Yeast Growth Research Articles

Genome-wide identification of metal tolerance protein genes in Quercus dentata and their roles in response to various heavy metal stresses

Metal tolerance protein (MTP) is a cation transporter that plays an important role in tolerance to heavy metal stress. However, thus far, there has been no genome-wide investigation of the MTP gene family in Quercus plants. Quercus dentata is one of the main constructive species of forest in northern China. It has strong tolerance to a variety of heavy metal stresses. In this study, 25 MTPs were identified from the Q. dentata genome and classified into three subfamilies and seven groups according to their sequence characteristics and phylogenetic relationships. Both tandem and segmental duplication events contributed to the expansion of the QdMTP gene family. Interestingly, all 10 tandem duplication events contributed to the expansion of the Mn-CDF subfamily. The expression of Mn-CDF subfamily members in different organs and tissues of Q. dentata was different, and they responded differently to manganese, iron, zinc and cadmium stress treatments. QdMTP10.7, a member of the Mn-CDF subfamily, enhanced yeast growth under manganese, zinc and iron stresses. The subcellular localization in tobacco leaf epidermis cells showed that QdMTP10.7 was located in vacuoles. These data generated from this study provide an important foundation to elucidate the biological roles of QdMTP genes related to heavy metal tolerance in Q. dentata.

Kinetics of Riboflavin Production by Hyphopichia wangnamkhiaoensis under Varying Nutritional Conditions.

Riboflavin, an essential vitamin for humans, is extensively used in various industries, with its global demand being met through fermentative processes. Hyphopichia wangnamkhiaoensis is a novel dimorphic yeast species capable of producing riboflavin. However, the nutritional factors affecting riboflavin production in this yeast species remain unknown. Therefore, we conducted a kinetic study on the effects of various nutritional factors-carbon and energy sources, nitrogen sources, vitamins, and amino acids-on batch riboflavin production by H. wangnamkhiaoensis. Batch experiments were performed in a bubble column bioreactor to evaluate cell growth, substrate consumption, and riboflavin production. The highest riboflavin production was obtained when the yeast growth medium was supplemented with glucose, ammonium sulfate, biotin, and glycine. Using these chemical components, along with the mineral salts from Castañeda-Agullo's culture medium, we formulated a novel, low-cost, and effective culture medium (the RGE medium) for riboflavin production by H. wangnamkhiaoensis. This medium resulted in the highest levels of riboflavin production and volumetric productivity, reaching 16.68 mg/L and 0.713 mg/L·h, respectively, within 21 h of incubation. These findings suggest that H. wangnamkhiaoensis, with its shorter incubation time, could improve the efficiency and cost-effectiveness of industrial riboflavin production, paving the way for more sustainable production methods.

Microbiological Profile of Phytoestrogen Rich Supplement and Its Impact on Gut-microbiome Composition in Drosophila melanogaster

Phytoestrogen-rich diet alters the composition of gut microbiota by enhancing the growth of beneficial bacteria and decreasing the microbial load of pathogenic organisms. Drosophila is an invertebrate model system used for research studies, as it shares 70% genetic homology with humans. The present study aimed to analyse microbiological profile of phytoestrogen rich supplement and its impact on gut-microbiome composition in Drosophila melanogaster. The phytoestrogen rich supplement was mixed with formula 424 plain and flies were exposed to it. Gut of flies was dissected and cell suspension was prepared. Bacterial colonies were developed by streaking method. Gram staining was performed to differentiate the bacterial cells and further gut microbiome composition (Acetobacteraceae and Lactobacillales taxa) was analysed by 16S rRNA sequencing. The microbiological analysis was carried out to ascertain the microbial load of the developed product for consumption. The total bacterial count and coliform counts of the phytoestrogen rich supplement were <10 CFU/g. Also, the developed supplement exhibited minimal yeast and mold growth (<1 CFU/g). Gram staining showed gram positive (Bacilli and cocci). 16S rRNA sequencing showed significance with mild variation in similarity. It confirmed the presence of Bacillus paramycoids. The developed supplement has showed improved gut microbiome composition in the Drosophila. In future, studies can be extended to humans to analyse the efficacy of the supplement in the gut microbiome composition.

Characterization of human aquaporin ion channels in a yeast expression system as a tool for novel ion channel discovery.

Aquaporin (AQP) channels found in all domains of life are transmembrane proteins which mediate passive transport of water, glycerol, signaling molecules, metabolites, and charged solutes. Discovery of new classes of ion-conducting AQP channels has been slow, likely reflecting time- and labor-intensive methods required for traditional electrophysiology. Work here defines a sensitive mass-throughput system for detecting AQP ion channels, identified by rescue of cell growth in the K+-transport-defective yeast strain CY162 following genetic complementation with heterologously expressed cation-permeable channels, using the well characterized human AQP1 channel for proof of concept. Results showed AQP1 conferred transmembrane permeability to cations which rescued survival in CY162 yeast. Comprehensive testing showed that growth response properties fully recapitulated AQP1 pharmacological agonist and antagonist profiles for activation, inhibition, dose-dependence, and structure-function relationships, demonstrating validity of the yeast screening tool for AQP channel identification and drug discovery efforts. This method also provided new information on divalent cation blockers of AQP1, pH sensitivity of antagonists, and ion permeability of human AQP6. Site-directed mutagenesis of AQP1 channel regulatory domains confirmed that yeast growth rescue was mediated by the introduced channels. Optical monitoring with a lithium-sensitive photoswitchable probe in living cells independently demonstrated monovalent cation permeability of AQP1 channels in yeast plasma membrane. Ion channel properties of AQP1 expressed in yeast were consistent with those of AQP1 expressed in Xenopus laevis oocyte and K+-transport defective Escherichia coli. Outcomes here establish a powerful new approach for efficient screening of phylogenetically diverse AQPs for yet untested functions as cation channels.

Cordia dichotoma fruit mucilage and flaxseed oil nanoemulsion loaded bioactive film: Synergistic approach to enhance antimicrobial properties and bread shelf-life

Recently, non-conventional sources of mucilage have gained significant attention due to their exceptional techno-functional properties and easy availability. This study aimed to utilize Cordia dichotoma fruit mucilage (CDM) for fabricating bioactive films to evaluate the shelf-life of bread. For the development of nanoemulsion, different concentrations of flaxseed oil (1–5% w/w) were employed. Selection was carried out based on droplet size, encapsulation efficiency, and antimicrobial activity. The selected nanoemulsion (CDM-FO1) was subsequently used to develop CDM-based films with the incorporation of carboxymethyl cellulose (CMC) and sodium alginate (SA). Results revealed that the droplet size of the nanoemulsions was significantly increased from 101.25 ± 4.68 nm to 205.15 ± 7.14 nm with increasing flaxseed oil concentration. However, the addition of CMC and SA improved the tensile strength (13.97 ± 0.34 MPa) of the CDM-F11 film compared to control films CDM (6.98 ± 0.68 MPa) and CDM-FC (7.76 ± 1.65 MPa). The CDM-F11 film exhibited excellent barrier properties compared to the control film. The control film showed higher yeast and mold growth, starting at 0.84 ± 0.57 Log CFU/g on the 3rd day and increased to 3.12 ± 0.39 Log CFU/g by the 9th day. In contrast, the CDM-FC film demonstrated better antimicrobial properties, with microbial growth at 0.52 ± 0.39 Log CFU/g on the 3rd day and 2.84 ± 0.38 Log CFU/g on the 9th day. Overall, CDM film loaded with flaxseed oil nanoemulsion can be effectively used as a sustainable food packaging material with enhanced antimicrobial efficiency for bread preservation.

Impact of Sweet Orange Peel Essential Oil on the Nutrient and Oxidative Stability of Aquafeed During the Storage Condition

The main goal of this study was to assess the qualitative composition of essential oil (P), a natural extract produced from wasted orange peels in the citrus processing sector. Additionally, the study aimed to investigate the alterations in fish feeds when this essential oil is incorporated and stored. The essential oil was extracted from the orange peels provided for the study using the hydrodistillation method and the Clevenger apparatus. The study determined the volatile components of the oil with 100% accuracy. The primary constituent was identified as D-limonene, comprising 59.27% of the total composition. Subsequently, experimental feed groups were established by including the essential oil in the feed rations at ratios of ‰0 (P0), ‰0.5 (P5), ‰1 (P10), and ‰3 (P30). Under storage conditions, the feeds were stored in feed sacks for 60 days. Periodic samples were collected during storage and subjected to nutritional, microbiological, structural, and oxidation tests. The results of the study show that P had no protective effect in fish diets against the growth of yeasts, molds, and other mesophilic aerobic bacteria (p > 0.05). It was shown that the nutritional values varied over time during storage. However, this variation was not substantially correlated with the amount of P in the diets (p > 0.05). There was no discernible impact of the addition of P on the structural characteristics of the feed grains. Nevertheless, the inclusion of P substantially impeded the process of lipid oxidation in the diet (p<0.05). After the two-month storage period, it was shown that adding at least 1 ‰ of P to the fish meals prevented the peroxide generated from oxidation in the feed from exceeding acceptable levels.

In Vitro Inhibitory Effects and Co-Aggregation Activity of Lactobacilli on Candida albicans

Lactobacilli are considered important probiotics for the prevention of some infections. In this study, the antifungal effect of both cells and cell-free supernatants of twenty-three strains of lactobacilli were investigated against Candida albicans by co-aggregation, agar diffusion assay, agar spot assay and co-culture assay. In all cases, a fungistatic effect was recorded. In the agar diffusion assay and agar spot assay, an effect was established primarily for heterofermentative species via the production of lactic acid. The anti-Candida effect was higher with microbial suspension than with cultural supernatants in the co-culture assay. A strain-specific reduction in the yeast growth up to 28.9% in MRS broth and up to 17.1% in BHI broth was observed. Cells of Limosilactobacillus fermentum LLF-01 and Limosilactobacillus reuteri LLR-K67 showed the highest activity in both model systems. For all strains, a lower reduction up to 9.7% was recorded with cultural supernatants. L. fermentum LLF-01 showed the highest ability of co-aggregation (64.8%) with C. albicans, followed by Lactobacillus acidophilus LLA-01, Lactobacillus gasseri LLG-V74, Lactobacillus delbrueckii subsp. bulgaricus LLB-02 and two strains of Lactobacillus delbrueckii subsp. lactis LLL-14 and LLL-F18. The present study showed the potential of several strains of lactobacilli to affect the population of C. albicans in vitro. The combination of cultures with proved anti-Candida and co-aggregation activity in a probiotic formula may have a positive effect for the prevention of yeast overgrowth in the gut and hence for the suppression of candidiasis.

A Droplet-Based Microfluidic Platform for High-Throughput Culturing of Yeast Cells in Various Conditions.

Yeast plays a significant role in a variety of fields. In particular, it is extensively used as a model organism in genetics and cellular biology studies, and is employed in the production of vaccines, pharmaceuticals, and biofuels. Traditional "bulk"-based studies on yeast growth often overlook cellular variability, emphasizing the need for single-cell analysis. Micro-droplets, tiny liquid droplets with high surface-area-to-volume ratios, offer a promising platform for investigating single or a small number of cells, allowing precise control and monitoring of individual cell behaviors. Microfluidic devices, which facilitate the generation of micro-droplets, are advantageous due to their reduced volume requirements and ability to mimic in vivo micro-environments. This study introduces a custom-designed microfluidic device to encapsulate yeasts in micro-droplets under various conditions in a parallel manner. The results reveal that optimal glucose concentrations promoted yeast growth while cycloheximide and Cu2+ ions inhibited it. This platform enhances yeast cultivation strategies and holds potential for high-throughput single-cell investigations in more complex organisms.

Different Nitrogen Consumption Patterns in Low Temperature Fermentations in the Wine Yeast Saccharomyces cerevisiae.

Nowadays, the wine industry carries out fermentations at low temperatures because this oenological practice clearly improves the aromatic complexity of the final wines. In addition, nitrogen content of the must also influences the quality of the wine. In this study, we carried out a phenotypic and fermentative analysis of two industrial wine Saccharomyces cerevisiae strains (P5 and P24) at 15 and 28 °C and three nitrogen concentrations (60, 140 and 300 mg N/L) in synthetic must. Our results show that both parameters, temperature and nitrogen, are interrelated and clearly determine the competitiveness of the wine strains and their ability to adapt at low temperatures. The best adapted strain at low temperatures decreased its competitiveness at lower nitrogen concentrations. In addition, our results show that it is not only the quantity of nitrogen transported that is important but also the quality of the nitrogen source used for wine yeast adaptation at low temperatures. The presence of some amino acids, such as arginine, branched chain amino acids, and some aromatic amino acids can improve the growth and fermentation activity of wine yeasts at low temperatures. These results allow us to better understand the basis of wine yeast adaptation to fermentation conditions, providing important information for winemakers to help them select the most appropriate yeast strain, thus reducing the economic costs associated with long and sluggish fermentations. The correlation between some amino acids and better yeast fermentation performance could be used in the future to design inactive dry yeast enriched in some of these amino acids, which could be added as a nutritional supplement during low temperature fermentations.

Pesticides and winemaking: A comprehensive review of conventional and emerging approaches.

The use of pesticides in viticulture may play a crucial role in ensuring the health and quality of grapes. This review analyzes the most common pesticides used, illustrating their classification and toxicity, and their variations throughout the winemaking process. Fungicides are generally harmless or mildly toxic, whereas insecticides are classified as either highly or moderately hazardous. Potential alternatives to synthetic pesticides in wine production are also reviewed, thereby including biopesticides and biological agents. Analytical methods for detecting and quantifying pesticide residues in wine are then described, including liquid chromatography and gas chromatography coupled with mass spectrometry. This review also discusses the impact of the winemaking process on pesticide content. Pesticides with strong hydrophobicity were more likely to accumulate in solid byproducts, whereas hydrophilic pesticides were distributed more in the liquid phase. Grape's skin contains lipids, so hydrophobic pesticides adsorb strongly on grape surfaces and the clarification has been shown to be effective in the reduction of hydrophobic compounds. Therefore, the final wine could have more quantities of hydrophilic pesticides. Alcoholic fermentation has been shown to be crucial in pesticide dissipation. However, pesticide residues in wine have been shown an antagonistic effect on yeasts, affecting the safety and quality of wine products. Therefore, proteomic and genomic analyses of yeast growth are reviewed to understand the effects of pesticides on yeast during fermentation. The last section describes new effective methods used in removing pesticides from grapes and wine, thereby improving product quality and reducing harmful effects.

Assessing predictions on fitness effects of missense variants in HMBS in CAGI6.

This paper presents an evaluation of predictions submitted for the "HMBS" challenge, a component of the sixth round of the Critical Assessment of Genome Interpretation held in 2021. The challenge required participants to predict the effects of missense variants of the human HMBS gene on yeast growth. The HMBS enzyme, critical for the biosynthesis of heme in eukaryotic cells, is highly conserved among eukaryotes. Despite the application of a variety of algorithms and methods, the performance of predictors was relatively similar, with Kendall's tau correlation coefficients between predictions and experimental scores around 0.3 for a majority of submissions. Notably, the median correlation (≥ 0.34) observed among these predictors, especially the top predictions from different groups, was greater than the correlation observed between their predictions and the actual experimental results. Most predictors were moderately successful in distinguishing between deleterious and benign variants, as evidenced by an area under the receiver operating characteristic (ROC) curve (AUC) of approximately 0.7 respectively. Compared with the recent two rounds of CAGI competitions, we noticed more predictors outperformed the baseline predictor, which is solely based on the amino acid frequencies. Nevertheless, the overall accuracy of predictions is still far short of positive control, which is derived from experimental scores, indicating the necessity for considerable improvements in the field. The most inaccurately predicted variants in this round were associated with the insertion loop, which is absent in many orthologs, suggesting the predictors still heavily rely on the information from multiple sequence alignment.

Genome-Wide Isolation of VIN Gene Family and Functional Identification of HpVIN4 in Red Pitaya (Hylocereus polyrhizus)

Soluble sugars, including glucose, fructose and sucrose, are the most important determinants that affect the flavor and quality of red pitaya (Hylocereus polyrhizus) fruit. Vacuolar invertase (VIN), which catalyzes sucrose hydrolysis into glucose and fructose, is a key type of enzyme responsible for soluble sugar metabolism in plant growth and development. Herein, we conducted genome-wide identification, gene expression analysis, subcellular localization and an enzymatic properties assay for the VIN-encoding genes from red pitaya. During red pitaya fruit development towards ripening, the enzymatic activities of VIN showed an up-regulated trend towards ripening. In total, four isoforms (HpVIN1–4) of the VIN-encoding gene were identified from the pitaya genome. Sequence alignment results revealed that the HpVIN1, HpVIN3 and HpVIN4 proteins contained essential motifs for targeting the vacuole and conserved motifs or residues responsible for sucrose binding and hydrolysis. Gene expression pattern analyses revealed that the level of HpVIN4 was obviously increasing during fruit development and acted as the most abundant VIN isoform towards ripening. Subcellular localization detection via transient expression in Arabidopsis thaliana mesophyll protoplasts revealed that the HpVIN4 protein was localized in the vacuole. Growth complementation tests of heterologous expression in the invertase-deficient baker’s yeast strain suggested that the HpVIN4 protein had a sucrose hydrolysis activity and could restore the yeast growth in vivo. The identification of enzymatic properties in vitro demonstrated that the HpVIN4 protein could degrade sucrose into glucose and fructose with an optimum pH of 4.0. Specifically, the HpVIN4 protein had an estimated Km value of 5.15 ± 1.03 mmol·L−1 for sucrose hydrolysis. Ultimately, this study provides a comprehensive understanding of the potential roles of VINs during fruit development and towards ripening and provides functional gene resources for regulating soluble sugar accumulation in red pitaya fruit.

The Impact of Cyclodextrins on the Physiology of Candida boidinii: Exploring New Opportunities in the Cyclodextrin Application.

Cyclodextrins, commonly used as excipients in antifungal formulations to improve the physicochemical properties and availability of the host molecules, have not been systematically studied for their effects and bioactivity without a complex active substance. This paper evaluates the effects of various cyclodextrins on the physiology of the test organism Candida boidinii. The research examines their impact on yeast growth, viability, biofilm formation and morphological changes. Native ACD, BCD, randomly methylated α- and β-CD and quaternary ammonium α-CD and β-CD were investigated in the 0.5-12.5 mM concentration range in both static and dynamic systems. The study revealed that certain cyclodextrins exhibited notable antifungal effects (up to ~69%) in dynamic systems; however, the biofilm formation was enhanced in static systems. The magnitude of these effects was influenced by several variables, including the size of the internal cavity, the concentration and structure of the cyclodextrins, and the contact time. Furthermore, the study found that CDs exhibited distinct effects in both static and dynamic systems, potentially related to their tendency to form aggregates. The findings suggest that cyclodextrins may have the potential to act as antifungal agents or growth promoters, depending on their structure and surrounding environments.

Engineering a GPCR-based yeast biosensor for a highly sensitive melatonin detection from fermented beverages

Melatonin is a multifunctional molecule with diverse biological roles that holds great value as a health-promoting bioactive molecule in any food product and yeast’s ability to produce it has been extensively demonstrated in the last decade. However, its quantification presents costly analytical challenges due to the usual low concentrations found as the result of yeast metabolism. This study addresses these analytical challenges by optimizing a yeast biosensor based on G protein-coupled receptors (GPCR) for melatonin detection and quantitation. Strategic genetic modifications were employed to significantly enhance its sensitivity and fluorescent signal output, making it suitable for detection of yeast-produced melatonin. The optimized biosensor demonstrated significantly improved sensitivity and fluorescence, enabling the screening of 101 yeast strains and the detection of melatonin in various wine samples. This biosensor’s efficacy in quantifying melatonin in yeast growth media underscores its utility in exploring melatonin production dynamics and potential applications in functional food development. This study provides a new analytical approach that allows a rapid and cost-effective melatonin analysis to reach deeper insights into the bioactivity of melatonin in fermented products and its implications for human health. These findings highlight the broader potential of biosensor technology in streamlining analytical processes in fermentation science.

Antimicrobial and Antifungal Effects of Essential Oils from Origanum vulgare, Lavandula officinalis, and Syzygium aromaticum on Bacterial Strains through Gaseous Contact

Background: Hospital-acquired infections, also known as nosocomial infections, affect thousands of patients worldwide. In Algeria, these infections raise significant concerns and give rise to numerous questions due to hygiene deficits and non-compliance with preventive measures. Objective: The main objective of this study was to investigate the antibacterial and antifungal properties of Origanum vulgare, Lavandula officinalis, and Syzygium aromaticum, as well as their combinations, to discover new antibacterial and antifungal agents to combat nosocomial infections related to hospital and medical care environments. Materials and Methods: Essential oils were obtained by a Clevenger-type apparatus and analyzed using GC and GC/MS. The antimicrobial and antifungal activity was tested against several bacteria responsible for nosocomial infections in in-vitro tests using the gas contact method in sealed containers. Results: The essential oil extracted from S. aromaticum buds was primarily composed of eugenol (59.4%), E-β-caryophyllene (16.5%), and eugenyl acetate (10.5%). The analysis of the chemical composition of L. officinalis essential oil identified 1,8-cineole (22.8%), β-pinene (12.4%), and linalool (8.5%) as the main compounds. As for O. vulgare essential oil, its major components were carvacrol (72.6%) and thymol (10.5%). The results revealed that the essential oils of L. officinalis, O. vulgare and S. aromaticum, as well as their combinations, used at a concentration of 1/10, had a remarkable effect on the E. coli, P. aeruginosa and K. pneumoniae strains. In contrast, they had a limited effect on the Gram-positive strain, S. aureus. The antifungal effectiveness of the essential oil of O. vulgare, as well as the combination of O. vulgare and S. aromaticum, has been remarkable against the C. albicans species, leading to a complete inhibition of yeast growth Conclusion: Our results indicate that our essential oils have a broad spectrum of antimicrobial activity, which makes them particularly useful as disinfectants in health facilities and could be used as a valuable medicine to control nosocomial infections and avoid infections acquired during a stay in a health facility such as respiratory tract infections and urinary tract infections.

Med3-mediated NADPH generation to help Saccharomyces cerevisiae tolerate hyperosmotic stress.

Hyperosmotic stress tolerance is crucial for Saccharomyces cerevisiae in producing value-added products from renewable feedstock. The limited understanding of its tolerance mechanism has impeded the application of these microbial cell factories. Previous studies have shown that Med3 plays a role in hyperosmotic stress in S. cerevisiae. However, the specific function of Med3 in hyperosmotic stress tolerance remains unclear. In this study, we showed that the deletion of the mediator Med3 impairs S. cerevisiae growth under hyperosmotic stress. Phenotypic analyses and yeast two-hybrid assays revealed that Med3 interacts with the transcription factor Stb5 to regulate the expression of the genes gnd1 and ald6, which are involved in NADPH production under hyperosmotic stress conditions. The deletion of med3 resulted in a decrease in intracellular NADPH content, leading to increased oxidative stress and elevated levels of intracellular reactive oxygen species under hyperosmotic stress, thereby impacting bud formation. These findings highlight the significant role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.IMPORTANCEHyperosmotic stress tolerance in the host strain is a significant challenge for fermentation performance in industrial production. In this study, we showed that the S. cerevisiae mediator Med3 is essential for yeast growth under hyperosmotic conditions. Med3 interacts with the transcription factor Stb5 to regulate the expression of genes involved in the NADPH-generation system during hyperosmotic stress. Adequate NADPH ensures the timely removal of excess reactive oxygen species and supports bud formation under these conditions. This work highlights the crucial role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.

NtZIP5A/B is involved in the regulation of Zn/Cu/Fe/Mn/Cd homeostasis in tobacco.

Plants grow in soils with varying concentrations of microelements, often in the presence of toxic metals e.g. Cd. To cope, they developed molecular mechanisms to regulate metal cross-homeostasis. Understanding underlying complex relationships is key to improving crop productivity. Recent research suggests that the Zn and Cd uptake protein NtZIP5A/B [Zinc-regulated, Iron-regulated transporter-like Proteins (ZIPs)] from tobacco (Nicotiana tabacum L. v. Xanthi) is involved in the regulation of a cross-talk between the two metals. Here, we support this conclusion by showing that RNAi-mediated silencing of NtZIP5A/B resulted in a reduction of Zn accumulation and that this effect was significantly enhanced by the presence of Cd. Our data also point to involvement of NtZIP5B in regulating a cross-talk between Cu, Fe, and Mn. Using yeast growth assays, Cu (but not Fe or Mn) was identified as a substrate for NtZIP5B. Furthermore, GUS-based analysis showed that the tissue-specific activity of the NtZIP5B promoter was different in each of the Zn-/Cu-/Fe-/Mn deficiencies applied with/without Cd. The results indicate that NtZIP5B is involved in maintaining multi-metal homeostasis under conditions of Zn, Cu, Fe, and Mn deficiency, and also in the presence of Cd. It was concluded that the protein regulates the delivery of Zn and Cu specifically to targeted different root cells depending on the Zn/Cu/Fe/Mn status. Importantly, in the presence of Cd, the activity of the NtZIP5B promoter is lost in meristematic cells and increased in mature root cortex cells, which can be considered a manifestation of a defense mechanism against its toxic effects.

Phosphate chelation over calcium impacts yeast growth and lipid production from short-chain fatty acids-rich media

Some oleaginous yeasts have the ability to produce microbial oils from alternative carbon sources, such as short-chain fatty acids (SCFAs). Nevertheless, there is still a lack of information about the possible effects that media nutrients have on yeast metabolisms when using SCFAs. For instance, inorganic phosphate (PO43-) has been reported to promote yeast growth in literature but its chelating effect over other elements such as calcium (Ca2+) is often not considered in fermentation processes while limitation of nitrogen is probably the most studied. Attending at the need to better understand the role of PO43-, this work assessed the lipid production capacity of Yarrowia lipolytica ACA DC 50109, both in synthetic and real SCFAs-rich media, at different SCFAs concentrations and PO43-:Ca2+ ratios. Reducing PO43-:Ca2+ ratio was identified to be an important factor to improve yeast growth, reaching the highest lipid content (52.7 ± 0.9 % w/w) and lipid yield (0.31 ± 0.01 w/w) in media without PO43-. These results demonstrated the importance of Ca2+ availability in the medium and nutrients interactions in yeast growth that are often underestimated.

Sustainable succinic acid production from lignocellulosic hydrolysates by engineered strains of Yarrowia lipolytica at low pH

Succinic acid (SA) is a valuable C4 platform chemical with diverse applications. Lignocellulosic biomass represents an abundant and renewable carbon resource for microbial production of SA. However, the presence of toxic compounds in pretreated lignocellulosic hydrolysates poses challenges to cell metabolism, leading to inefficient SA production. Here, engineered Yarrowia lipolytica Hi-SA2 was shown to utilize glucose and xylose from corncob hydrolysate to produce 32.6 g/L SA in shaking flasks. The high concentration of undetoxified hydrolysates significantly inhibited yeast growth and SA biosynthesis, with furfural identified as the key inhibitor. Through overexpressing glutathione synthetase encoding gene YlGsh2, the tolerance of engineered strain to furfural and toxic hydrolysate was significantly improved. In a 5-L bioreactor, Hi-SA2-YlGsh2 strain produced 45.34 g/L SA within 32 h, with a final pH of 3.28. This study provides a sustainable process for bio-based SA production, highlighting the efficient SA synthesis from lignocellulosic biomass through low pH fermentation.

A random mutagenesis screen enriched for missense mutations in bacterial effector proteins.

To remodel their hosts and escape immune defenses, many pathogens rely on large arsenals of proteins (effectors) that are delivered to the host cell using dedicated translocation machinery. Effectors hold significant insight into the biology of both the pathogens that encode them and the host pathways that they manipulate. One of the most powerful systems biology tools for studying effectors is the model organism, Saccharomyces cerevisiae. For many pathogens, the heterologous expression of effectors in yeast is growth inhibitory at a frequency much higher than housekeeping genes, an observation ascribed to targeting conserved eukaryotic proteins. Abrogation of yeast growth inhibition has been used to identify bacterial suppressors of effector activity, host targets, and functional residues and domains within effector proteins. We present here a yeast-based method for enriching for informative, in-frame, missense mutations in a pool of random effector mutants. We benchmark this approach against three effectors from Legionella pneumophila, an intracellular bacterial pathogen that injects a staggering >330 effectors into the host cell. For each protein, we show how in silico protein modeling (AlphaFold2) and missense-directed mutagenesis can be combined to reveal important structural features within effectors. We identify known active site residues within the metalloprotease RavK, the putative active site in SdbB, and previously unidentified functional motifs within the C-terminal domain of SdbA. We show that this domain has structural similarity with glycosyltransferases and exhibits in vitro activity consistent with this predicted function.