Chemical transformation of the multibudding yeast, Aureobasidium pullulans.

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.

Production and characterization of extracellular liamocins produced from fungal strains of Aureobasidium spp.

Liamocins, a group of high-density glycolipids, are only produced by certain strains of the yeast-like fungi in the genus Aureobasidium. Until now, few studies have focused on the surfactant properties of liamocins produced from the highly diverse tropical strains of Aureobasidium. Therefore, the aims of this research were to screen the liamocin production from tropical strains of Aureobasidium spp. and to characterize their surfactant properties. A total of 41 strains of Thai Aureobasidium spp. were screened for their ability to produce liamocins, and the products were detected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and thin-layer chromatography. Of those strains, 30 strains of Aureobasidium spp. tested were found to produce liamocins with yields ranging from 0.53 to 10.60 g/l. The nature of all crude liamocins was heterogeneous, with different compositions and ratios depending on the yeast strain. These liamocins exhibited relatively high emulsifying activity against vegetable oils tested, with an emulsification index of around 40-50%; the emulsion stability of some liamocins was up to 30 days. The obtained critical micelle concentration values were varied, with those ​​of liamocins produced from A. pullulans, A. melanogenum and A. thailandense falling in ranges from 7.70 to 119.78, 10.73 to > 1,000, and 68.56 to > 1,000 mg/l, respectively. The emulsification activity of liamocins was higher than that of the analytical grade rhamnolipids. These compounds showed strong surface tension reduction in a sodium chloride concentration range of 2-12% (w/v), pH values between 3 and 7, and temperatures between 4 and 121 °C. This is the first report of liamocins produced by A. thailandense.

Water-soluble biopolymers calcium polymalate derived from fermentation broth of Aureobasidium pullulans markedly alleviates osteoporosis and fatigue.

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 Ca2+. 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.

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-TiO2 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@TiO2 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.

Chemical Constituents from the deep-sea-derived Fungus Aureobasidium melanogenum LUO5.

Three new C10 and C12 aliphatic δ-lactones (1-3), three new fatty acid methyl esters (4-6), and eight known compounds (7-14) were isolated from the marine Aureobasidium sp. LUO5. Their structures were established by detailed analyses of the NMR, HRESIMS, optical rotation, and ECD data. All isolates were tested for their inhibitory effects on nitric oxide production in LPS-induced BV-2 cells. Notably, compound 4 displayed the strongest inhibitory effect with the IC50 value of 120.3 nM.

NsdD, a GATA-type transcription factor is involved in regulation and biosynthesis of macromolecules melanin, pullulan, and polymalate in Aureobasidium melanogenum.

In this study, an NSDD gene, which encoded a GATA-type transcription factor involved in the regulation and biosynthesis of melanin, pullulan, and polymalate (PMA) in Aureobasidium melanogenum, was characterized. After the NSDD gene was completely removed, melanin production by the Δnsd mutants was enhanced, while pullulan and polymalate production was significantly reduced. Transcription levels of the genes involved in melanin biosynthesis were up-regulated while expression levels of the genes responsible for pullulan and PMA biosynthesis were down-regulated in the Δnsdd mutants. In contrast, the complementation of the NSDD gene in the Δnsdd mutants made the overexpressing mutants restore melanin production and transcription levels of the genes responsible for melanin biosynthesis. Inversely, the complementation strains, compared to the wild type strains, showed enhanced pullulan and PMA yields. These results demonstrated that the NsdD was not only a negative regulator for melanin biosynthesis, but also a key positive regulator for pullulan and PMA biosynthesis in A. melanogenum. It was proposed how the same transcriptional factor could play a negative role in melanin biosynthesis and a positive role in pullulan and PMA biosynthesis. This study provided novel insights into the regulatory mechanisms of multiple A. melanogenum metabolites and the possibility for improving its yields of some industrial products through genetic approaches.

Engineering the reductive tricarboxylic acid pathway in Aureobasidium pullulans for enhanced biosynthesis of poly-L-malic acid.

Aureobasidium pullulans produced poly-L-malic acid (PMA) as the main metabolite in fermentation but with relatively low productivity and yield limiting its industrial application. In this study, A. pullulans ZX-10 was engineered to overexpress cytosolic malate dehydrogenase (MDH) and pyruvate carboxylase (PYC) and PMA synthetase (PMS) using a high-copy yeast episomal plasmid with the gpdA promoter from Aspergillus nidulans. Overexpressing endogenous PMS and heterologous MDH and PYC from Aspergillus oryzae respectively increased PMA production by 19 % - 37 % (0.64 - 0.74 g/g vs. 0.54 g/g for wild type) in shake-flask fermentations, demonstrating the importance of the reductive tricarboxylic acid (rTCA) pathway in PMA biosynthesis. A. pullulans co-expressing MDH and PYC produced 96.7 g/L PMA at 0.90 g/L∙h and 0.68 g/g glucose in fed-batch fermentation, which were among the highest yield and productivity reported. The engineered A. pullulans with enhanced rTCA pathway is advantageous and promising for PMA production.

Green process for isolation and purification of poly(ß-L-malic acid) from Aureobasidium spp. by an integrated ion exchange and membrane separation.

Poly (ß-L-malic acid) (PMLA) is a biopolymer used in food and medical fields. However, the industrial processes are susceptible to the pollution of CaSO4 waste and organic solvent owing to the heavy use of CaCO3 in fermentation process and organic solvents in isolation process. This study developed an organic solvent and CaSO4 -free process for the industrial-scale production of PMLA. Firstly, calcium ion was removed at pH 9.2 by pH adjustment with Na2CO3, and the generated CaCO3 was reused in the fermentation process. Then, the D296 resin was selected to isolate the PMLA from the Ca2+-free broth, where the adsorption data were both primely described by the Freundlich and Langmuir equation, while Freundlich model better fit the process than Langmuir equation, indicating that it was non-monolayer adsorption of PMLA on the resin. Meanwhile, a three-step gradient elution with phosphate buffer (i.e., 0.2 mol/L, pH 7.0) containing 0.1, 0.2 and 1 mol/L NaCl was developed to recover PMLA. Finally, a PES15 membrane was selected to recover the PMLA from the elution solution, which could be reused in the next cycle. As a result, the PMLA with a purity of 98.89 % was obtained with the developed green process. In the developed process, it removed the pollution of organic solvent and calcium waste for the biosynthesis of PMLA on an industrial scale, which also offers a sustainable and green route for the biosynthesis of other carboxylic acids.

Fractionation and characterization of poly(ß-L-malic acid) produced by Aureobasidium melanogenum ipe-1.

Poly (ß-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries, whose functions primarily depend upon its molecular size and chemical structure. Up to now, the fractionation and characterization of PMLA produced by Aureobasidium spp. were still unclear. In this study, the product from A. melanogenum ipe-1 was effectively fractionated using 300 and 50 kDa membranes. During the filtration, the mechanisms of membrane fouling were illegible since the PMLA can both reject and permeate the membrane, while the main fouling mechanism varied between standard blocking and complete blocking during the diafiltration. After fractionation, 14.0, 8.4 and 77.6 % of the PMLAs with Mws of 75,134, 21,344 and 10,056 Da were distributed in the 300 kDa retentate after diafiltrating, 50 kDa retentate after diafiltrating, and the 50 kDa permeate, respectively. The Mw/Mns of the PMLAs were 4.12, 1.92, and 1.12 in the three fractions. Based on characteristic spectra of NMR, HPLC and FTIR, the product was not usual L-malic acid monomers, but glucose-terminated PMLA. The glucose was located at the terminal hydroxyl of PMLA. These results would serve as a valuable guide for process design and practical operation in subsequent industrial application.

Anti-inflammatory effects of ß-1,3-1,6-glucan derived from black yeast Aureobasidium pullulans in RAW264.7 cells.

ß-glucan derived from the black yeast Aureobasidium pullulans (A. pullulans) is one of the natural products attracting attention due to its high potential for application as a functional food and pharmaceutical. Our team of researchers obtained a highly soluble, low-molecular-weight ß-glucan from the fermentation culture medium of A. pullulans via mechanochemical ball milling method, that is, the low-molecular-weight A. pullulans-fermented ß-D-glucan (LMW-AP-FBG). We investigated the anti-inflammatory effect of LMW-AP-FBG using lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells) in the current study. LMW-AP-FBG altered LPS-stimulated inflammatory responses by reducing the release of inflammatory mediators such as nitric oxide (NO), interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α. As well, the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways were downregulated by LMW-AP-FBG. Furthermore, LMW-AP-FBG markedly reduced LPS-induced expression of cell surface molecules, CD14, CD86, and MHC class II, which mediate macrophage activation. These findings suggest that LMW-AP-FBG can be used as an effective immune modulator to attenuate the progression of inflammatory disease.

The GATA type transcriptional factors regulate pullulan biosynthesis in Aureobasidium melanogenum P16.

Aureobasidium melanogenum P16, the high pullulan producer, had only one GATA type transcriptional activator AreA and one GATA type transcriptional repressor AreB. It was found that 2.4 g/L of (NH4)2SO4 had obvious nitrogen repression on pullulan biosynthesis by A. melanogenum P16. Removal of the AreB gene could make the disruptant DA6 produce 34.8 g/L pullulan while the P16 strain only produced 28.8 g/L pullulan at the efficient nitrogen condition. Further both removal of the native AreA gene and overexpression of the mutated AreAS628-S678 gene with non-phosphorylatable residues could render the transformant DEA12 to produce 39.8 g/L pullulan. The transcriptional levels of most of the genes related to pullulan biosynthesis in the transformant DEA12 were greatly enhanced. The mutated AreAS628-S678 was localized in the nuclei of the transformant DEA12 while the native AreA was distributed in the cytoplasm in A. melanogenum P16. This meant that nitrogen repression on pullulan biosynthesis in the transformant DEA12 was indeed significantly relieved. This was the first time to report that the GATA type transcriptional factors of nitrogen catabolite repression system could regulate pullulan biosynthesis in Aureobasidium spp.

Microbial Synthesis of (S)- and (R)-Benzoin in Enantioselective Desymmetrization and Deracemization Catalyzed by Aureobasidium pullulans Included in the Blossom Protect™ Agent.

In this study, we examined the Aureobasidium pullulans strains DSM 14940 and DSM 14941 included in the Blossom Protect™ agent to be used in the bioreduction reaction of a symmetrical dicarbonyl compound. Both chiral 2-hydroxy-1,2-diphenylethanone antipodes were obtained with a high enantiomeric purity. Mild conditions (phosphate buffer [pH 7.0, 7.2], 30 °C) were successfully employed in the synthesis of (S)-benzoin using two different methodologies: benzyl desymmetrization and rac-benzoin deracemization. Bioreduction carried out with higher reagent concentrations, lower pH values and prolonged reaction time, and in the presence of additives, enabled enrichment of the reaction mixture with (R)-benzoin. The described procedure is a potentially useful tool in the synthesis of chiral building blocks with a defined configuration in a simple and economical process with a lower environmental impact, enabling one-pot biotransformation.

Correlation between the synthesis of pullulan and melanin in Aureobasidium pullulans.

The content of pullulan and melanin in 500 mutants of Aureobasidum pullulans obtained by ultraviolet mutagenesis were examined and statistically analyzed, and a strong positive correlation was found between them. The result was further confirmed by culturing wild type strain As3.3984 in different media. Then we constructed melanin-deletion mutant As-Δalb1 and pullulan-deletion mutant As-Δpul. As-Δalb1 was a melanin-free strain with the yield of pullulan decreased by 41.01%. The supplementation of melanin in the culture of As-Δalb1 increased the production of pullulan. As-Δpul synthesized neither pullulan nor melanin and recovered melanin synthesis by adding pullulan to the medium. The results suggested that high concentration- of pullulan induced morphological transformation and synthesis of melanin, and melanin promoted the synthesis of pullulan. The pullulan biosynthetic genes, upt, pgm, ugp, and pul, were down-regulated, while the negative regulatory gene of pullulan synthesis, creA, was up-regulated by melanin deficiency.

Polymalate (PMA) biosynthesis and its molecular regulation in Aureobasidium spp.

It has been well documented that different strains of Aureobasidium spp. can synthesize and secrete over 30.0 g/L of polymalate (PMA) and the produced PMA has many potential applications in biomaterial, medical and food industries. The substrates for PMA biosynthesis include glucose, xylose, fructose, sucrose and glucose or fructose or xylose or sucrose-containing natural materials from industrial and agricultural wastes. Malate, the only monomer for PMA biosynthesis mainly comes from TCA cycle, cytosolic reduction TCA pathway and the glyoxylate cycle. The PMA synthetase (a NRPS) containing A like domain, T domain and C like domain is responsible for polymerization of malate into PMA molecules by formation of ester bonds between malates. PMA biosynthesis is regulated by the transcriptional activator Crz1 from Ca2+ signaling pathway, the GATA-type transcription factor Gat1 from nitrogen catabolite repression and the GATA-type transcription factor NsdD.

Biological Activity of High-Purity ß-1,3-1,6-Glucan Derived from the Black Yeast Aureobasidium pullulans: A Literature Review.

The black yeast Aureobasidium pullulans produces abundant soluble ß-1,3-1,6-glucan-a functional food ingredient with known health benefits. For use as a food material, soluble ß-1,3-1,6-glucan is produced via fermentation using sucrose as the carbon source. Various functionalities of ß-1,3-1,6-glucan have been reported, including its immunomodulatory effect, particularly in the intestine. It also exhibits antitumor and antimetastatic effects, alleviates influenza and food allergies, and relieves stress. Moreover, it reduces the risk of lifestyle-related diseases by protecting the intestinal mucosa, reducing fat, lowering postprandial blood glucose, promoting bone health, and healing gastric ulcers. Furthermore, it induces heat shock protein 70. Clinical studies have reported the antiallergic and triglyceride-reducing effects of ß-1,3-1,6-glucan, which are indicators of improvement in lifestyle-related diseases. The primary and higher-order structures of ß-1,3-1,6-glucan have been elucidated. Specifically, it comprises a single highly-branched glucose residue with the ß-1,6 bond (70% or more) on a backbone of glucose with 1,3-ß bonds. ß-Glucan shows a triple helical structure, and studies on its use as a drug delivery system have been actively conducted. ß-Glucan in combination with anti-inflammatory substances or fullerenes can be used to target macrophages. Based on its health functionality, ß-1,3-1,6-glucan is an interesting material as both food and medicine.

Purification of a killer toxin from Aureobasidium pullulans for the biocontrol of phytopathogens.

The objectives of the present study were to purify and assess the killer toxin effect produced by Aureobasidium pullulans under casual agents of green mold (Penicillum digitatum) and sour rot (Geotrichum citri-aurantii). Initially, different methods of protein precipitation were tested. The proteolytic activity and the presence of proteins acting on cell wall receptors, ß-1,3-glucanase and chitinase were determined, and toxin purification was conducted by Sephadex G-75 gel exclusion chromatography and cellulose chromatography (medium fibers). Subsequently, purification was confirmed by polyacrylamide gel electrophoresis, and the detection of killer activity was performed in solid YEPD-methylene blue buffered with citrate-phosphate (0.1 M, pH 4.6). Toxin identification was performed by liquid chromatography-mass spectrometry. The results showed that the best protein precipitation method was 2:1 ethanol (vol/vol ethanol/supernatant). It was possible to observe the presence of enzymes with proteolytic activity, including ß-1,3-glucanase and chitinase. During the purification process, it was verified that the killer toxin produced by the yeast has a low-molecular-weight protein belonging to the ubiquitin family, which presents killer activity against P. digitatum and G. citri-aurantii.

Multiple biosurfactant production by Aureobasidium pullulans strain YTP6-14 in aqueous and heavy oil layers.

Aureobasidium pullulans YTP6-14 was demonstrated to be an excellent multiple biosurfactant producer utilizing cheap carbon sources available in Thailand, including glycerol and cassava flour hydrolysate. A. pullulans YTP6-14 maximally produced 1.81 g/l biosurfactant in an aqueous layer (BS-AQ) in a medium containing glycerol, and 7.37 or 6.37 g/l biosurfactant in a heavy oil layer (BS-HO) in cassava flour hydrolysate or a glucose containing medium, respectively. Each BS-AQ and BS-HO had critical micelle concentration values of 41.32 mg/l and 13.51 mg/l, and both biosurfactants formed a stable food oil emulsion and reduced the amount of biofilms formed by Streptococcus sobrinus and Streptococcus mutans. BS-AQ and BS-HO were mainly composed of liamocins or exophilins and massoia lactone, respectively.

Volatile organic compounds produced by Aureobasidium pullulans induce electrolyte loss and oxidative stress in Botrytis cinerea and Alternaria alternata.

Aureobasidium pullulans is a yeast-like fungus that produces volatile organic compounds (VOCs) with antifungal properties. VOCs have the potential to trigger the production of intracellular reactive oxygen species (ROS), lipid peroxidation and electrolyte loss in microorganisms. The relationship among A. pullulans VOCs, induced ROS accumulation and electrolyte leakage was investigated in Botrytis cinerea and Alternaria alternata in vitro. Exposure to a mixture of A. pullulans VOCs: ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol, resulted in electrolyte leakage in both B. cinerea and A. alternata. Fluorescence microscopy using 2',7'-dichlorofluorescein diacetate indicated triggered ROS accumulation in exposed fungal mycelia and the presence of the superoxide radical was evident by intense red fluorescence with dihydroethidium. Partial inhibition of enzymes of the mitochondrial respiratory chain complex I of B. cinerea and A. alternata by pre-treatment with rotenone reduced ROS accumulation in hypha exposed to A. pullulans VOCs and reversed the VOCs inhibition of fungal growth. Scanning electron micrographs revealed that B. cinerea and A. alternata hypha exposed to A. pullulans VOCs had altered cell wall structures. Our findings give insights into the potential mechanisms involved in the antifungal properties of A. pullulans in the suppression of B. cinerea and A. alternata growth in vitro.

Pullulan biosynthesis and its regulation in Aureobasidium spp.

It has been well known that different strains of Aureobasidium spp. can yield a large amount of pullulan. Although pullulan has wide applications in various sectors of biotechnology, its biosynthesis and regulation were not resolved. Lately, the molecular mechanisms of pullulan biosynthesis and regulation have been elucidated and their genes and encoding proteins have been identified using the genome-wide mutant analysis. It is found that a multidomain AmAgs2 is the key enzyme for pullulan biosynthesis and the alternative primers are required for its biosynthesis. Pullulan biosynthesis is regulated by glucose repression and signaling pathways. Elucidation of such a biosynthetic pathway and regulation is of significance in biotechnology. Therefore, the present review article mainly summaries the recent research proceedings in this field, hoping to promote further endeavors on enhanced pullulan production and improved chemical properties of pullulan via molecular modifications of the producers by using synthetic biology approaches.

Glycerol, trehalose and vacuoles had relations to pullulan synthesis and osmotic tolerance by the whole genome duplicated strain Aureobasidium melanogenum TN3-1 isolated from natural honey.

In our previous study, it was found that Aureobasidium melanogenum TN3-1 was a high pullulan producing and osmotic tolerant yeast-like fungal strain. In this study, the HOG1 signaling pathway controlling glycerol synthesis, glycerol, trehalose and vacuoles were found to be closely related to its pullulan biosynthesis and high osmotic tolerance. Therefore, deletion of the key genes for the HOG1 signaling pathway, glycerol and trehalose biosynthesis and vacuole formation made all the mutants reduce pullulan biosynthesis and increase sensitivity of the growth of the mutants to high glucose concentration. Especially, abolishment of both the VSP11 and VSP12 genes which controlled the fission/fusion balance of vacuoles could cause big reduction in pullulan production (less than 7.4 ± 0.4 g/L) by the double mutant ΔDV-5 and increased sensitivity to high concentration glucose, while expression of the VSP11 gene in the double mutant ΔDV-5 made the transformants EV-2 restore pullulan production and tolerance to high concentration glucose. But cell growth of them were the similar. The double mutant ΔDV-5 had much bigger vacuoles and less numbers of vacuoles than the transformant EV-2 and its wild type strain TN3-1 while it grew weakly on the plate with 40% (w/v) glucose while the transformant EV-2 and its wild type strain TN3-1 could grow normally on the plate even with 60% (w/v) glucose. The double mutant ΔDV-5 also had high level of pigment and its cells were swollen. This was the first time to give the evidence that glycerol, trehalose and vacuoles were closely related to pullulan biosynthesis and high osmotic tolerance by A. melanogenum.