Filamentous Strains Research Articles

Engineering the filamentous fungus Penicillium oxalicum for rapid, low-background and efficient protein expression

Filamentous fungi are widely used in the field of recombinant protein expression due to their well-established protein modification systems and excellent secretion capacities. Although Penicillium oxalicum has been developed as an expression host, its potential for efficient and convenient protein production has not been fully exploited. In this study, we obtained an engineered strain by dominant activation of the G protein PGA3 using a point-mutation method based on the low extracellular background P. oxalicum host Δ13A-OamyR. This genetically modified strain, OamyR-QL, with faster cell growth and a more efficient Pamy15A promoter, will be used to construct a novel expression system. The relevant genes and pathways involved in the response to the G protein dominant activation in the engineered strain were revealed by RNA sequencing. Moreover, the transcription activator AmyR was overexpressed in OamyR-QL, resulting in a dramatically enhanced efficiency of the Pamy15A promoter. The construction of an efficient, low-background system by utilizing the G protein-AmyR regulatory pathway provides not only a theoretical reference for the genetic engineering of other filamentous fungal strains, but also a preferable option for the efficient and high purity expression of recombinant proteins in filamentous fungi.

Common but Nonpersistent Acquisitions of Plant Viruses by Plant-Associated Fungi.

Investigating a virus’s host range and cross-infection is important for better understanding the epidemiology and emergence of viruses. Previously, our research group discovered a natural infection of a plant RNA virus, cumber mosaic virus (genus Cucumovirus, family Bromoviridae), in a plant pathogenic basidiomycetous fungus, Rhizoctonia solani, isolated from a potato plant grown in the field. Here, we further extended the study to investigate whether similar cross-infection of plant viruses occurs widely in plant-associated fungi in natural conditions. Various vegetable plants such as spinach, leaf mustard, radish, celery, and other vegetables that showed typical virus-like diseases were collected from the fields in Shandong Province, China. High-throughput sequencing revealed that at least 11 known RNA viruses belonging to different genera, including Potyvirus, Fabavirus, Polerovirus, Waikavirus, and Cucumovirus, along with novel virus candidates belonging to other virus genera, infected or associated with the collected vegetable plants, and most of the leaf samples contained multiple plant viruses. A large number of filamentous fungal strains were isolated from the vegetable leaf samples and subjected to screening for the presence of plant viruses. RT-PCR and Sanger sequencing of the PCR products revealed that among the 169 fungal strains tested, around 50% were carrying plant viruses, and many of the strains harbored multiple plant viruses. The plant viruses detected in the fungal isolates were diverse (10 virus species) and not limited to particular virus genera. However, after prolonged maintenance of the fungal culture in the laboratory, many of the fungal strains have lost the virus. Sequencing of the fungal DNA indicated that most of the fungal strains harboring plant viruses were related to plant pathogenic and/or endophytic fungi belonging to the genera Alternaria, Lecanicillium, and Sarocladium. These observations suggest that the nonpersistent acquisition of plant viruses by fungi may commonly occur in nature. Our findings highlight a possible role for fungi in the life cycle, spread, and evolution of plant viruses.

Purification, Biochemical and Kinetic Characterization of a Novel Alkaline sn-1,3-Regioselective Triacylglycerol Lipase from Penicilliumcrustosum Thom Strain P22 Isolated from Moroccan Olive Mill Wastewater.

A novel extracellular lipase from a filamentous fungus Ascomycota strain, P22, was isolated from olive mill wastewater, then purified and characterized. This strain was identified as Penicillium crustosum Thom based on sequencing analyses. Penicillium crustosum Thom strain P22 lipase (PCrL) was purified 63-fold to homogeneity using ammonium sulfate precipitation and chromatography on a Q-Sepharose Fast Flow column, with a total yield of 34%. The purified PCrL had a molecular mass of 28 kDa, estimated by SDS-PAGE. The 20 NH2-terminal amino-acid residues showed a high degree of homology with those of other Penicillium lipases. The specific activity of PCrL at pH 9 and 37 °C were found to be 5000 and 10,000 U/mg on olive oil and trioctanoin emulsions, respectively. PCrL exhibited clear regioselectivity toward the sn-1 position of the surface-coated triglycerides which were esterified with α-eleostearic acid at the sn-1/3 position. PCrL was completely inhibited by 53 µM of Orlistat, 5 mM of phenylmethylsulfonylfluoride, and 2 mM of diiodopropyl fluorophosphate, suggesting that it belonged to the serine lipase family. PCrL showed high activity and stability in the presence of water-immiscible organic solvents, surfactant, and oxidizing agents, and showed considerable compatibility with commercial laundry detergents. Washing performance analysis revealed that it could effectively remove oil stains. Hence, PCrL has several attractive properties that make it a promising potential candidate for detergent formulations.

Differential near-infrared imaging of heterocysts using single-walled carbon nanotubes

The internalization of near-infrared (NIR) optical nanoprobes in photosynthetic microbes can be exploited for applications ranging from energy conversion to biomolecule delivery. However, the intrinsic, species-dependent properties of microbial cell walls, including their surface charge density, composition, thickness, and elasticity, can severely impact nanoprobe uptake and affect the cellular response. An examination of the interaction of the optical nanoprobe in various species and its impact on cell viability is, therefore, imperative for the development of new imaging technologies. Herein, we extend the technology recently developed for internalizing fluorescent single-walled carbon nanotubes (SWCNTs) in prokaryotes, specifically unicellular Synechocystis sp. PCC 6803, to a filamentous cyanobacterial strain, Nostoc punctiforme. Using a combination of NIR fluorescence, scanning electron microscopy (SEM), and Raman spectroscopy, we investigate uptake in vegetative cells as well as differentiated heterocysts. We demonstrate a strong dependence of long-term cell integrity, activity, and viability on SWCNT surface functionalization. We further show differential uptake of SWCNTs across a single filament, with positively charged functionalized SWCNTs preferentially localizing within the heterocysts of the filament. This cell dependency of the nanoparticle internalization motivates the use of SWCNTs as a NIR stain for monitoring cell differentiation.Graphical abstract

Phenolic compounds and antioxidant activity of Lippia graveolens Kunth residual leaves fermented by two filamentous fungal strains in solid-state process

The residual leaves from essential oil extraction from Mexican oregano (Lippia graveolens) have a potential as a source of bioactive molecules (e.x. flavonoids) that could be extracted by solid state fermentation (SSF) process. In this study, the residual leaves were subjected to a SSF (120 h) by two microorganism Trichoderma harzianum and Rhizopus oryzae evaluating the phenolic compounds (Total polyphenols and flavonoids), the antioxidant activity (DPPH● / FRAP), the bioactivities (α-amylase inhibition and antimicrobial against Escherichia coli), the cytotoxic effect (erythrocytes) and a characterization (HPLC-MS). The results showed that the total polyphenols for T.harzarium did not show statistical differences (first 48 h), and R. oryzae fermentation obtained a higher context (48 h). Also, the higher flavonoid release was present at 48 and 96 h (T. harzianum and R. oryzae). The antioxidant activity decreased in the last fermentation hours, the a-amylase reported a higher inhibition value (29.97 ± 1.97 %) and antimicrobial capacities; R. oryzae (96 h) did not register a cytotoxic effect. The characterization determined the presence of valuable molecules as ferulic acid 4-O-glucoside, phloridzin and luteolin. The fermentation allowed the recovery of bioactive phenolic compounds that could be an alternative for the revalorization of oregano waste.

N-acetylation of toxic aromatic amines by fungi: Strain screening, cytotoxicity and genotoxicity evaluation, and application in bioremediation of 3,4-dichloroaniline

Aromatic amines (AA) are one of the most commonly used classes of compounds in industry and the most common pollutants found in both soil and water. 3,4-Dichloaniline (3,4-DCA) is a persistent residue of the phenylurea herbicide in the environment. In this study, we used a colorimetric method as a new approach to screen 12 filamentous fungal strains of the genera Aspergillus, Chaetomium, Cladosporium, and Mucor to assess their capacity to perform AA N-acetylation since it is considered a potential tool in environmental bioremediation. Subsequently, the selected strains were biotransformed with different AA substrates to evaluate the product yield. The strains Aspergillus niveus 43, Aspergillus terreus 31, and Cladosporium cladosporioides showed higher efficiencies in the biotransformation of 3,4-DCA at 500 µM into its N-acetylated product. These fungal strains also showed great potential to reduce the phytotoxicity of 3,4-DCA in experiments using Lactuca sativa seeds. Furthermore, N-acetylation was shown to be effective in reducing the cytotoxic and genotoxic effects of 3,4-DCA and other AA in the immortalized human keratinocyte (HaCaT) cell line. The isolated products after biotransformation showed that fungi of the genera Aspergillus and Cladosporium appeared to have N-acetylation as the first and main AA detoxification mechanism. Finally, A. terreus 31 showed the highest 3,4-DCA bioremediation potential, and future research can be carried out on the application of this strain to form microbial consortia with great potential for the elimination of toxic AA from the environment.

1,2,4-Triazole derivatives as novel and potent antifungal agents: Design, synthesis and biological evaluation

Fungal infections are still threatening human health due to resistance to existing drugs, therefore, the design and development of novel antifungal agents is to be necessary and also, is interesting topic for medicinal chemist. Azole derivatives are one of the promising antifungal agents, which exert their activities through inhibition of cytochrome P450 14α-demethylase (CYP51). In this regard, a new series of 1,2,4-triazole derivatives (7a-i) were designed, synthesized and confirmed with IR, 1HNMR, 13CNMR and Mass spectrum. The antifungal activity of these compounds were investigated against several yeasts (candida species), filamentous and clinical strains using the CLSI method. Furthermore, to measure the cytotoxic activity, MTT assay was also done against MRC-5 as normal human fibroblasts cell line. Our results represented that most of the compounds had appropriate activity ranging from 0.5-256 µg/mL, especially, compounds 7g and 7h were found to be more potent against yeast strains and clinical strain of Fluconazole-resistant and Fluconazole-sensitive with MIC values of 0.5 µg/mL compared to the Fluconazole as control drug. Subsequently, molecular docking studies were performed to find the binding energy and interaction mode of these compounds in the active site of 14α-demethylase enzyme as plausible target of azole compounds. According to in vitro antifungal assay and in silico ADME predictions, compounds 7g and 7h can be considered as potent candidates for further studies.

Carbon Nanotube Uptake in Photosynthetic Bacteria for Near-infrared Imaging and Enhancing Bioelectricity Generation in Living Photovoltaics

The internalization of fluorescent single-walled carbon nanotubes (SWCNTs) in photosynthetic microbes can be exploited for applications ranging from energy conversion and imaging to biomolecule delivery. The internalization of these nanoparticles are largely controlled by the permeability of the surrounding microbial cell walls, which vary in surface charge density, composition, thickness, and elasticity across different species of microbes. Since these properties play a critical role in controlling internalization, the extension of SWCNT-based technologies to prokaryotes thus requires a comparative assessment of uptake across different species.Herein, we extend a technology previously developed for internalizing SWCNTs in prokaryotes, specifically unicellular Synechocystis sp. PCC 6803, to Nostoc punctiforme, a filamentous cyanobacterial strain known to differentiate into heterocysts under nitrogen starvation. Heterocysts are specialized compartments within the filament that are capable of nitrogen fixation. These compartments possess cell walls that are distinct from vegetative cells, conferring heterocysts with relatively reduced permeability and increased resistance to mechanical and osmotic stress. Using a combination of near-infrared (NIR) fluorescence, scanning electron microscopy (SEM), and Raman spectroscopy, we investigate the effects of different cell-wall architectures and SWCNT functionalizations on internalization. We specifically examine the effects of these distinct SWCNT-cell interactions on long-term cell integrity, activity, and viability. We further show that local variations in membrane structure can dictate the extent of SWCNT association and uptake in Nostoc cells. This dependency of nanoparticle internalization on cell type can be exploited for the development of novel cell-specific staining and imaging technologies.

Hydroxylation of Progesterone and Its Derivatives by the Entomopathogenic Strain Isaria farinosa KCh KW1.1.

Progesterone biotransformation is worth studying because of the high industrial value of its derivatives. This study investigated the catalytic ability of the entomopathogenic filamentous fungus strain Isaria farinosa KCh KW1.1 to transform progesterone derivatives: 11α-hydroxyprogesterone, 17α-hydroxyprogesterone, 16α,17α-epoxyprogesterone and pregnenolone. In the culture of Isaria farinosa KCh KW1.1, 11α-hydroxyprogesterone was effectively transformed into only one product: 6β,11α-dihydroxyprogesterone. Transformation of 17α-hydroxyprogesterone gave three hydroxy derivatives: 6β,17α-dihydroxyprogesterone, 12β,17α-dihydroxyprogesterone and 6β,12β,17α-trihydroxyprogesterone. Two products: 6β-hydroxy-16α,17α-epoxyprogesterone and 6β,11α-dihydroxy-16α,17α-epoxyprogesterone, were obtained from the 16α,17α-epoxyprogesterone transformation. We isolated two compounds from the biotransformation medium with pregnenolone: 11α-hydroxy-7-oxopregnenolone and 5α,6α-epoxy-3β,11α-dihydroxypregnan-7,20-dione. In this study, we observed only mono- and dihydroxy derivatives of the tested substrates, and the number of obtained products for each biotransformation did not exceed three.

Production of filamentous fungal biomass with increased oil content using olive oil as a carbon source

AbstractBACKGROUNDVegetable oil is more difficult for microorganisms to degrade compared with carbohydrates and protein. Oil‐rich waste creates serious environmental and health concerns if it remains untreated. The aim of the work reported here was to evaluate the effects on the nutritional composition of filamentous fungal biomass by growing it in pure olive oil as a carbon source.RESULTSThe growth of different filamentous fungal strains (Aspergillus oryzae,Neurospora intermediaandRhizopus oryzae) was investigated in pure olive oil. A pre‐germination stage of either glucose or yeast extract was necessary for successful fungal growth in olive oil.A. oryzaeshowed superior performance in comparison withN. intermediaandR. oryzaein terms of biomass growth. The obtained biomass fromA. oryzaeandN. intermediawas analyzed for protein, fat, ash and alkali‐insoluble material, where the presence of olive oil had a steering effect on biomass growth (16 g L−1with oilversus4 g L−1without oil). Nutritional composition of the fungal biomass ofA. oryzaecontained 0.33% fat and 48% protein when cultivated in medium without olive oil supplementation, while 31% fat and 14% protein contents were observed in the presence of olive oil‐containing medium. Similar trends for fat and protein contents were observed for the biomass ofN. intermedia. Moreover, microscopy confirmed the presence of oil globules inside the fungal cells.CONCLUSIONSFat composition of fungal biomass can be steered through addition of olive oil, which increases the versatility of the produced biomass for various applications, namely in feed, food and biofuel production. © 2022 The Authors.Journal of Chemical Technology and Biotechnologypublished by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Antifungal Activity and In Silico Studies on 2-Acylated Benzo- and Naphthohydroquinones.

The high rates of morbidity and mortality due to fungal infections are associated with a limited antifungal arsenal and the high toxicity of drugs. Therefore, the identification of novel drug targets is challenging due to the several resemblances between fungal and human cells. Here, we report the in vitro antifungal evaluation of two acylphenols series, namely 2-acyl-1,4-benzo- and 2-acyl-1,4-naphthohydroquinones. The antifungal properties were assessed on diverse Candida and filamentous fungi strains through the halo of inhibition (HOI) and minimal inhibitory concentration (MIC). The antifungal activities of 2-acyl-1,4-benzohydroquinone derivatives were higher than those of the 2-acyl-1,4-naphthohydroquinone analogues. The evaluation indicates that 2-octanoylbenzohydroquinone 4 is the most active member of the 2-acylbenzohydroquinone series, with MIC values ranging from 2 to 16 μg/mL. In some fungal strains (i.e., Candida krusei and Rhizopus oryzae), such MIC values of compound 4 (2 and 4 μg/mL) were comparable to that obtained by amphotericin B (1 μg/mL). The compound 4 was evaluated for its antioxidant activity by means of FRAP, ABTS and DPPH assays, showing moderate activity as compared to standard antioxidants. Molecular docking studies of compound 4 and ADMET predictions make this compound a potential candidate for topical pharmacological use. The results obtained using the most active acylbenzohydroquinones are promising because some evaluated Candida strains are known to have decreased sensitivity to standard antifungal treatments.

Effect of Voriconazole on Biofilm of Filamentous Species Isolated from Keratitis

Background: Biofilm formation is a vital feature of the pathogenesis in filamentous fungi. Objectives: Herein, we investigated in vitro antifungal pattern of biofilm of filamentous species keratitis isolates, and the effect of different concentrations of voriconazole on biofilm morphology changes using scanning electron microscopy. Methods: Here 25 ocular fungal isolates were included (Fusarium solani; (n = 15) and Aspergillus flavus; (n = 10). We determined the biofilm formation of isolates in terms of their susceptibilities to different antifungals on sessile cells. This procedure was performed according to CLSI-M38-3rd. Biofilm morphology changes revealed with scanning electron microscopy (SEM). Results: Twelve F. solani strains displayed the capacity to form biofilms from patients wearing contact lenses, while 8 A. flavus strains were recovered from cornea scrapings of trauma. Itraconazole, posaconazole and natamycin had the maximum activity against biofilms of all tested filamentous strains (MIC ranging from 0.031 - 0.25 µg/mL, 0.031 - 0.25 µg/mL and 2 - 4 µg/mL), respectively. Three F. solani and one A. flavus strains showed the high MIC values against voriconazole (MIC ≥ 1 µg/mL). Posaconazole revealed the lowest MIC values against biofilms of two strains (MIC ranging from 0.031 - 0.25 µg/mL); however, no significant difference was observed for itraconazole (P > 0.05). The efficacy of 16 µg/mL and 4 µg/mL of voriconazole was confirmed for biofilm of F. solani and A. flavus, respectively. The considerable changes in the morphologies of improved biofilms were seen by SEM. Conclusions: For the successful treatment of fungal biofilm infections, it was necessary to have knowledge of the mechanism of action, penetration rates, and therapeutic concentrations of drugs.

Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria.

Cyanobacteria have raised great interest in biotechnology due to their potential for a sustainable, photosynthesis-driven production of fuels and value-added chemicals. This has led to a concomitant development of molecular tools to engineer the metabolism of those organisms. In this regard, however, even cyanobacterial model strains lag behind compared to their heterotrophic counterparts. For instance, replicative shuttle vectors that allow gene transfer independent of recombination into host DNA are still scarce. Here, we introduce the pSOMA shuttle vector series comprising 10 synthetic plasmids for comprehensive genetic engineering of Synechocystis sp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 and pCB2.4, each combined with a replicon from Escherichia coli, different selection markers as well as features facilitating molecular cloning and the insulated introduction of gene expression cassettes. We made use of genes encoding green fluorescent protein (GFP) and a Baeyer-Villiger monooxygenase (BVMO) to demonstrate functional gene expression from the pSOMA plasmids in vivo. Moreover, we demonstrate the expression of distinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between the two pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into the filamentous model strain Anabaena sp. PCC 7120 is generally possible, which is encouraging to further explore the range of cyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays an attractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups and to perform extensive metabolic engineering of cyanobacteria.

Concomitant knockout of target and transporter genes in filamentous fungi by genome co-editing.

In most countries, genetically modified microorganisms are not approved for use for fermentation in the food industry. Therefore, random mutagenesis and subsequent screening are performed to improve the productivities of valuable metabolites and enzymes as well as other specific functions in an industrial microbial strain. In addition, targeted gene knockout is performed by genetic recombination using its enzyme genes as selectable markers to maintain self‐cloning status. However, random mutagenesis has a drawback as it does not guarantee improvement of the targeted function. Conversely, self‐cloning is rarely used to breed an industrial microbial strain. This is probably because a self‐cloning strain is similar to a genetically modified strain, as both undergo homologous recombination, although exogenous genes are not introduced. In this article, I discuss the usefulness of genome editing technology as a substitute for conventional techniques to breed filamentous fungal strains. This article particularly focusses on “genome co‐editing,” a genome editing technology used for knocking out two genes concomitantly, as reported in Magnaporthe grisea and Aspergillus oryzae. Especially, when genome co‐editing is applied to a target gene and a membrane transporter gene that aid the entry of toxic compounds into cells, the resulting clone can be categorized as an autotrophic and non‐genetically modified clone. Such a clone should easily apply to industrial fermentation without being restricted by a genetically modified status. Genome co‐editing will also be used to construct mutant strains with multiple target gene knockouts by eliminating multiple membrane transporter genes. This could substantially improve the productivities of valuable metabolites and enzymes in a stepwise manner. Thus, genome co‐editing is considered a potentially powerful method to knockout single or multiple target genes that can contribute to the breeding of filamentous fungal strains in the food industry.

Apple leaf macro- and micronutrient content as affected by soil treatments with fertilizers and microorganisms

The effects of bacterial and fungal inocula on the nutritional status of ‘Sampion’ apple trees was evaluated in three-years study (2018–2020). The experiment was set up in a two-factor system: (i) fertilization (without fertilization or mineral fertilization with nitrogen, phosphorus, potassium); (ii) application of beneficial microorganisms (without application, application of fungi or bacteria). The mixture of beneficial soil fungi contained two species: Aspergillus niger and Purpureocillium lilacinum. The mixture of beneficial bacteria contained three strains of Bacillus (Bacillus sp., Bacillus amyloliquefaciens and Paenibacillus polymyxa). The analysis of mineral content and leaf greenness (SPAD index) was performed on the samples collected from the trees in August. The lack of mineral fertilization caused a significant decrease in the nitrogen content of apple leaves, which resulted in their lighter color (lower SPAD index). The application of filamentous fungi or bacterial strains had a modifying effect on the mineral composition of apple leaves, what became evident especially in the third year of the study. An increase in the concentration of nitrogen, calcium, magnesium, sulphur and most microelements (except for boron) was found as a result of the influence of tree mineral fertilization and microorganisms applied to the soil. In the absence of mineral fertilization of the trees, the applied microbial inocula resulted in the significant increase of concentration of most macro- and microelements in the leaf tissue (except for magnesium and boron).

Identification of novel extracellular putative chitinase and hydrolase from Geomyces sp. B10I with the biodegradation activity towards polyesters

Cold-adapted filamentous fungal strain Geomyces sp. B10I has been reported to decompose polyesters such as poly(e-caprolactone) (PCL), poly(butylene succinate) (PBS) and poly(butylene succinate-co-butylene adipate) (PBSA). Here, we identified the enzymes of Geomyces sp. B10I, which appear to be responsible for its biodegradation activity. We compared their amino acid sequences with sequences of well-studied fungal enzymes. Partial purification of an extracellular mixture of the two enzymes, named hydrGB10I and chitGB10I, using ammonium sulfate precipitation and ionic exchange chromatography gave 14.16-fold purity. The amino acid sequence of the proteins obtained from the MALDI-TOF analysis determined the molecular mass of 77.2 kDa and 46.5 kDa, respectively. Conserved domain homology analysis revealed that both proteins belong to the class of hydrolases; hydrGB10I belongs to the glycosyl hydrolase 81 superfamily, while chitGB10I contains the domain of the glycosyl hydrolase 18 superfamily. Phylogenetic analysis suggests a distinct nature of the hydrGB10I and chitGB10I of Geomyces sp. B10I when compared with other fungal polyester-degrading enzymes described to date.

Impact of silver nanoparticles size on SERS for detection and identification of filamentous fungi

Using the proper size of nanoparticles as an active substrate, Surface-enhanced Raman scattering (SERS) can provide a reliable technique for detecting and identifying fungi, including Alternaria alternata, Aspergillus flavus, Fusarium verticilliodes, and Aspergillus parasiticus that have been associated to biodeterioration and biodegradation of cultural heritage materials. In this research spherical silver nanoparticles (AgNPs) of average size of 10, 30 and 60 nm were synthesized using the wet chemical method with good yield and their size and shape distributions were examined using small-angle X-ray scattering (SAXS). The protocol for fungi sample preparation proved to be critical for producing high-quality and reproducible SERS spectra. We found that the effect of AgNPs on SERS signal enhancement is size dependent under the same experimental conditions; the SERS intensity of fungal strains using 60 nm achieved up to 2.3x105 enhancement, about twice as intense as those produced with 30 nm, and 10 nm produced a minor broad weak peak barely discernible around 1400 cm−1, similar to the NR spectra profile in the 550–1700 cm−1 spectral region, and the SERS signals using 60 nm showed high reproducibility, with less than 20% variance. Furthermore, we used principal component analysis (PCA) to statistically classify the SERS spectrum into four separate clusters with 99 percent variability so that the four fungal strains could be clearly detected and identified. The SERS technique, in combination with the PCA developed in this study, provides a simple, rapid, accurate, and cost-effective analytical tool for detecting and identifying filamentous fungal strains.

Rapid Enzymatically Reduction of Zincum Gluconicum for the Biomanufacturing of Zinc Oxide Nanoparticles by Mycoextracellular Filtrate of Penicillium Digitatum (Pdig-B3) as a Soft Green Technique.

Molds have been used as micro-biofactories for biomanufacturing of metal oxide nanoparticles (MetNps) since they are effortless, immaculate, safe, non-poisonous, vital-biocompatible, and environmentally acceptable. The present study aimed to explore the bioindustry, mold screening protocol, and characterization of zinc oxide nanoparticles (ZnONPs) using a diverse filamentous Green mold (FiGM) isolated from spoiled citrus fruits. Eight filamentous Penicillium Digitatum mold strains had been obtained and subjected to investigate the capability of ZnONPs biosynthesis by fungal extracellular free-cell filtrate. P. digitatum (P-digB3) obtained the peak of ZnONps (379 nm) detected by the UV-visible spectrophotometry and was found as a significantly optimum strain in the highest quantity (mean±SD: 0.0138±0.001 gm/100 ml) and the smallest average NPs size. The ZnONPs were characterized by UV-visible scanning spectrophotometry, Atomic Force Microscopy, X-RD, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM). The final average size of ZnONPs was obtained at 65.46 nm with diversified shapes and dimensions. The present study concluded the high capabilities of fungi (FiGMs) as eco-friendly and cheap bio-nano factories to manufacture ZnONPs with great nano-level average size, which may consider new boost sources for use in many nano-sectors and applications.

Prospection of Psychrotrophic Filamentous Fungi Isolated from the High Andean Paramo Region of Northern Ecuador: Enzymatic Activity and Molecular Identification.

The isolation of filamentous fungal strains from remote habitats with extreme climatic conditions has led to the discovery of a series of enzymes with attractive properties that can be useful in various industrial applications. Among these, cold-adapted enzymes from fungi with psychrotrophic lifestyles are valuable agents in industrial processes aiming towards energy reduction. Out of eight strains isolated from soil of the paramo highlands of Ecuador, three were selected for further experimentation and identified as Cladosporium michoacanense, Cladosporium sp. (cladosporioides complex), and Didymella sp., this last being reported for the first time in this area. The secretion of seven enzymes, namely, endoglucanase, exoglucanase, β-D-glucosidase, endo-1,4-β-xylanase, β-D-xylosidase, acid, and alkaline phosphatases, were analyzed under agitation and static conditions optimized for the growth period and incubation temperature. Cladosporium strains under agitation as well as incubation for 72 h mostly showed the substantial activation for endoglucanase reaching up to 4563 mU/mL and xylanase up to 3036 mU/mL. Meanwhile, other enzymatic levels varied enormously depending on growth and temperature. Didymella sp. showed the most robust activation at 8 °C for endoglucanase, β-D-glucosidase, and xylanase, indicating an interesting profile for applications such as bioremediation and wastewater treatment processes under cold climatic conditions.

A recombinant SARS-CoV-2 receptor-binding domain expressed in an engineered fungal strain of Thermothelomyces heterothallica induces a functional immune response in mice

Since the beginning of the COVID-19 pandemic, the development of effective vaccines against this pathogen has been a priority for the scientific community. Several strategies have been developed including vaccines based on recombinant viral protein fragments. The receptor-binding domain (RBD) in the S1 subunit of S protein has been considered one of the main targets of neutralizing antibodies. In this study we assess the potential of a vaccine formulation based on the recombinant RBD domain of SARS-CoV-2 expressed in the thermophilic filamentous fungal strain Thermothelomyces heterothallica and the hepatitis B virus (HBV) core protein. Functional humoral and cellular immune responses were detected in mice. To our knowledge, this is the first report on the immune evaluation of a biomedical product obtained in the fungal strain T. heterothallica. These results together with the intrinsic advantages of this expression platform support its use for the development of biotechnology products for medical purpose.