtt109—responsible for installing H3S10ph and H3K56ac—are excluded from the nucleus in the context of FUS proteinopathy. Furthermore, we found that Ipl1 colocalizes with FUS, but does not bind it directly. We identified Nop1 and Rrp5, a histone methyltransferase and rRNA biogenesis protein, respectively, as FUS binding partners involved in the growth suppression phenotype connected to FUS proteinopathy. We propose that the nuclear exclusion of Ipl1 through indirect interaction with FUS drives the dysregulation of H3S10ph as well as H3K14ac via crosstalk. We found that the knockdown of Nop1 interferes with these processes. In a parallel mechanism, Rtt109 mislocalization results in reduced levels of H3K56ac. Our results highlight the contribution of epigenetic mechanisms to ALS/FTD and identify novel targets for possible therapeutic intervention. Full article

We have previously shown that 2-thiouridine (S2U), either as a single nucleoside or as an element of RNA chain, is effectively desulfurized under applied in vitro oxidative conditions. The chemically induced desulfuration of S2U resulted in two products: 4-pyrimidinone nucleoside (H2U) and uridine (U). Recently, we investigated whether the desulfuration of S2U is a natural process that also occurs in the cells exposed to oxidative stress or whether it only occurs in the test tube during chemical reactions with oxidants at high concentrations. Using different types of eukaryotic cells, such as baker’s yeast, human cancer cells, or modified HEK293 cells with an impaired antioxidant system, we confirmed that 5-substituted 2-thiouridines are oxidatively desulfurized in the wobble position of the anticodon of some tRNAs. The quantitative LC-MS/MS-MRMhr analysis of the nucleoside mixtures obtained from the hydrolyzed tRNA revealed the presence of the desulfuration products of mcm5S2U: mcm5H2U and mcm5U modifications. We also observed some amounts of immature cm5S2U, cm5H2U and cm5U products, which may have indicated a disruption of the enzymatic modification pathway at the C5 position of 2-thiouridine. The observed process, which was triggered by oxidative stress in the living cells, could impair the function of 2-thiouridine-containing tRNAs and alter the translation of genetic information. Full article

The use of probiotics has been gaining popularity in terms of inclusion into human diets over recent years. Based on properties exerted by these organisms, several benefits have been elucidated and conferred to the host. Bacteria have been more commonly used in probiotic preparations compared to yeast candidates; however, yeast exhibit several beneficial properties, such as the prevention and treatment of diarrhea, the production of antimicrobial agents, the prevention of pathogen adherence to intestinal sites, the maintenance of microbial balance, the modulation of the immune system, antibiotic resistance, amongst others. Saccharomyces boulardii is by far the most studied strain; however, the potential for the use of other yeast candidates, such as Kluyveromyces lactis and Debaryomyces hansenii, amongst others, have also been evaluated in this review. Furthermore, a special focus has been made regarding the production considerations for yeast-based probiotics and their formulation into different delivery formats. When drafting this review, evidence suggests that the use of yeasts, both wild-type and genetically modified candidates, can extend beyond gut health to support skin, the respiratory system, and overall immune health. Hence, this review explores the potential of yeast probiotics as a safe, effective strategy for preventative health in humans, highlighting their mechanisms of action, clinical applications, and production considerations. Full article

Biocatalysis is a green and sustainable technology based on the use of natural substances to catalyze chemical reactions. Humans have been unconsciously using biocatalysis for thousands of years to produce food and alcoholic beverages, but it is only since the 19th century that we have begun to understand its fundamentals and its enormous potential. In fact, advances in our knowledge of enzymes and metabolic pathways and, in recent decades, the introduction of tools such as bioinformatics, DNA sequencing and protein engineering have made biocatalysis a key strategy in fine chemistry and for the production of active pharmaceutical ingredients. In addition, the discovery of new microorganisms adapted to adverse conditions has also been crucial in advancing this avenue. The present review focuses on the use of unconventional yeasts and their enzymes in the most interesting reactions where biocatalysis is applied. It highlights the advantages of using these microorganisms in industrial chemical processes due to their particular phenotypes, such as their ability to withstand high temperatures and pressures, as well as acidic or alkaline environments, high substrate loads, presence of organic solvents, etc. All this results in a wider range of possible substrates and higher efficiency. Examples of the most important reactions in which their use has been described are included, considering both catalysis by wild-type whole cells or their isolated enzymes and their genetically modified variants. All this information will help to understand the current relevance of unconventional yeasts and their enzymes in biocatalysis. Full article

Cassava (Manihot esculenta Crantz) is used as a biomass energy material and an effective supplement for food and feed. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step of lignin biosynthesis and is responsible for various stresses. However, systematic investigations of the CAD gene family in cassava have been poorly understood. In this study, a genome-wide survey and bioinformatics analysis of CAD gene family was performed, transcriptomics, qRT-PCR, gene silencing and stress of yeast cell were used for excavate and validate the candidate MeCADs gene. 36 MeCADs genes unevenly distributed across 12 chromosomes were identified. Through phylogenetic analyses alongside their Arabidopsis counterparts, these MeCADs were divided into four groups, each containing a similar structure and conserved motifs. Interestingly, transcriptome data analysis revealed that 32 MeCAD genes were involved in the postharvest physiological deterioration (PPD) process, whereas 27 MeCAD genes showed significant changes. Additionally, the relative quantitative analysis of 6 MeCAD genes demonstrated that they were sensitive to PPD, suggesting that they may be involved in the regulation of PPD. Silencing MeCAD13 and MeCAD28 further showed that lignin content significantly decreased in the leaves. The wound-stress tolerance of transgenic yeast cells was enhanced after transformation with MeCAD13 and MeCAD28. MeCAD13 and MeCAD28 may play positive roles in lignin biosynthesis and PPD response, respectively. These results provided a systematic functional analysis of MeCADs in cassava and paved a new way to genetically modify lignin biosynthesis and PPD tolerance. Full article

The estimation of parameters in a wine fermentation model provides the opportunity to predict the rate and concentration outcomes, to strategically intervene to change the conditions, and to forecast the rates of heat and carbon dioxide release. The chosen parameters of the fermentation model are the initial assimilable nitrogen concentration and yeast properties (lag time, viability constant, and specific maintenance rate). This work evaluates six methods for parameter estimation: Bard, Bayesian Optimization, Particle Swarm Optimization, Differential Evolution, Genetic Evolution, and a modified Direct Grid Search technique. The benefits and drawbacks of the parameter computational methods are discussed, as well as a comparison of numerical integration methods (Euler, Runge–Kutta, backward differential formula (BDF), and Adams/BDF). A test set of density-time data for five white and five red commercial wine fermentations across vintage, grape cultivar, fermentation temperature, inoculated yeast strain, and fermentor size was used to evaluate the parameter estimation methods. A Canonical Variate Analysis shows that the estimation methods are not significantly different from each other while, in the parameter space, each of the fermentations were significantly different from each other. Full article

The addition of pectinase enzymes during the maceration stage of grape skins in order to improve the extraction yields and color of red wines is a common practice in many wineries. The objective of this work was to study in depth the changes that occurred in the aminoacidic, volatile, and phenolic compositions of Cabernet Sauvignon wines fermented with a Saccharomyces cerevisiae strain genetically modified with the gene encoding for endopolygalacturonase (PGU1) in transcriptional fusion with the promoter of the phosphoglycerate kinase (PGK1) gene, both from S. cerevisiae origin. A higher yield extraction of wine was obtained in wines fermented with the modified strain (PW), increasing by around 6.1% compared to the control wine (CW). Moreover, there was a 40% decrease in the malic acid content in the PW, thus suggesting that this modified yeast could be investigated as a malic acid-reducing agent. There were slight differences in other aroma volatile compounds studied as well as in the phenolic content. However, there was a considerable increase in the amino acid content in the PW. Full article

This review aims to create a communication tool for low-alcohol and nonalcoholic wine production, catering to scientists, educators, students, and wine producers in the field. With health concerns regarding alcohol consumption, the need for information on low-alcohol wines is essential. This paper outlines the methods for the pre-fermentation (leaf area reduction, early grape harvest, grape must dilution, filtration of grape juice and addition of glucose oxidase), mid-fermentation (employing non-saccharomyces yeasts, using genetically modified yeasts through metabolic engineering, and controlling yeast nutrition), and post-fermentation (nanofiltration and reverse osmosis, osmotic distillation, pervaporation, spinning cone column, vacuum distillation, and multi-stage membrane-based systems) stages and their effects on wine quality. It also presents evidence of the impact of alcoholic, low-alcohol, and nonalcoholic wines on cardiovascular health. Finally, the potential market for low-alcohol and nonalcoholic wines is discussed. Key findings indicate a shift toward low-alcohol alternatives due to health, economic, and social factors and consumer interest in healthier lifestyles. Low-alcohol and nonalcoholic wines offer health benefits, particularly cardiovascular health, presenting an opportunity for winemakers to cater to a health-conscious market. From an economic perspective, the low-alcohol and nonalcoholic wine market is poised to grow and diversify its revenue streams. The development of high-quality low-alcohol and nonalcoholic wines, which can command premium prices, enhances profitability. The changing regulatory landscape in Europe, with a focus on transparency in alcohol labeling and nutritional information, aligns with the new consumer preferences and regulatory standards. Full article

The exploration of space is becoming more feasible, and with this comes the possibility of performing fermentations in microgravity. Our study explores the potential effects of microgravity on a standard brewing model fermentation. As the fermentation of barley wort has been studied for centuries, there is an established foundation of knowledge with which to compare any changes that occur under microgravity. A modified ASBC miniature fermentation protocol (Yeast-14) was conducted within a Yuri 2.0 microgravity chamber to examine the response of Saccharomyces pastorianus to simulated microgravity conditions. Our findings reveal that yeast exhibited accelerated growth rates under microgravity compared to standard conditions. Additionally, the microgravity environment led to significantly lower levels of volatile compounds such as isoamyl acetate and 2-phenethyl acetate in the final product. Further genetic analysis showed significant downregulation of the ATF2 gene in the control group, potentially the mechanism behind the observed decrease in volatile compounds. These results show that while fermentation under microgravity is likely to eventually be commonplace, there may be changes in rate and gene expression that are beneficial or detrimental to the final product depending upon the desired characteristics. Full article

Hydroxytyrosol (HT) is a well-known compound for its bioactive properties. It is naturally present in olives, olive oil, and wine. Its presence in wines is partly due to its production during alcoholic fermentation by yeast through a hydroxylation of tyrosol formed through the Ehrlich pathway. This work aims to explore the influence of yeast assimilable nitrogen (YAN) and glucose content as precursors of HT formation during alcoholic fermentation. Commercial Saccharomyces cerevisiae QA23 and its metabolically engineered strain were used to ferment synthetic must. Each strain was tested at two different YAN concentrations (210 and 300 mg L⁻¹) and two glucose concentrations (100 and 240 g L⁻¹). This work confirms that the less YAN and the more glucose, the higher the HT content, with fermentations carried out with the metabolically engineered strain being the ones with the highest HT content (0.6 mg L⁻¹). Full article

Reversed-flow gas chromatography (R.F.G.C.) was employed to assess the impact of genetic modification on Saccharomyces cerevisiae yeast strains during the process of alcoholic fermentation, utilizing fig syrup. Multiple fermentations were carried out at various temperatures to evaluate the influence of genetic modifications on yeast strain efficiency. The study involved a wild-type yeast strain, W303, as a control and two genetically modified strains, W_M4_533 and W_M4_558, sharing the same genetic background as the wild type. Notably, the genetic modifications in the Msn4p transcription factor involved the substitution of serine residues with alanine at positions 533 and 558, resulting in the development of psychrophilic or ethanol-resistant strains. Utilizing the R.F.G.C. method enabled the differentiation of the duration of alcoholic fermentation phases, providing insights correlated to the yeast cell life cycle. The values of rate constants (k) for each phase, conducted with both wild-type and genetically modified cells using RFGC, aligned with the existing literature. Additionally, the calculation of activation energies for distinct phases revealed lower values for genetically modified strains compared to wild-type strains. This decrease in activation energies suggests enhanced efficiency in the alcoholic fermentation process for the genetically modified strains. Full article

The valorization of byproducts from the sugarcane industry represents a potential alternative method with a low energy cost for the production of metabolites that are of commercial and industrial interest. The production of exopolysaccharides (EPSs) was carried out using the yeast Suhomyces kilbournensis isolated from agro-industrial sugarcane, and the products and byproducts of this agro-industrial sugarcane were used as carbon sources for their recovery. The effect of pH, temperature, and carbon and nitrogen sources and their concentration in EPS production by submerged fermentation (SmF) was studied in 170 mL glass containers of uniform geometry at 30 °C with an initial pH of 6.5. The resulting EPSs were characterized with Fourier-transform infrared spectroscopy (FT-IR). The results showed that the highest EPS production yields were 4.26 and 44.33 g/L after 6 h of fermentation using sucrose and molasses as carbon sources, respectively. Finally, an FT-IR analysis of the EPSs produced by S. kilbournensis corresponded to levan, corroborating its origin. It is important to mention that this is the first work that reports the production of levan using this yeast. This is relevant because, currently, most studies are focused on the use of recombinant and genetically modified microorganisms; in this scenario, Suhomyces kilbournensis is a native yeast isolated from the sugar production process, giving it a great advantage in the incorporation of carbon sources into their metabolic processes in order to produce levan sucrose, which uses fructose to polymerize levan. Full article

There is currently growing interest in the creation of artificial microbial consortia, especially in the field of developing and applying various bioremediation processes. Heavy metals, dyes, synthetic polymers (microplastics), pesticides, polycyclic aromatic hydrocarbons and pharmaceutical agents are among the pollutants that have been mainly targeted by bioremediation based on various consortia containing fungi (mycelial types and yeasts). Such consortia can be designed both for the treatment of soil and water. This review is aimed at analyzing the recent achievements in the research of the artificial microbial consortia that are useful for environmental and bioremediation technologies, where various fungal cells are applied. The main tendencies in the formation of certain microbial combinations, and preferences in their forms for usage (suspended or immobilized), are evaluated using current publications, and the place of genetically modified cells in artificial consortia with fungi is assessed. The effect of multicomponence of the artificial consortia containing various fungal cells is estimated, as well as the influence of this factor on the functioning efficiency of the consortia and the pollutant removal efficacy. The conclusions of the review can be useful for the development of new mixed microbial biocatalysts and eco-compatible remediation processes that implement fungal cells. Full article