Pilot scale production of high-content mycoprotein using Rhizopus microsporus var. oligosporus by submerged fermentation and agro-industrial by-products.

While mycoprotein has gained traction as a human food source, its potential as a nutrient for animals remains largely unexplored. The mycoprotein-producing Rhizopus microsporus var. oligosporus, a fungus traditionally used for human food in Indonesia, is promising. It could revolutionise animal nutrition once it is Generally Recognized as Safe (GRAS) and is a biosafety level 1 (BSL1) organism. To enhance sustainably, we propose using sugar cane molasses (SM) and corn steep liquor (CSL) as nutrient sources. Also, we investigated the growth of R. microsporus var. oligosporus in five 14 L external-loop airlift bioreactors using CSL as the sole nutrient source. After 96 h of fermentation, at 25 °C and 0.5 vvm, the mycelium produced had an average biomass yield of 38.34 g L-1, with 70.18 % (m v-1) crude protein (mycoprotein). This bioprocess, which is scalable and economically viable, produces high amounts of mycoprotein for animal feed using CSL, a cost-effective agro-industrial by-product, providing a practical solution to the growing demand for animal protein.

Predicting of novel homoserine dehydrogenase inhibitors against Paracoccidioides brasiliensis: integrating in silico and in vitro approaches.

Aim: To search for potential inhibitors to homoserine dehydrogenase (HSD) in Paracoccidioides brasiliensis the causative agent of paracoccidioidomycosis, an infection with a high mortality rate in Brazil.Materials & methods: The enzyme was modeled and used in the virtual screening of the compounds. The library was first screened by the Autodock, in which 66 molecules were better ranked than substrate, and then, also evaluated by the Molegro and Gold programs.Results: The HS23 and HS87 molecules were selected in common by the three programs, and ADME/Tox evaluation indicates they are not toxic. The molecular dynamics of PbHSD bonded to ligands showed stable complexes until 50 ns. To validate the results, compounds were purchased for assays of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), synergic profile with Amphotericin B (AmB) and cytotoxicity. The two molecules presented MIC of 32 µg/ml and MFC of 64 µg/ml against the P. brasiliensis (strain Pb18). They also showed synergistic activity with AmB and a lack of toxicity against Hela and Vero cell lines.Conclusion: These results suggest that the HS23 and HS87 are promising candidates as PbHSD inhibitors and may be used as hits for the development of new drugs against paracoccidioidomycosis.

[Box: see text].

In silico approach revealed the membrane receptor PHO36 as a new target for synthetic anticandidal peptides.

Aim: Synthetic antimicrobial peptides (SAMPs) present the potential to fight systemic fungal infections. Here, the PHO36 receptor from Candida albicans was analyzed by in silico tools as a possible target for three anticandidal SAMPs: RcAlb-PepIII, PepGAT and PepKAA.Materials & methods: Molecular docking, dynamics and quantum biochemistry were employed to understand the individual contribution of amino acid residues in the interaction region.Results: The results revealed that SAMPs strongly interact with the PHO36 by multiple high-energy interactions. This is the first study to employ quantum biochemistry to describe the interactions between SAMPs and the PHO36 receptor.Conclusion: This work contributes to understanding and identifying new molecular targets with medical importance that could be used to discover new drugs against systemic fungal infections.

Here, computers helped us find new proteins in Candida albicans that may guide the development of new medicines.

Identification of Potential New Genes Related to the SREBP Pathway in Xanthophyllomyces dendrorhous.

The sterol regulatory element-binding protein (SREBP) pathway is an integral cellular mechanism that regulates lipid homeostasis, in which transcriptional activator SREBPs regulate the expression of various genes. In the carotenogenic yeast Xanthophyllomyces dendrorhous, Sre1 (the yeast SREBP homolog) regulates lipid biosynthesis and carotenogenesis, among other processes. Despite the characterization of several components of the SREBP pathway across various eukaryotes, the specific elements of this pathway in X. dendrorhous remain largely unknown. This study aimed to explore the potential regulatory mechanisms of the SREBP pathway in X. dendrorhous using the strain CBS.cyp61- as a model, which is known to have Sre1 in its active state under standard culture conditions, resulting in a carotenoid-overproducing phenotype. This strain was subjected to random mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), followed by a screening methodology that focused on identifying mutants with altered Sre1 activation phenotypes. Single-nucleotide polymorphism (SNP) analysis of 20 selected mutants detected 5439 single-nucleotide variants (SNVs), narrowing them down to 1327 SNPs of interest after a series of filters. Classification based on SNP impact identified 116 candidate genes, including 49 genes with high impact and 68 genes with deleterious moderate-impact mutations. BLAST, InterProScan, and gene ontology enrichment analyses highlighted 25 genes as potential participants in regulating Sre1 in X. dendrorhous. The key findings of this study include the identification of genes potentially encoding proteins involved in protein import/export to the nucleus, sterol biosynthesis, the ubiquitin-proteasome system, protein regulatory activities such as deacetylases, a subset of kinases and proteases, as well as transcription factors that could be influential in SREBP regulation. These findings are expected to significantly contribute to the current understanding of the intricate regulation of the transcription factor Sre1 in X. dendrorhous, providing valuable groundwork for future research and potential biotechnological applications.

Synergic Effect of the Antimicrobial Peptide ToAP2 and Fluconazole on Candida albicans Biofilms.

Candida albicans is one of the agents of invasive candidiasis, a life-threatening disease strongly associated with hospitalization, particularly among patients in intensive care units with central venous catheters. This study aimed to evaluate the synergistic activity of the antifungal peptide ToAP2 combined with fluconazole against C. albicans biofilms grown on various materials. We tested combinations of different concentrations of the peptide ToAP2 with fluconazole on C. albicans biofilms. These biofilms were generated on 96-well plates, intravenous catheters, and infusion tubes in RPMI medium at two maturation stages. Scanning electron microscopy and atomic force microscopy were employed to assess the biofilm structure. We also evaluated the expression of genes previously proven to be involved in C. albicans biofilm formation in planktonic and biofilm cells after treatment with the peptide ToAP2 using qPCR. ToAP2 demonstrated a synergistic effect with fluconazole at concentrations up to 25 µM during both the early and mature stages of biofilm formation in 96-well plates and on medical devices. Combinations of 50, 25, and 12.5 µM of ToAP2 with 52 µM of fluconazole significantly reduced the biofilm viability compared to individual treatments and untreated controls. These results were supported by substantial structural changes in the biofilms observed through both scanning and atomic force microscopy. The gene expression analysis of C. albicans cells treated with 25 µM of ToAP2 revealed a decrease in the expression of genes associated with membrane synthesis, along with an increase in the expression of genes involved in efflux pumps, adhesins, and filamentation. Our results highlight the efficacy of the combined ToAP2 and fluconazole treatment against C. albicans biofilms. This combination not only shows therapeutic potential but also suggests its utility in developing preventive biofilm tools for intravenous catheters.

Response of Fusarium oxysporum soil isolate to amphotericin B and fluconazole at the proteomic level.

Fusarium oxysporum is a cross-kingdom pathogen that infects humans, animals, and plants. The primary concern regarding this genus revolves around its resistance profile to multiple classes of antifungals, particularly azoles. However, the resistance mechanism employed by Fusarium spp. is not fully understood, thus necessitating further studies to enhance our understanding and to guide future research towards identifying new drug targets. Here, we employed an untargeted proteomic approach to assess the differentially expressed proteins in a soil isolate of Fusarium oxysporum URM7401 cultivated in the presence of amphotericin B and fluconazole. In response to antifungals, URM7401 activated diverse interconnected pathways, such as proteins involved in oxidative stress response, proteolysis, and lipid metabolism. Efflux proteins, antioxidative enzymes and M35 metallopeptidase were highly expressed under amphotericin B exposure. Antioxidant proteins acting on toxic lipids, along with proteins involved in lipid metabolism, were expressed during fluconazole exposure. In summary, this work describes the protein profile of a resistant Fusarium oxysporum soil isolate exposed to medical antifungals, paving the way for further targeted research and discovering new drug targets.

Expression patterns of Mal genes and association with differential maltose and maltotriose transport rate of two Saccharomyces pastorianus yeasts.

Beer brewing is a well-known process that still faces great challenges, such as the total consumption of sugars present in the fermentation media. Lager-style beer, a major worldwide beer type, is elaborated by Saccharomyces pastorianus (Sp) yeast, which must ferment high maltotriose content worts, but its consumption represents a notable problem, especially among Sp strains belonging to group I. Factors, such as fermentation conditions, presence of maltotriose transporters, transporter copy number variation, and genetic regulation variations contribute to this issue. We assess the factors affecting fermentation in two Sp yeast strains: SpIB1, with limited maltotriose uptake, and SpIB2, known for efficient maltotriose transport. Here, SpIB2 transported significantly more maltose (28%) and maltotriose (32%) compared with SpIB1. Furthermore, SpIB2 expressed all MAL transporters (ScMALx1, SeMALx1, ScAGT1, SeAGT1, MTT1, and MPHx) on the first day of fermentation, whereas SpIB1 only exhibited ScMalx1, ScAGT1, and MPH2/3 genes. Some SpIB2 transporters had polymorphic transmembrane domains (TMD) resembling MTT1, accompanied by higher expression of these transporters and its positive regulator genes, such as MAL63. These findings suggest that, in addition to the factors mentioned above, positive regulators of Mal transporters contribute significantly to phenotypic diversity in maltose and maltotriose consumption among the studied lager yeast strains.IMPORTANCEBeer, the third most popular beverage globally with a 90% market share in the alcoholic beverage industry, relies on Saccharomyces pastorianus (Sp) strains for lager beer production. These strains exhibit phenotypic diversity in maltotriose consumption, a crucial process for the acceptable organoleptic profile in lager beer. This diversity ranges from Sp group II strains with a notable maltotriose-consuming ability to Sp group I strains with limited capacity. Our study highlights that differential gene expression of maltose and maltotriose transporters and its upstream trans-elements, such as MAL gene-positive regulators, adds complexity to this variation. This insight can contribute to a more comprehensive analysis needed to the development of controlled and efficient biotechnological processes in the beer brewing industry.

Fungal endo and exochitinase production, characterization, and application for Candida biofilm removal.

Chitinases are promising enzymes for a multitude of applications, including chitooligosaccharide (COS) synthesis for food and pharmaceutical uses and marine waste management. Owing to fungal diversity, fungal chitinases may offer alternatives for chitin degradation and industrial applications. The rapid reproduction cycle, inexpensive growth media, and ease of handling of fungi may also contribute to reducing enzyme production costs. Thus, this study aimed to identify fungal species with chitinolytic potential and optimize chitinase production by submerged culture and enzyme characterization using shrimp chitin. Three fungal species, Coriolopsis byrsina, Trichoderma reesei, and Trichoderma harzianum, were selected for chitinase production. The highest endochitinase production was achieved in C. byrsina after 168 h cultivation (0.3 U mL- 1). The optimal temperature for enzyme activity was similar for the three fungal species (up to 45 and 55 ºC for endochitinases and exochitinases, respectively). The effect of pH on activity indicated maximum hydrolysis in acidic pH (4-7). In addition, the crude T. reesei extract showed promising properties for removing Candida albicans biofilms. This study showed the possibility of using shrimp chitin to induce chitinase production and enzymes that can be applied in different industrial sectors.

Role of ERG11 and MDR1 genes in cycloheximide and multidrug resistance in Candida species.

Candida species are amongst the commensals of the mucosal surfaces of the human body which include the oral cavity, vagina, and intestinal mucosa. Fungal infections are on the rise worldwide. The overall burden of infections due to fungi is difficult to estimate because the majority of them remain undiagnosed. The present study aims to determine the burden of antifungal resistance in low socioeconomic country, Pakistan and the frequency of ERG11 and MDR1 genes involved. A total of 636 Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Antifungal resistance was determined against Fluconazole, Itraconazole, Ketoconazole, and Nystatin via disk diffusion technique. Most resistance was observed against Fluconazole followed by Itraconazole, Ketoconazole, and Nystatin. The Candida isolates recovering from CVP tip and tissue have a high resistance profile. Candida species resistant to at least two antifungals were chosen for further ERG11 and MDR1 detection through real-time PCR. Among 255 Candida isolates, 240 contained ERG11 gene while MDR1 gene is present in 149 Candida isolates. The isolates carrying both genes were tested by the broth microdilution technique for the susceptibility against cycloheximide, all of them were able to grow in cycloheximide. The genetic determinants of antifungal resistance such as ERG11 and MDR1 are as important in the multidrug resistance against a variety of compounds and antifungal drugs.

Enzymes and biosurfactants of industrial interest produced by culturable fungi present in sediments of Boeckella Lake, Hope Bay, north-east Antarctic Peninsula.

This study characterized cultivable fungi present in sediments obtained from Boeckella Lake, Hope Bay, in the north-east of the Antarctic Peninsula, and evaluated their production of enzymes and biosurfactants of potential industrial interest. A total of 116 fungal isolates were obtained, which were classified into 16 genera within the phyla Ascomycota, Basidiomycota and Mortierellomycota, in rank. The most abundant genera of filamentous fungi included Pseudogymnoascus, Pseudeurotium and Antarctomyces; for yeasts, Thelebolales and Naganishia taxa were dominant. Overall, the lake sediments exhibited high fungal diversity and moderate richness and dominance. The enzymes esterase, cellulase and protease were the most abundantly produced by these fungi. Ramgea cf. ozimecii, Holtermanniella wattica, Leucosporidium creatinivorum, Leucosporidium sp., Mrakia blollopis, Naganishia sp. and Phenoliferia sp. displayed enzymatic index > 2. Fourteen isolates of filamentous fungi demonstrated an Emulsification Index 24% (EI24%) ≥ 50%; among them, three isolates of A. psychrotrophicus showed an EI24% > 80%. Boeckella Lake itself is in the process of drying out due to the impact of regional climate change, and may be lost completely in approaching decades, therefore hosts a threatened community of cultivable fungi that produce important biomolecules with potential application in biotechnological processes.

Candida parapsilosis: Heterogeneous and strain-specific expression of secreted aspartic proteases (Sapp1 and Sapp2).

The increasing prevalence of Candida parapsilosis as a causative agent of fungal infections underscores the need to comprehensively understand its virulence factors. Secreted aspartic proteases (Saps) play a significant role in adhesion events, promoting biofilm formation, causing tissue damage and evading the host's immune response. In C. parapsilosis, three Saps have been identified: Sapp1, Sapp2 and Sapp3. The present study investigates the production dynamics of Sapp1 and Sapp2 across 10 clinical isolates of C. parapsilosis using various approaches. Each fungal isolate demonstrated the capability to utilize bovine serum albumin (BSA) as the sole nitrogen source, as evidenced by its degradation in a cell-free culture medium, forming low molecular mass polypeptides. Interestingly, the degradation of different proteinaceous substrates, such as BSA, human serum albumin (HSA), gelatin and hemoglobin, was typically isolate-dependent. Notably, higher proteolysis of HSA compared to BSA, gelatin and hemoglobin was observed. A quantitative assay revealed that the cleavage of a peptide fluorogenic substrate (cathepsin D) was isolate-specific, ranging from 44.15 to 270.61 fluorescence arbitrary units (FAU), with a mean proteolysis of 150.7 FAU. The presence of both Sapp1 and Sapp2 antigens on the cell surface of these fungal isolates was confirmed through immunological detection employing specific anti-Sapp1 and anti-Sapp2 antibodies. The surface levels of Sapp1 were consistently higher, up to fourfold, compared to Sapp2. Similarly, higher levels of Sapp1 than Sapp2 were detected in fungal secretions. This study provides insights into the dynamic expression and regulation of Sapps in C. parapsilosis, highlighting a known virulence factor that is considered a potential target for drug development against this increasingly prominent pathogen.

The fungal pathogen Candida parapsilosis can secrete aspartic proteases (Sapps) as part of its arsenal of virulence factors. We demonstrated that Sapps were able to cleave key host proteins, and the production of Sapp1 and Sapp2 antigens was typically dependent on the fungal isolate when grown in both planktonic- and biofilm-forming cells.

Utilization of Fusarium Solani lipase for enrichment of polyunsaturated Omega-3 fatty acids.

Omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), offer numerous health benefits. Enriching these fatty acids in fish oil using cost-effective methods, like lipase application, has been studied extensively. This research aimed to investigate F. solani as a potential lipase producer and compare its efficacy in enhancing polyunsaturated omega-3 fatty acids with commercial lipases. Submerged fermentation with coconut oil yielded Lipase F2, showing remarkable activity (215.68 U/mL). Lipase F2 remained stable at pH 8.0 (activity: 93.84 U/mL) and active between 35 and 70 °C, with optimal stability at 35 °C. It exhibited resistance to various surfactants and ions, showing no cytotoxic activity in vitro, crucial for its application in the food and pharmaceutical industries. Lipase F2 efficiently enriched EPA and DHA in fish oil, reaching 22.1 mol% DHA and 23.8 mol% EPA. These results underscore the economic viability and efficacy of Lipase F2, a partially purified enzyme obtained using low-cost techniques, demonstrating remarkable stability and resistance to diverse conditions. Its performance was comparable to highly pure commercially available enzymes in omega-3 production. These findings highlight the potential of F. solani as a promising lipase source, offering opportunities for economically producing omega-3 and advancing biotechnological applications in the food and supplements industry.

Drug to genome to drug: a computational large-scale chemogenomics screening for novel drug candidates against sporotrichosis.

Sporotrichosis is recognized as the predominant subcutaneous mycosis in South America, attributed to pathogenic species within the Sporothrix genus. Notably, in Brazil, Sporothrix brasiliensis emerges as the principal species, exhibiting significant sapronotic, zoonotic and enzootic epidemic potential. Consequently, the discovery of novel therapeutic agents for the treatment of sporotrichosis is imperative. The present study is dedicated to the repositioning of pharmaceuticals for sporotrichosis therapy. To achieve this goal, we designed a pipeline with the following steps: (a) compilation and preparation of Sporothrix genome data; (b) identification of orthologous proteins among the species; (c) identification of homologous proteins in publicly available drug-target databases; (d) selection of Sporothrix essential targets using validated genes from Saccharomyces cerevisiae; (e) molecular modeling studies; and (f) experimental validation of selected candidates. Based on this approach, we were able to prioritize eight drugs for in vitro experimental validation. Among the evaluated compounds, everolimus and bifonazole demonstrated minimum inhibitory concentration (MIC) values of 0.5 µg/mL and 4.0 µg/mL, respectively. Subsequently, molecular docking studies suggest that bifonazole and everolimus may target specific proteins within S. brasiliensis- namely, sterol 14-α-demethylase and serine/threonine-protein kinase TOR, respectively. These findings shed light on the potential binding affinities and binding modes of bifonazole and everolimus with their probable targets, providing a preliminary understanding of the antifungal mechanism of action of these compounds. In conclusion, our research advances the understanding of the therapeutic potential of bifonazole and everolimus, supporting their further investigation as antifungal agents for sporotrichosis in prospective hit-to-lead and preclinical investigations.

Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus.

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins' involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Elucidating solvent effects on lipase-catalyzed peroxyacid synthesis through activity-based kinetics and molecular dynamics.

Peroxyacid synthesis is the first step in Prilezhaev epoxidation, which is an industrial method to form epoxides. Motivated by the development of a kinetic model as a tool for solvent selection, the effect of solvent type and acid chain length on the lipase-catalyzed peroxyacid synthesis was studied. A thermodynamic activity-based ping-pong kinetic expression was successfully applied to predict the effect of the reagent loadings in hexane. The activity-based reaction quotients provided a prediction of solvent-independent equilibrium constants. However, this strategy did not achieve satisfactory estimations of initial rates in solvents of higher polarity. The lack of compliance with some assumptions of this methodology could be confirmed through molecular dynamics calculations i.e. independent solvation energies and lack of solvent interaction with the active site. A novel approach is proposed combining the activity-based kinetic expression and the free binding energy of the solvent with the active site to predict kinetics upon solvent change. Di-isopropyl ether generated a strong interaction with the enzyme's active site, which was detrimental to kinetics. On the other hand, toluene or limonene gave moderate interaction with the active site rendering improved catalytic yield compared with less polar solvents, a finding sharpened when peroctanoic acid was produced.

Antifungal resistance: why are we losing this battle?

The emergence of fungal pathogens and changes in the epidemiological landscape are prevalent issues in clinical mycology. Reports of resistance to antifungals have been reported. This review aims to evaluate molecular and nonmolecular mechanisms related to antifungal resistance. Mutations in the ERG genes and overexpression of the efflux pump (MDR1, CDR1 and CDR2 genes) were the most reported molecular mechanisms of resistance in clinical isolates, mainly related to Azoles. For echinocandins, a molecular mechanism described was mutation in the FSK genes. Furthermore, nonmolecular virulence factors contributed to therapeutic failure, such as biofilm formation and selective pressure due to previous exposure to antifungals. Thus, there are many public health challenges in treating fungal infections.

[Box: see text].

Assessment of Fungal Lytic Enzymatic Extracts Produced Under Submerged Fermentation as Enhancers of Entomopathogens' Biological Activity.

The application of enzymes in agricultural fields has been little explored. One potential application of fungal lytic enzymes (chitinases, lipases, and proteases) is as an additive to current biopesticides to increase their efficacy and reduce the time of mortality. For this, a screening of lytic overproducer fungi under submerged fermentation with a chemical-defined medium was performed. Then, the enzymatic crude extract (ECE) was concentrated and partially characterized. This characterization consisted of measuring the enzymatic activity (lipase, protease and, chitinase) and determining the enzyme stability after storage at temperatures of - 80, - 20 and, 4 °C. And lastly, the application of these concentrated enzymatic crude extracts (C-ECE) as an enhancer of spores-based fungal biopesticide was proven. Beauveria were not as good producers of lytic enzymes as the strains from Trichoderma and Metarhizium. The isolate M. robertsii Mt015 was selected for the co-production of chitinases and proteases; and the isolate T. harzianum Th180 for co-production of chitinases, lipases, and proteases. The C-ECE of Mt015 had a protease activity of 18.6 ± 1.1 U ml-1, chitinase activity of 0.28 ± 0.01 U ml-1, and no lipase activity. Meanwhile, the C-ECE of Th180 reached a chitinase activity of 0.75 U ml-1, lipase activity of 0.32 U ml-1, and protease activity of 0.24 U ml-1. Finally, an enhancing effect of the enzymatic extracts of M. robertsii (66.7%) and T. harzianum (43.5%) on the efficacy of B. bassiana Bv064 against Diatraea saccharalis larvae was observed. This work demonstrates the non-species-specific enhancing effect of enzymatic extracts on the insecticidal activity of conidial-based biopesticides, which constitutes a contribution to the improvement of biological control agents' performance.

Enhancing Selenium Accumulation in Rhodotorula mucilaginosa Strain 6S Using a Proteomic Approach for Aquafeed Development.

It is known that selenium (Se) is an essential trace element, important for the growth and other biological functions of fish. One of its most important functions is to contribute to the preservation of certain biological components, such as DNA, proteins, and lipids, providing protection against free radicals resulting from normal metabolism. The objective of this study was to evaluate and optimize selenium accumulation in the native yeast Rhodotorula mucilaginosa 6S. Sodium selenite was evaluated at different concentrations (5-10-15-20-30-40 mg/L). Similarly, the effects of different concentrations of nitrogen sources and pH on cell growth and selenium accumulation in the yeast were analyzed. Subsequently, the best cultivation conditions were scaled up to a 2 L reactor with constant aeration, and the proteome of the yeast cultured with and without sodium selenite was evaluated. The optimal conditions for biomass generation and selenium accumulation were found with ammonium chloride and pH 5.5. Incorporating sodium selenite (30 mg/L) during the exponential phase in the bioreactor after 72 h of cultivation resulted in 10 g/L of biomass, with 0.25 mg total Se/g biomass, composed of 25% proteins, 15% lipids, and 0.850 mg total carotenoids/g biomass. The analysis of the proteomes associated with yeast cultivation with and without selenium revealed a total of 1871 proteins. The results obtained showed that the dynamic changes in the proteome, in response to selenium in the experimental medium, are directly related to catalytic activity and oxidoreductase activity in the yeast. R. mucilaginosa 6S could be an alternative for the generation of selenium-rich biomass with a composition of other nutritional compounds also of interest in aquaculture, such as proteins, lipids, and pigments.

A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

MIG1, TUP1 and NRG1 mediated yeast to hyphal morphogenesis inhibition in Candida albicans by ganciclovir.

Candida albicans is a polymorphic human fungal pathogen and the prime etiological agent responsible for candidiasis. The main two aspects of C. albicans virulence that have been suggested are yeast-to-hyphal (Y-H) morphological transitions and biofilm development. Anti-fungal agents targeting these virulence attributes enhances the antifungal drug development process. Repositioning with other non-fungal drugs offered a one of the new strategies and a potential alternative option to counter the urgent need for antifungal drug development. In the current study, an antiviral drug ganciclovir was screened as an antifungal agent against ATCC 90028, 10231 and clinical isolate (C1). Ganciclovir at 0.5 mg/ml concentration reduced 50% hyphal development on a silicon-based urinary catheter and was visualized using scanning electron microscopy. Ganciclovir reduced ergosterol biosynthesis in both strains and C1 isolate of C. albicans in a concentration-dependent manner. Additionally, a gene expression profile study showed that ganciclovir treatment resulted in upregulation of hyphal-specific repressors MIG1, TUP1, and NRG1 in C. albicans. Additionally, an in vivo study on the Bombyx mori silkworm model further evidenced the virulence inhibitory ability of ganciclovir (0.5 mg/ml) against C. albicans. This is the first report that explore the novel anti-morphogenic activities of ganciclovir against the pathogenic C. albicans strains, along with clinical isolates. Further, ganciclovir may be considered for therapeutic purpose after combinations with standard antifungal agents.