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Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression.Fungal Biology and Biotechnology Jun 2024Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other...
BACKGROUND
Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains.
RESULTS
The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy.
CONCLUSION
The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.
PubMed: 38824542
DOI: 10.1186/s40694-024-00175-4 -
Medical Mycology Case Reports Jun 2024Spontaneous mycosis caused by is documented in roach in a cyprinid-prevalent water reservoir in Czechia. Gross pathological lesions included pale gills and...
Spontaneous mycosis caused by is documented in roach in a cyprinid-prevalent water reservoir in Czechia. Gross pathological lesions included pale gills and splenomegaly, as revealed during necropsy examination. Histological examination showed extensive foci with fungal elements in the kidney. The isolated fungus was identified through phenotypic and molecular characterization, including phylogeny. This report represents the first case of infection in fish and cold-blooded vertebrates, to the best of our knowledge.
PubMed: 38799503
DOI: 10.1016/j.mmcr.2024.100652 -
Bioresources and Bioprocessing Aug 2023Aureobasidium pullulans (A. pullulans) has a wide range of applications. Ultraviolet (UV) rays from the sun can cause skin photoaging. In order to explore the protective...
BACKGROUND
Aureobasidium pullulans (A. pullulans) has a wide range of applications. Ultraviolet (UV) rays from the sun can cause skin photoaging. In order to explore the protective effect and application potential of A. pullulans lysate on UV-damaged human skin fibroblasts (HSF) and HaCaT Cells, this study investigates the anti-aging and anti-inflammatory effects of A. pullulans lysate as well as the mechanism of anti-oxidative stress at the cellular and molecular levels through cytotoxicity experiments, enzyme-linked immunosorbent assays (ELISA), and real-time quantitative PCR (RT-qPCR).
RESULTS
The experimental results have shown that the A. pullulans lysate can effectively reduce the loss of extracellular matrix components (EMC), such as collagen and hyaluronic acid (HA). It is also capable of scavenging excess reactive oxygen species (ROS) from the body, thereby increasing the activity of catalase, decreasing the overexpression of intracellular matrix metalloproteinases (MMPs), enhancing the gene expression of metalloproteinase inhibitors (TIMPs), and decreasing the level of inflammatory factors, reducing UV-induced apoptosis of HaCaT cells. Meanwhile, oxidative stress homeostasis is also regulated through the Nrf2/Keap1 and MAPK signaling pathways.
CONCLUSIONS
This study shows that the A. pullulans lysate has the potential to resist photoaging.
PubMed: 38647892
DOI: 10.1186/s40643-023-00678-9 -
International Journal of Food... Jun 2024Taggiasca table olives are typical of Liguria, a Northwestern Italian region, produced with a spontaneous fermentation carried out by placing the raw drupes directly...
Monitoring the yeasts ecology and volatiles profile throughout the spontaneous fermentation of Taggiasca cv. table olives through culture-dependent and independent methods.
Taggiasca table olives are typical of Liguria, a Northwestern Italian region, produced with a spontaneous fermentation carried out by placing the raw drupes directly into brine with a salt concentration of 8-12 % w/v. Such concentrations limit the development of unwanted microbes and favor the growth of yeasts. This process usually lasts up to 8 months. Yeasts are found throughout the entire fermentation process and they are mainly involved in the production of volatile organic compounds, which strongly impact the quality of the final product. The aim of this study was to evaluate the dynamics of autochthonous yeasts in brines and olives in a spontaneous process with no lye pre-treatment or addition of acids in the fermenting brine with 10 % NaCl (w/v) in two batches during 2021 harvest. Three hundred seventy-three yeast colonies were isolated, characterized by rep-PCR and identified by the D1/D2 region of the 26S rRNA gene sequencing. Mycobiota was also studied by 26S rRNA gene metataxonomics, while metabolome was assessed through GC-MS analysis. Traditional culture-dependent methods showed the dominance of Candida diddensiae, Wickerhamomyces anomalus, Pichia membranifaciens and Aureobasidium pullulans, with differences in species distribution between batches, sampling time and type of sample (olives/brines). Amplicon-based sequencing confirmed the dominance of W. anomalus in batch 1 throughout the entire fermentation, while Cyteromyces nyonsensis and Aureobasidium spp. were most abundant in the fermentation in batch 2. Volatilome results were analyzed and correlated to the mycobiota data, confirming differences between fermentation stages. Given the high appreciation for this traditional food, this study helps elucidate the mycobiota associated to Taggiasca cv. table olives and its relationship with the quality of the final product.
Topics: Fermentation; Olea; Volatile Organic Compounds; Yeasts; Food Microbiology; Italy; Salts
PubMed: 38615425
DOI: 10.1016/j.ijfoodmicro.2024.110688 -
Frontiers in Bioengineering and... 2024Polyol lipids (a.k.a. liamocins) produced by the polyextremotolerant, yeast-like fungus are amphiphilic molecules with high potential to serve as biosurfactants. So...
Polyol lipids (a.k.a. liamocins) produced by the polyextremotolerant, yeast-like fungus are amphiphilic molecules with high potential to serve as biosurfactants. So far, cultivations of have been performed in media with complex components, which complicates further process optimization due to their undefined composition. In this study, we developed and optimized a minimal medium, focusing on biosurfactant production. Firstly, we replaced yeast extract and peptone in the best-performing polyol lipid production medium to date with a vitamin solution, a trace-element solution, and a nitrogen source. We employed a design of experiments approach with a factor screening using a two-level-factorial design, followed by a central composite design. The polyol lipid titer was increased by 56% to 48 g L, and the space-time yield from 0.13 to 0.20 g L h in microtiter plate cultivations. This was followed by a successful transfer to a 1 L bioreactor, reaching a polyol lipid concentration of 41 g L. The final minimal medium allows the investigation of alternative carbon sources and the metabolic pathways involved, to pinpoint targets for genetic modifications. The results are discussed in the context of the industrial applicability of this robust and versatile fungus.
PubMed: 38511129
DOI: 10.3389/fbioe.2024.1379707 -
Molecular Biology of the Cell Apr 2024is a ubiquitous fungus with a wide variety of morphologies and growth modes including "typical" single-budding yeast, and interestingly, larger multinucleate yeast than...
is a ubiquitous fungus with a wide variety of morphologies and growth modes including "typical" single-budding yeast, and interestingly, larger multinucleate yeast than can make multiple buds in a single cell cycle. The study of promises to uncover novel cell biology, but currently tools are lacking to achieve this goal. Here, we describe initial components of a cell biology toolkit for , which is used to express and image fluorescent probes for nuclei as well as components of the cytoskeleton. These tools allowed live-cell imaging of the multinucleate and multibudding cycles, revealing highly synchronous mitoses in multinucleate yeast that occur in a semiopen manner with an intact but permeable nuclear envelope. These findings open the door to using this ubiquitous polyextremotolerant fungus as a model for evolutionary cell biology.
Topics: Saccharomyces cerevisiae; Ascomycota; Aureobasidium; Cytoskeleton
PubMed: 38446617
DOI: 10.1091/mbc.E23-10-0388 -
Acta Myologica : Myopathies and... 2023Muscular dystrophies other than Duchenne muscular dystrophy (DMD) are genetic diseases characterized by increasing muscle weakness, loss of ambulation, and ultimately...
BACKGROUND
Muscular dystrophies other than Duchenne muscular dystrophy (DMD) are genetic diseases characterized by increasing muscle weakness, loss of ambulation, and ultimately cardiac and respiratory failure. There are currently no effective therapeutics available. Having demonstrated the efficacy of a N-163 strain of Aureobasidium Pullulans (Neu-REFIX) produced B-1, 3-1,6-Glucan in pre-clinical and clinical studies of Duchenne muscular dystrophy (DMD) earlier, we assessed the effectiveness of this novel Beta glucan in the other muscular dystrophies in the present study.
METHODS
In this 60-day study, six patients with muscular dystrophies other than DMD consumed one 8g gel of Neu-REFIX beta-glucan along with their usual standard of care treatment regimen, and their biomarkers of relevance to muscle function such as serum calcium (SC), creatine phosphokinase (CPK), and alkaline phosphatase (ALP) levels along with functional improvement criteria, which is, Medical research council (MRC) scale and North Star Ambulatory assessment (NSAA), assessed at baseline and following the intervention.
RESULTS
After the intervention, the SC levels significantly decreased from a mean baseline value of 9.28 mg/dL to 8.31 mg/dL (p-value = 0.02). With a p-value of 0.29, the mean CPK value dropped from 2192.33 IU/L to 1567.5 IU/L. Following the intervention, the ALP levels dropped from 200.33 to 75.5 U/L (p-value = 0.15). MRC scale improved in three out of six patients. NSAA remained stable. There were no adverse effects.
CONCLUSION
This study has proven the safety of Neu REFIX beta-glucan food supplement and its efficacy in improving both plasma biomarkers and functional parameters of muscle in a short duration of 2 months. Further validation by evaluation of muscle function for a longer duration is recommended to confirm the efficacy of Neu-REFIX food supplement as a potential adjuvant DMT in muscular dystrophies.
Topics: Humans; Muscular Dystrophy, Duchenne; beta-Glucans; Biomarkers; Muscles; Muscle Weakness
PubMed: 38406382
DOI: 10.36185/2532-1900-312 -
Microorganisms Feb 2024Post-harvest decay of fresh table grapes causes considerable annual production losses. The main fungal agents of decay both in pre- and post-harvest are , spp., spp.,...
Post-harvest decay of fresh table grapes causes considerable annual production losses. The main fungal agents of decay both in pre- and post-harvest are , spp., spp., spp., and spp. To date, the use of agrochemicals and SO are the main methods to control grape molds in pre- and postharvest, respectively. Significant improvements, however, have already been made in to apply innovative and more environmentally sustainable control strategies, such as Biological Control Agents (BCAs), which can reduce disease severity in both pre- and post-harvest. In this study, 31 new non- yeast strains, isolated from berries of native Apulian table grape genotypes, were tested for their in vivo effectiveness against grey mold of table grapes, resulting in two ('N22_I1' and 'S13_I3'), one ('N22_I3'), one ('OLB_9.1_VL') and one ('OLB_9.1_BR') yeast strains that were marked as efficient and good BCAs. Their mechanisms of action were characterized through in vitro assays, and additional characteristics were evaluated to assess the economic feasibility and viability for future technological employment. Their effectiveness was tested by reducing the working concentration, their antagonistic effect on a wide range of fungal pathogens, their ability to survive in formulations with long shelf life, and their safety to human health.
PubMed: 38399744
DOI: 10.3390/microorganisms12020340 -
Toxins Feb 2024Different preventive strategies are needed to minimize the intake risks of mycotoxins, including zearalenone (ZEN). The aim of this study was to determine the ZEN...
Different preventive strategies are needed to minimize the intake risks of mycotoxins, including zearalenone (ZEN). The aim of this study was to determine the ZEN adsorption ability of an autolyzed biomass preparation of polymorphic yeast A.p.-3. The evaluation of the antitoxic properties of the preparation was also performed in relation to yeast (ATCC 2366, ATCC 7090 and ATCC 9763) used as a model cell exposed to a toxic ZEN dose. The preparation at a dose of 5 mg/mL showed the adsorption of ZEN present in model systems at concentrations between 1 μg/mL to 100 μg/mL. The highest degree of adsorption was established for ZEN concentrations of 1 μg/mL and 5 μg/mL, becoming limited at higher doses of the toxin. Based on the Langmuir model of adsorption isotherms, the predicted maximum ZEN adsorption was approx. 190 µg/mL, regardless of pH. The growth of three strains of yeast cells in the medium with ZEN at concentrations within the range of 1.56 μg/mL-100 μg/mL was analyzed to determine the minimum inhibitory concentration. The growth of all tested strains was especially limited by high doses of ZEN, i.e., 50 and 100 μg/mL. The protective effect of the tested preparation was noted in relation to yeast cells exposed to toxic 100 μg/mL ZEN doses. The highest yeast cell growth (app. 36% percentage) was noted for a ATCC 9763 strain compared to the medium with ZEN but without preparation. More detailed tests determining the antitoxic mechanisms of the preparation are planned in the future, including cell culture bioassays and animal digestive tract models.
Topics: Animals; Zearalenone; Saccharomyces cerevisiae; Adsorption; Biomass; Aureobasidium
PubMed: 38393183
DOI: 10.3390/toxins16020105 -
Journal of Fungi (Basel, Switzerland) Feb 2024Verticillium wilt of olive (VWO) caused by is considered a major olive () disease in Mediterranean-type climate regions. The lack of effective chemical products against...
Verticillium wilt of olive (VWO) caused by is considered a major olive () disease in Mediterranean-type climate regions. The lack of effective chemical products against VWO makes it necessary to search for alternatives such as biological control. The main goal of this study was to evaluate the effect of six spp. strains as biological control agents (BCAs) against VWO. All of them were molecularly characterized by sequencing 16S or 23S rRNA genes and via phylogenetic analysis. Their effect was evaluated in vitro on the mycelial growth of (isolates V004 and V323) and on microsclerotia (MS) viability using naturally infested soils. Bioassays in olive plants inoculated with were also conducted to evaluate their effect against disease progress. In all the experiments, the reference BCAs FO12 and AP08 were included for comparative purposes. The six strains were identified as spp., and they were considered as potential new species. All the BCAs, including strains, showed a significant effect on mycelial growth inhibition for both isolates compared to the positive control, with FO12 being the most effective, followed by AP08, while the spp. strains showed an intermediate effect. All the BCAs tested also showed a significant effect on the inhibition of germination of MS compared to the untreated control, with FO12 being the most effective treatment. Irrigation treatments with strain CBQ-EBa-21 or FO12 were significantly more effective in reducing disease severity and disease progress in olive plants inoculated with compared to the remaining treatments. This study represents the first approach to elucidating the potential effect of strains against VWO.
PubMed: 38392810
DOI: 10.3390/jof10020138