Taxonomy, comparative genomics and evolutionary insights of Penicillium ucsense: a novel species in series Oxalica.

The genomes of two Penicillium strains were sequenced and studied in this study: strain 2HH was isolated from the digestive tract of Anobium punctatum beetle larva in 1979 and the cellulase hypersecretory strain S1M29, derived from strain 2HH by a long-term mutagenesis process. With these data, the strains were reclassified and insight is obtained on molecular features related to cellulase hyperproduction and the albino phenotype of the mutant. Both strains were previously identified as Penicillium echinulatum and this investigation indicated that these should be reclassified. Phylogenetic and phenotype data showed that these strains represent a new Penicillium species in series Oxalica, for which the name Penicillium ucsense is proposed here. Six additional strains (SFC101850, SFCP10873, SFCP10886, SFCP10931, SFCP10932 and SFCP10933) collected from the marine environment in the Republic of Korea were also classified as this species, indicating a worldwide distribution of this new taxon. Compared to the closely related strain Penicillium oxalicum 114-2, the composition of cell wall-associated proteins of P. ucsense 2HH shows five fewer chitinases, considerable differences in the number of proteins related to ß-D-glucan metabolism. The genomic comparison of 2HH and S1M29 highlighted single amino-acid substitutions in two major proteins (BGL2 and FlbA) that can be associated with the hyperproduction of cellulases. The study of melanin pathways shows that the S1M29 albino phenotype resulted from a single amino-acid substitution in the enzyme ALB1, a precursor of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Our study provides important knowledge towards understanding species distribution, molecular mechanisms, melanin production and cell wall biosynthesis of this new Penicillium species.

Mycotechnology to remove of metals from tannery and galvanic effluents - Fungal species from the Amazon and Atlantic Forest show high efficiency.

Metals are considered one of the biggest environmental problems, due to their toxicity and the complexity of removal. This study evaluated the bioaccumulation capacity of water contaminating metals by fungal isolates of Lentinus and Panus species, to elucidate the bioremediation processes of metal contaminated effluents. Initially, tests were performed with fungal isolates using a mixture of metals, aluminum, iron, copper, lead, chromium, nickel and zinc. Lentinus crinitus 154L.21 was the most promising fungus for the removal of metals in the mixture. Based on these data, the potential application of this fungus for the treatment of galvanic and tannery effluents was evaluated. For galvanic effluent, no detectable copper, chromium, and nickel was removed; however, for tannery effluents, reductions in aluminum concentrations from 204.1 to 3.7 mg L-1 (≅98% removal), chromium from 1199.6 to 20.4 mg L-1 (≅98% removal) and iron from 22.6 mg L-1 (100% removal) to an amount lower than the detection limit were observed. These data indicated that L. crinitus 154L.21 removes metals from industrial effluents, being an important route for bioremediation processes.

Production of antimicrobial metabolites against pathogenic bacteria and yeasts by Fusarium oxysporum in submerged culture processes.

The antimicrobial activity of the metabolites produced by Fusarium oxysporum PR-33 in submerged culture was evaluated against Gram-positive and Gram-negative bacteria and yeasts. Metabolites were determined by HPLC-DAD-MS/MS. An extract was obtained following the removal of mycelium by centrifugation and lyophilisation of the supernatant. The compounds in this extract demonstrated broad-spectrum antimicrobial action, with rates of inhibition between 60 and 80%, depending on the species and extract tested. The major compounds of the extracts were identified as fusarinolic acid and its isomer [56.9% flask extract (FE)] and 59.2% bioreactor extract (BE), dehydrofusaric acid (35.7% FE and 31.6% BE), and fusaric acid (6.5% FE and 1.1% BE). Fusaric acid has been shown to be responsible for antimicrobial activity. The cytotoxicity of the extracts was evaluated in culture of HEK-293 and SH-SY5Y animal cells and toxicity of these extracts was verified even in the lowest tested concentrations. Therefore, our results indicate that the compounds identified exhibit potential as antimicrobial agents.

Fermentation of hexoses and pentoses from sugarcane bagasse hydrolysates into ethanol by Spathaspora hagerdaliae.

The present study evaluated 13 strains of yeast for ethanol and xylitol production from xylose. Among them, Spathaspora hagerdaliae UFMG-CM-Y303 produced ethanol yields (YP/S) of 0.25 g g- 1 and 0.39 g g- 1 under aerobic and microaerophilic conditions, respectively, from a mixture of glucose and xylose in flasks. A pH of 5.0 and an inoculum of 3.0 × 108 cells mL- 1r resulted in the highest ethanol yields. These conditions were tested in a bioreactor for fermenting a medium containing an enzymatic hydrolysate of sugarcane bagasse with 15.5 g L- 1 of glucose and 3 g L- 1 of xylose, and achieved a YP/S of 0.47 g g- 1, in relation to total available sugar. These results suggest that S. hagerdaliae UFMG-CM-Y303 has potential for use in second-generation ethanol studies.

Production of ethanol and xylitol by Trametes membranacea.

The potential to produce ethanol and xylitol from xylose by the macro basidiomycete Trametes membranacea was evaluated. All strains studied showed ethanol and xylitol production. The highest ethanol production of xylose was obtained by T. membranacea strain TM158/10 with 5.65 ± 0.21 g/L at pH 4 and 28 °C with 288 h of fermentation and 5.59 ± 0.05 g/L ethanol concentration at pH 5 and 24 °C with 360 h of fermentation. When the conversion was carried out using sugars generated from enzymatic hydrolysis of sugarcane bagasse, there were higher yields from 74 to 15% for ethanol and xylitol, respectively. Although the ethanol and xylitol production need to be optimized, this study showed for the first time the possibility of using T. membranacea for the simultaneous xylitol and ethanol production from pentose sugars, allowing for the possibility of using all released sugars during the hydrolysis of lignocelluloses.

Influence of different chemical pretreatments of elephant grass (Pennisetum purpureum, Schum.) used as a substrate for cellulase and xylanase production in submerged cultivation.

This study evaluated the potential use of elephant grass biomass, a highly productive species, for cellulase and xylanase production by the cellulolytic mutant Penicillium echinulatum 9A02S1 in submerged cultivation, using untreated biomass, biomass pretreated with different concentrations of NaOH, H2SO4 or NH4OH, or biomass pretreated with H2O at 121 °C. For filter paper activity, all cultivation carried out with pretreated elephant grass under the evaluated conditions showed superior activity when compared with the control (untreated elephant grass). The activities of endoglucanases and ß-glucosidases were higher in the cultivation prepared from pretreated samples than the control made with cellulose (Celuflok(®)). Without pretreatment, elephant grass can be used for xylanase production, enabling similar activities to those obtained in the cultivation with cellulose, reducing the enzyme production cost. These results indicate that the pretreatment of elephant grass, especially when pretreated with H2SO4, may be used as a partial or total replacement for cellulose to cellulase production, and untreated elephant grass may be used for xylanase production.

Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the ß-actin gene (actb), ß-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR.

Secretion of laccase and manganese peroxidase by Pleurotus strains cultivate in solid-state using Pinus spp. sawdust.

Pleurotus species secrete phenol oxidase enzymes: laccase (Lcc) and manganese peroxidase (MnP). New genotypes of these species show potential to be used in processes aiming at the degradation of phenolic compounds, polycyclic aromatic hydrocarbons and dyes. Hence, a screening of some strains of Pleurotus towards Lcc and MnP production was performed in this work. Ten strains were grown through solid-state fermentation on a medium based on Pinus spp. sawdust, wheat bran and calcium carbonate. High Lcc and MnP activities were found with these strains. Highest Lcc activity, 741 ± 245 U gdm(-1) of solid state-cultivation medium, was detected on strain IB11 after 32 days, while the highest MnP activity occurred with strains IB05, IB09, and IB11 (5,333 ± 357; 4,701 ± 652; 5,999 ± 1,078 U gdm(-1), respectively). The results obtained here highlight the importance of further experiments with lignocellulolytic enzymes present in different strains of Pleurotus species. Such results also indicate the possibility of selecting more valuable strains for future biotechnological applications, in soil bioremediation and biological biomass pre-treatment in biofuels production, for instance, as well as obtaining value-added products from mushrooms, like phenol oxidase enzymes.

Pre-clinical evidence for the therapeutic effect of Pitaya (Hylocereus lemairei) on diabetic intestinal microenvironment.

Intestinal glucose absorption plays a central role in the regulation of glucose plasmatic; however, current clinical management does not target the gut for treating diabetes. This study evaluated the effects of peel and pulp aqueous extract from Hylocereus lemairei on human enterocytes under high glucose concentration. Anti-hyperglycemic and antiobesity activities in vitro were also evaluated. Extracts did not cause cytotoxicity at 1 to 500 µg/mL. Moreover, they were effective in attenuating oxidative stress (DCFH-DA assay) and inflammation (•ON production) caused by high glucose. Intestinal enzymes (α- glucosidase and pancreatic lipase) were inhibited by pulp and peel extracts (>60% and >95%, respectively). Extracts exhibited a redox capacity superior to ascorbic and chlorogenic acids, presenting high phenolic content, mainly anthocyanins. The main compounds for both extracts were chlorogenic acid and naringin, and peel stood both qualitatively and quantitatively. Data suggest red Pitaya has potential as a new medicine for diabetes.

ß-(1 → 3)(1 → 6)glucan from Schizophyllum commune 227E.32: High yield production via glucose/xylose co-metabolization.

A co-metabolization of xylose and glucose by Schizophyllum commune 227E.32 wild mushroom for exopolysaccharide (EPS) production is presented. Cultivations performed with S. commune 227E.32 at different xylose concentrations demonstrated that the concentration of 50 g·L-1 of xylose achieved the highest EPS production, around 4.46 g·L-1. Scale-up in a stirred tank reactor (STR) was performed. 10 % inoculum showed the highest cost/benefit ratio regarding sugar conversion and EPS production (Y P/S = 0.90 g·g-1), achieving 1.82 g·L-1 of EPS. Isolation, purification, and characterization were conducted with EPS produced in flasks and STR. GC-MS analysis showed glucose as main monosaccharide constituents for both isolates. 13C NMR and HSQC-edited showed that both EPS isolated consisted of a ß-D-Glcp (1 â†’ 3) main chain, partially substituted at O-6 with nonreducing ß-D-Glcp ends on every third residue, similar to ß-D-glucan isolated from S. commune basidiomes known as schizophyllan (SPG). The Mw was determined by GPC to 1.5 × 106 Da (flasks) and 1.1 × 106 Da (STR). AFM topographs revealed a semi-flexible appearance of the ß-D-glucan, consistent with the triple helical structures adopted by SPG and overall contour length consistent with a high molar mass.

Effects of bioactive compounds from Pleurotus mushrooms on COVID-19 risk factors associated with the cardiovascular system.

Mushrooms are a group of fungi with great diversity and ultra-accelerated metabolism. As a consequence, mushrooms have developed a protective mechanism consisting of high concentrations of antioxidants such as selenium, polyphenols, ß-glucans, ergothioneine, various vitamins and other bioactive metabolites. The mushrooms of the Pleurotus genus have generated scientific interest due to their therapeutic properties, especially related to risk factors connected to the severity of coronavirus disease 2019 (COVID-19). In this report, we highlight the therapeutic properties of Pleurotus mushrooms that may be associated with a reduction in the severity of COVID-19: antihypertensive, antihyperlipidemic, antiatherogenic, anticholesterolemic, antioxidant, anti-inflammatory and antihyperglycemic properties. These properties may interact significantly with risk factors for COVID-19 severity, and the therapeutic potential of these mushrooms for the treatment or prevention of this disease is evident. Besides this, studies show that regular consumption of Pleurotus species mushrooms or components isolated from their tissues is beneficial for immune health. Pleurotus species mushrooms may have a role in the prevention or treatment of infectious diseases either as food supplements or as sources for pharmacological agents.

Pleurotus albidus (Agaricomycetes) Antioxidant Action Induces Vasodilation in Aorta Arteries: The Influence of the NADPH/NOS Balance.

The main objective of this work was to evaluate whether Pleurotus albidus extract exerts influences on aorta artery tone by its antioxidant properties. The hearts and aortic arteries of male Wistar rats were removed for use in biochemical analysis and vascular reactivity. Both tissues were exposed to P. albidus extract at different concentrations for 30 min and were then exposed to a free radical generation system for 30 min. The extract reduced lipid peroxidation levels and increased catalase and glutathione peroxidase activity in cardiac tissue. In the aorta, P. albidus extract demonstrated a direct vasodilatory effect, which was associated with a reduction in nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity and an increase in sulfhydryl levels and nitric oxide synthase (NOS) activity. Our findings suggest that P. albidus extract has regulatory potential on aorta arteries, regulating the balance of NOX/NOS enzymes and then influencing vessel tone. Further studies are needed to determine the protective mechanisms of the extract.

Analysis of carbohydrate-active enzymes and sugar transporters in Penicillium echinulatum: A genome-wide comparative study of the fungal lignocellulolytic system.

Penicillium echinulatum 2HH is an ascomycete well known for its production of cellulolytic enzymes. Understanding lignocellulolytic and sugar uptake systems is essential to obtain efficient fungi strains for the production of bioethanol. In this study we performed a genome-wide functional annotation of carbohydrate-active enzymes and sugar transporters involved in the lignocellulolytic system of P. echinulatum 2HH and S1M29 strains (wildtype and mutant, respectively) and eleven related fungi. Additionally, signal peptide and orthology prediction were carried out. We encountered a diverse assortment of cellulolytic enzymes in P. echinulatum, especially in terms of ß-glucosidases and endoglucanases. Other enzymes required for the breakdown of cellulosic biomass were also found, including cellobiohydrolases, lytic cellulose monooxygenases and cellobiose dehydrogenases. The S1M29 mutant, which is known to produce an increased cellulase activity, and the 2HH wild type strain of P. echinulatum did not show significant differences between their enzymatic repertoire. Nevertheless, we unveiled an amino acid substitution for a predicted intracellular ß-glucosidase of the mutant, which might contribute to hyperexpression of cellulases through a cellodextrin induction pathway. Most of the P. echinulatum enzymes presented orthologs in P. oxalicum 114-2, supporting the presence of highly similar cellulolytic mechanisms and a close phylogenetic relationship between these fungi. A phylogenetic analysis of intracellular ß-glucosidases and sugar transporters allowed us to identify several proteins potentially involved in the accumulation of intracellular cellodextrins. These may prove valuable targets in the genetic engineering of P. echinulatum focused on industrial cellulases production. Our study marks an important step in characterizing and understanding the molecular mechanisms employed by P. echinulatum in the enzymatic hydrolysis of lignocellulosic biomass.

Lignin nanoparticles enter the scene: A promising versatile green tool for multiple applications.

Strategies to take advantage of residual lignin from industrial processes are well regarded in the field of green chemistry and biotechnology. Quite recently, researchers transformed lignin into nanomaterials, such as nanoparticles, nanofibers, nanofilms, nanocapsules and nanotubes, attracting increasing attention from the scientific community. Lignin nanoparticles are seen as green way to use high-value renewable resources for application in different fields because recent studies have shown they are non-toxic in reasonable concentrations (both in vitro and in vivo assays), inexpensive (a waste generated in the biorefinery, for example, from the bioethanol platform) and potentially biodegradable (by fungi and bacteria in nature). Promising studies have tested lignin nanoparticles for antioxidants, UV-protectants, heavy metal absorption, antimicrobials, drugs carriers, gene delivery systems, encapsulation of molecules, biocatalysts, supercapacitors, tissue engineering, hybrid nanocomposites, wound dressing, and others. These nanoparticles can be produced from distinct lignin types and by different chemical/physical/biological methods, which will result in varied characteristics for their morphology, shape, size, yield and stability. Therefore, taking into account that the theme "lignin nanoparticles" is a trending topic, this present review is emerging and has the discuss the current status, covering from concepts, the formation mechanism, synthesis methods and applications, to the future perspectives and challenges linked to lignin-based nanomaterials, aiming at the viability and commercialization of this biotechnological product.

Polyphenolic Content and Antioxidant Activity of Mycelia and Basidiomes of Oyster Mushrooms Pleurotus spp. (Agaricomycetes) from Brazil.

This study aimed to examine the antioxidant activity, total phenolic content, and phenolic profile of nine strains of Pleurotus spp. isolated in southern Brazil. Basidiomes were obtained from a solid-state culture in medium containing Pinus sp. sawdust (SCM-PSW), coffee grounds (CG), or organic grape waste (OGW). Mycelia were obtained from submerged culture-potato dextrose broth (MSC-PDB). Basidiomes had the highest total phenolic content (between 31.30 ± 0.26 and 47.00 ± 0.12 mg gallic acid equivalents [GAE]/g) compared with mycelia (between 8.15 ± 0.26 and 15.96 ± 0.82 mg GAE/g). Antioxidant activity of the basidiomes showed an IC50 value between 5.36 ± 0.27 (88F.13) and 10.68 ± 0.22 mg/mL (189H.3). Mushrooms produced in the OGW and CG media had higher total phenolic content than those from MSC, indicating that they can serve as sources of bioactive compounds on culture media. These findings show the potential of natural wastes to be used as a strategy for increasing secondary metabolites in edible mushrooms, proposing an interesting approach for the nutraceutical and pharmaceutical industries.

Chemical features and antioxidant profile by Schizophyllum commune produced on different agroindustrial wastes and byproducts of biodiesel production.

Schizophyllum commune VE_07 was produced in different culture media containing pine sawdust (PS), grape residue (GR), cotton cake (CC) and jatropha seed cake (JC). The content of phenolics and antioxidant activity were determined for the substrates and mushrooms produced. The content of ß-glucans and the composition of S. commune were also evaluated. The medium formulated with 94% grape residue enabled the highest values of yield, biological efficiency, and productivity. Mushrooms grown in this condition showed the highest value (13.14%) of ß-glucans. The contents of proteins and dietary fibre were 16.59% and 59.61%, respectively. Mushrooms grown in cotton cake showed the highest phenolic content (291.51 ± 1.83 mg GAE/ 100 g mushroom) and antioxidant activity (58.15 ± 0.86 DPPH % scavenging). The results obtained indicate that substrate composition affected the production of S. commune and its chemical composition.

Lipid-lowering effect of Pinus sp. sawdust and Pycnoporus sanguineus mycelium in streptozotocin-induced diabetic rats.

The purpose of this study was to investigate the effect of diet supplementations on biochemical, hematological, and redox metabolism parameters in streptozotocin-induced diabetic rats. Healthy male Wistar rats and streptozotocin-induced diabetic rats were provided diets supplemented with 20% of Pinus sp. sawdust or Pycnoporus sanguineus mycelium for 4 weeks. Diabetic rats treated with both Pinus sp. sawdust- and P. sanguineus mycelium-supplemented diets presented a significant decrease in non-HDL cholesterol of 38.43% and 33.53% and triglycerides of 70.03% and 69.81%, respectively, compared to diabetic control. As far as we know, this is the first report of a significant decrease in serum lipids attributed to these supplementations. Even though with the alterations in hematological and redox metabolism parameters related to these diet treatments, our data suggest that Pinus sp. sawdust and Pycnoporus sanguineus mycelium could be a useful a diet supplement to control diabetic dyslipidemia in animals. PRACTICAL APPLICATIONS: Pinus sp. sawdust is a residue from the wood industry that can be reused as a substrate to cultivate Pycnoporus sanguineus mycelium. Both species have specific and rich natural compounds. The results of the present study surprisingly showed that diets supplemented with the isolated substrate (Pinus sp. sawdust) and Pycnoporus sanguineus mycelium significantly decrease non-HDL cholesterol and triglycerides in induced diabetic animal model. These new natural approaches can be interesting to develop a nutraceutical to treat dyslipidemia.

Gene Regulatory Networks of Penicillium echinulatum 2HH and Penicillium oxalicum 114-2 Inferred by a Computational Biology Approach.

Penicillium echinulatum 2HH and Penicillium oxalicum 114-2 are well-known cellulase fungal producers. However, few studies addressing global mechanisms for gene regulation of these two important organisms are available so far. A recent finding that the 2HH wild-type is closely related to P. oxalicum leads to a combined study of these two species. Firstly, we provide a global gene regulatory network for P. echinulatum 2HH and P. oxalicum 114-2, based on TF-TG orthology relationships, considering three related species with well-known regulatory interactions combined with TFBSs prediction. The network was then analyzed in terms of topology, identifying TFs as hubs, and modules. Based on this approach, we explore numerous identified modules, such as the expression of cellulolytic and xylanolytic systems, where XlnR plays a key role in positive regulation of the xylanolytic system. It also regulates positively the cellulolytic system by acting indirectly through the cellodextrin induction system. This remarkable finding suggests that the XlnR-dependent cellulolytic and xylanolytic regulatory systems are probably conserved in both P. echinulatum and P. oxalicum. Finally, we explore the functional congruency on the genes clustered in terms of communities, where the genes related to cellular nitrogen, compound metabolic process and macromolecule metabolic process were the most abundant. Therefore, our approach allows us to confer a degree of accuracy regarding the existence of each inferred interaction.

Upscale and characterization of lignin-modifying enzymes from Marasmiellus palmivorus VE111 in a bioreactor under parameter optimization and the effect of inducers.

Testing different pHs, dissolved oxygen concentrations and temperatures, plus the addition of inducers, to optimize ligninolytic enzyme activity, resulted in increased production of laccases, total peroxidases and manganese peroxidases on the order of 2.1-fold, 4.6-fold and 10-fold, respectively; laccases reached 6588 U/mL, total peroxidases reached 3533 U/mL and manganese peroxidase achieved 60 U/mL. Furthermore, an increase in laccase volumetric productivity and in its specific activity was verified. The addition of inducers, such as copper sulphate and manganese sulphate, improved enzymatic activity. In addition, a new previously unidentified laccase isoform was documented by zymography. The present work successfully increased the production of ligninolytic enzymes.

New two-stage pretreatment for the fractionation of lignocellulosic components using hydrothermal pretreatment followed by imidazole delignification: Focus on the polysaccharide valorization.

The combination of hydrothermal pretreatment followed by delignification with imidazole was evaluated for the first time as a potential selective two-stage fractionation for elephant grass, aiming at obtaining pure fractions susceptible to conversion to high value-added products. In addition, the recovery of cellulose and hemicelluloses and enzymatic hydrolysis yield of pretreated elephant grass were evaluated. Hydrothermal pretreatment at 180 °C under non-isothermal conditions allowed obtaining a liquor rich mainly in xylo- and glucooligosaccharides, as well as pentoses. Subsequent treatment of the recovered solid fraction with imidazole at 140 °C for 182.5 min resulted in 83.8 wt% delignification and cellulose enrichment of 97.7 wt%. The solids obtained from the two-stage pretreatment process also permitted high glucan to glucose conversion through enzymatic hydrolysis using Cellic CTec2 (99.0 mol%) or an enzymatic complex of Penicillium echinulatum (96.3 mol%).