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The Journal of Cell Biology Oct 2024Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists...
Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists for its unconventional mode of proliferation in which multinucleate yeast cells make multiple buds within a single cell cycle. Here, we combine a chemical transformation method with genome-targeted homologous recombination to yield ∼60 transformants/μg of DNA in just 3 days. This protocol is simple, inexpensive, and requires no specialized equipment. We also describe vectors with codon-optimized green and red fluorescent proteins for A. pullulans and use these tools to explore novel cell biology. Quantitative imaging of a strain expressing cytosolic and nuclear markers showed that although the nuclear number varies considerably among cells of similar volume, total nuclear volume scales with cell volume over an impressive 70-fold size range. The protocols and tools described here expand the toolkit for A. pullulans biologists and will help researchers address the many other puzzles posed by this polyextremotolerant and morphologically plastic organism.
Topics: Aureobasidium; Cell Nucleus; Genetic Vectors; Green Fluorescent Proteins; Homologous Recombination; Luminescent Proteins; Red Fluorescent Protein; Transformation, Genetic; Genetic Techniques
PubMed: 38935076
DOI: 10.1083/jcb.202402114 -
Biochemical Genetics Jun 2024Endophytic fungi associated with plants may contain undiscovered bioactive compounds. Under standard laboratory conditions, most undiscovered compounds are inactive,...
Endophytic fungi associated with plants may contain undiscovered bioactive compounds. Under standard laboratory conditions, most undiscovered compounds are inactive, whereas their production could be stimulated under different cultivation conditions. In this study, six endophytic fungi were isolated from the bark of Koelreuteria paniculata in Quancheng Park, Jinan City, Shandong Province, one of which was identified as a new subspecies of Aureobasidium pullulans, named A. pullulans KB3. Additionally, metabolomic tools were used to screen suitable media for A. pullulans KB3 fermentation, and the results showed that peptone dextrose medium (PDM) was more beneficial to culture A. pullulans KB3 for isolation of novel compounds. Sphaerolone, a polyketone compound, was initially isolated from A. pullulans KB3 via scaled up fermentation utilizing PDM. Additionally, the whole-genome DNA of A. pullulans KB3 was sequenced to facilitate compound isolation and identify the biosynthesis gene clusters (BGCs). This study reports the multi-omics (metabolome and genome) analysis of A. pullulans KB3, laying the foundation for discovering novel compounds of silent BGCs and identifying their biosynthesis pathway.
PubMed: 38877158
DOI: 10.1007/s10528-024-10866-7 -
Fungal Biology Jun 2024Volatile organic compounds (VOCs) produced by yeasts can positively affect crops, acting as antifungals or biostimulants. In this study, Aureobasidium pullulans and...
Volatile organic compounds (VOCs) produced by yeasts can positively affect crops, acting as antifungals or biostimulants. In this study, Aureobasidium pullulans and Metschnikowia pulcherrima were evaluated as potential antagonists of Trichoderma spp., common fungal pathogen in mushroom cultivation. To assess the biocontrol ability and biostimulant properties of the selected yeast species, in vitro co-culture and VOCs exposure assays were conducted. In both assays, VOCs produced by Aureobasidium spp. showed the stronger antifungal activity with a growth inhibition up to 30 %. This result was further confirmed by the higher volatilome alcohol content revealed by solid phase microextraction-gas chromatography mass spectrometry (SPME/GC-MS). Overall, Aureobasidium strains can be potentially used as biocontrol agent in Pleorotus ostreatus and Cyclocybe cylindracea mycelial growth, without affecting their development as demonstrated by VOCs exposure assay and Fourier-transform infrared spectroscopy (FT-IR). Conversely, M. pulcherrima was characterized by a lower or absent antifungal properties and by a volatilome composition rich in isobutyl acetate, an ester often recognized as plant growth promoter. As confirmed by FT-IR, Lentinula mycelia exposed to M. pulcherrima VOCs showed a higher content of proteins and lipids, suggesting an improvement of some biochemical properties. Our study emphasizes that VOCs produced by specific yeast strains are potentially powerful alternative to synthetic fungicide in the vegetative growth of mushroom-forming fungi and also able to modify their biochemical composition.
Topics: Volatile Organic Compounds; Mycelium; Agaricales; Gas Chromatography-Mass Spectrometry; Antifungal Agents; Biological Control Agents; Metschnikowia; Antibiosis; Aureobasidium; Trichoderma; Solid Phase Microextraction
PubMed: 38876538
DOI: 10.1016/j.funbio.2024.05.007 -
Fungal Biology Jun 2024Post-harvest decay of fresh agricultural produce is a major threat to food security globally. Synthetic fungicides, commonly used in practice for managing the...
Antagonistic potential of endophytic fungal isolates of tomato (Solanum lycopersicum L.) fruits against post-harvest disease-causing pathogens of tomatoes: An in vitro investigation.
Post-harvest decay of fresh agricultural produce is a major threat to food security globally. Synthetic fungicides, commonly used in practice for managing the post-harvest losses, have negative impacts on consumers' health. Studies have reported the effectiveness of fungal isolates from plants as biocontrol agents of post-harvest diseases, although this is still poorly established in tomatoes (Solanum lycopersicum L. cv. Jasmine). In this study, 800 endophytic fungi were isolated from mature green and ripe untreated and fungicide-treated tomato fruits grown in open soil and hydroponics systems. Of these, five isolates (Aureobasidium pullulans SUG4.1, Coprinellus micaceus SUG4.3, Epicoccum nigrum SGT8.6, Fusarium oxysporum HTR8.4, Preussia africana SUG3.1) showed antagonistic properties against selected post-harvest pathogens of tomatoes (Alternaria alternata, Fusarium solani, Fusarium oxysporum, Geotrichum candidum, Rhizopus stolonifera, Rhizoctonia solani), with Lactiplantibacillus plantarum as a positive control. P. africana SUG3.1 and C. micaceus SUG4.3 significantly inhibited growth of all the pathogens, with antagonistic capabilities comparable to that exhibited by L. plantarum. Furthermore, the isolates produced an array of enzymes, including among others, amylase, cellulose and protease; and were able to utilize several carbohydrates (glucose, lactose, maltose, mannitol, sucrose). In conclusion, P. africana SUG3.1 and C. micaceus SUG4.3 may complement L. plantarum as biocontrol agents against post-harvest pathogens of tomatoes.
Topics: Solanum lycopersicum; Plant Diseases; Fruit; Endophytes; Fungi; Antibiosis; Biological Control Agents; Fungicides, Industrial
PubMed: 38876537
DOI: 10.1016/j.funbio.2024.05.006 -
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 -
International Journal of Biological... Jun 2024This study presents a novel and efficient approach for pullulan production using artificial neural networks (ANNs) to optimize semi-solid-state fermentation (S-SSF) on...
Optimization of pullulan production by Aureobasidium pullulans using semi-solid-state fermentation and artificial neural networks: Characterization and antibacterial activity of pullulan impregnated with Ag-TiO nanocomposite.
This study presents a novel and efficient approach for pullulan production using artificial neural networks (ANNs) to optimize semi-solid-state fermentation (S-SSF) on faba bean biomass (FBB). This method achieved a record-breaking pullulan yield of 36.81 mg/g within 10.82 days, significantly exceeding previous results. Furthermore, the study goes beyond yield optimization by characterizing the purified pullulan, revealing its unique properties including thermal stability, amorphous structure, and antioxidant activity. Energy-dispersive X-ray spectroscopy and scanning electron microscopy confirmed its chemical composition and distinct morphology. This research introduces a groundbreaking combination of ANNs and comprehensive characterization, paving the way for sustainable and cost-effective pullulan production on FBB under S-SSF conditions. Additionally, the study demonstrates the successful integration of pullulan with Ag@TiO nanoparticles during synthesis using Fusarium oxysporum. This novel approach significantly enhances the stability and efficacy of the nanoparticles by modifying their surface properties, leading to remarkably improved antibacterial activity against various human pathogens. These findings showcase the low-cost production medium, and extensive potential of pullulan not only for its intrinsic properties but also for its ability to significantly improve the performance of nanomaterials. This breakthrough opens doors to diverse applications in various fields.
Topics: Glucans; Neural Networks, Computer; Fermentation; Nanocomposites; Titanium; Anti-Bacterial Agents; Aureobasidium; Silver; Antioxidants; Fusarium
PubMed: 38714281
DOI: 10.1016/j.ijbiomac.2024.132109 -
International Journal of Biological... May 2024Osteoporosis is a prevalent condition characterized by bone loss and decreased skeletal strength, resulting in an elevated risk of fractures. Calcium plays a crucial...
Osteoporosis is a prevalent condition characterized by bone loss and decreased skeletal strength, resulting in an elevated risk of fractures. Calcium plays a crucial role in preventing and managing osteoporosis. However, traditional calcium supplements have limited bioavailability, poor solubility, and adverse effects. In this study, we isolated a natural soluble biopolymer, calcium polymalate (PMACa), from the fermentation broth of the fungus Aureobasidium pullulans, to investigate its potential as an anti-osteoporosis therapeutic agent. Characterization revealed that linear PMA-Ca chains juxtaposed to form a porous, rod-like state, in the presence of Ca. In vivo mouse models demonstrated that PMA-Ca significantly promoted the conversion of serum calcium into bone calcium, and stimulated bone growth and osteogenesis. Additionally, PMA-Ca alleviated exercise fatigue in mice by facilitating the removal of essential metabolites, such as serum lactate (BLA) and blood urea nitrogen (BUN), from their bloodstream. In vitro studies further showed that PMA-Ca strengthened osteoblast cell activity, proliferation, and mineralization. And PMA-Ca upregulated the expression of some genes involved in osteoblast differentiation, indicating a potential correlation between bone formation and PMACa. These findings indicate that soluble PMA-Ca has the potential to be a novel biopolymer-based calcium supplement with sustainable production sourced from the fermentation industry.
Topics: Animals; Mice; Fermentation; Osteoporosis; Calcium; Solubility; Biopolymers; Aureobasidium; Osteoblasts; Osteogenesis; Fatigue; Water; Cell Proliferation; Disease Models, Animal; Female; Cell Differentiation
PubMed: 38697412
DOI: 10.1016/j.ijbiomac.2024.132013 -
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 -
IUBMB Life Apr 2024Experimental evolution was carried out to investigate the adaptive responses of extremotolerant fungi to a stressful environment. For 12 cultivation cycles, the...
Experimental evolution was carried out to investigate the adaptive responses of extremotolerant fungi to a stressful environment. For 12 cultivation cycles, the halotolerant black yeasts Aureobasidium pullulans and Aureobasidium subglaciale were grown at high NaCl or glycerol concentrations, and the halophilic basidiomycete Wallemia ichthyophaga was grown close to its lower NaCl growth limit. All evolved Aureobasidium spp. accelerated their growth at low water activity. Whole genomes of the evolved strains were sequenced. No aneuploidies were detected in any of the genomes, contrary to previous studies on experimental evolution at high salinity with other species. However, several hundred single-nucleotide polymorphisms were identified compared with the genomes of the progenitor strains. Two functional groups of genes were overrepresented among the genes presumably affected by single-nucleotide polymorphisms: voltage-gated potassium channels in A. pullulans at high NaCl concentration, and hydrophobins in W. ichthyophaga at low NaCl concentration. Both groups of genes were previously associated with adaptation to high salinity. Finally, most evolved Aureobasidium spp. strains were found to have increased intracellular and decreased extracellular glycerol concentrations at high salinity, suggesting that the strains have optimised their management of glycerol, their most important compatible solute. Experimental evolution therefore not only confirmed the role of potassium transport, glycerol management, and cell wall in survival at low water activity, but also demonstrated that fungi from extreme environments can further improve their growth rates under constant extreme conditions in a relatively short time and without large scale genomic rearrangements.
PubMed: 38647201
DOI: 10.1002/iub.2825