Peroxide-Mediated Oxygenation of Organic Compounds by Fungal Peroxygenases

Unspecific peroxygenases (UPOs), whose sequences can be found in the genomes of thousands of filamentous fungi, many yeasts and certain fungus-like protists, are fascinating biocatalysts that transfer peroxide-borne oxygen (from H2O2 or R-OOH) with high efficiency to a wide range of organic substrates, including less or unactivated carbons and heteroatoms. A twice-proline-flanked cysteine (PCP motif) typically ligates the heme that forms the heart of the active site of UPOs and enables various types of relevant oxygenation reactions (hydroxylation, epoxidation, subsequent dealkylations, deacylation, or aromatization) together with less specific one-electron oxidations (e.g., phenoxy radical formation). In consequence, the substrate portfolio of a UPO enzyme always combines prototypical monooxygenase and peroxidase activities. Here, we briefly review nearly 20 years of peroxygenase research, considering basic mechanistic, molecular, phylogenetic, and biotechnological aspects.

A New Filter Based Cultivation Approach for Improving Aspergillus Identification using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct identification, ease of MSP creation, time consumption, cost and labour intensity. This method can be reliably applied for identification of clinically important Aspergilli and has potential for identification of other filamentous fungi.

Quantitative Monitoring of Mycelial Growth of Aspergillus fumigatus in Liquid Culture by Optical Density

Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi to apply the methods used for unicellular organisms such as yeast and bacteria.

Intron Retention Coupled with Nonsense-Mediated Decay is Involved in Cellulase Biosynthesis in Cellulolytic Fungi

Background Knowledge on regulatory networks associated with cellulase biosynthesis is prerequisite for exploitation of such regulatory systems in ehancing cellulase production with low cost. The biological functions of intron retention (IR) and nonsense-mediated mRNA decay (NMD) in filamentous fungi is lack of study, let alone their roles in cellulase biosynthesis. Result We found that major cellulase genes (cel7a, cel7b, and cel3a) exhibited concomitant decrease in IR rates and increase in their gene expression in T. reesei under cellulase-producing condition (cellulose and lactose) that was accompanied with a more active NMD pathway, as compared to non cellulase-producing condition (glucose). In the presence of the NMD pathway inhibitor that successfully repressed the NMD pathway, the mRNA levels of cellulase genes were sharply down-regulated, but the rates of IR in these genes were significantly up-regulated. Consistently, the cellulase activities were severely inhibited. In addition, the NMD pathway inhibitor caused the downregulated mRNA levels of two important genes of the target of rapamycin (TOR) pathway, trfkbp12 and trTOR1. The absence of gene trfkbp12 made the cellulase production in T. reesei more sensitive to the NMD pathway inhibitor. Conclusion All these findings suggest that the IR of cellulase genes regulates their own gene expression by coupling with the NMD pathway, which might involve the TOR pathway. Our results provide better understanding on intron retention, the NMD pathway, and cellulase production mechanism in filamentous fungi.

Yeast and Filamentous Fungi Microbial Communities in Organic Red Grape Juice: Effect of Vintage, Maturity Stage, SO2, and Bioprotection

Changes are currently being made to winemaking processes to reduce chemical inputs [particularly sulfur dioxide (SO2)] and adapt to consumer demand. In this study, yeast growth and fungal diversity were investigated in merlot during the prefermentary stages of a winemaking process without addition of SO2. Different factors were considered, in a two-year study: vintage, maturity level and bioprotection by the adding yeast as an alternative to SO2. The population of the target species was monitored by quantitative-PCR, and yeast and filamentous fungi diversity was determined by 18S rDNA metabarcoding. A gradual decrease of the α-diversity during the maceration process was highlighted. Maturity level played a significant role in yeast and fungal abundance, which was lower at advanced maturity, while vintage had a strong impact on Hanseniaspora spp. population level and abundance. The presence of SO2 altered the abundance of yeast and filamentous fungi, but not their nature. The absence of sulfiting led to an unexpected reduction in diversity compared to the presence of SO2, which might result from the occupation of the niche by certain dominant species, namely Hanseniaspora spp. Inoculation of the grape juice with non-Saccharomyces yeast resulted in a decrease in the abundance of filamentous fungi generally associated with a decline in grape must quality. Lower abundance and niche occupation by bioprotection agents were observed at the overripened stage, thus suggesting that doses applied should be reconsidered at advanced maturity. Our study confirmed the bioprotective role of Metschnikowia pulcherrima and Torulaspora delbrueckii in a context of vinification without sulfites.

Characterization of biodiversity of filamentous fungi in the soil of public spaces in João Pessoa, Northeast of Brazil

Os fungos são microrganismos eucarióticos ubíquos com presença de parede celular, sendo encontrados principalmente no solo, vegetais, água e no ar. Os fungos desempenham um papel importante no ciclo de decomposição da matéria orgânica. Ambientes públicos como praças e parques, destinados ao lazer da população, são locais onde também circula diariamente um grande número de pessoas e animais. Este estudo teve como objetivo avaliar a presença de fungos filamentosos no solo de praças e parques públicos da cidade de João Pessoa, Nordeste do Brasil. Amostras de solo de seis praças/parques foram coletadas e então processadas e cultivadas. Além disso, fungos queratinofílicos foram isolados usando cabelo estéril como substrato. Fungos filamentosos foram isolados de todas as amostras. O gênero mais frequente foi Aspergillus (100% das amostras), seguido por Trichophyton (66,7%) e Penicillium (33,3%). Aspergillus seção Nigri foi isolada em 66,7% das amostras, seguido por Aspergillus seção Flavi, Aspergillus seção Terrei e Trichophyton rubrum, que foram isoladas em 50% das amostras). Vários gêneros e espécies com potencial patogênico ao homem foram isolados de todos os pontos selecionados. Esses achados reforçam a importância do conhecimento da composição do solo dos espaços destinados ao uso público, contribuindo com informações à população, principalmente aos mais vulneráveis, quanto ao uso consciente desses ambientes para atividades recreativas.

Antifungal Susceptibility of Aspergillus flavus, Aspergillus ochraceus, and Fusarium graminearum to Ganoderma lucidum Extract

Background: Ganoderma lucidum is a well-known fungus that has been widely used in traditional medicine around the world, especially in East Asia, due to its various health promotion properties. Recently, researchers have drawn attention to the biologically active compounds found in this fungus, and this fungus has become very popular due to its pharmaceutical properties. Objectives: The aim of this study was to investigate the antifungal properties of the Iranian strain of G. lucidum as a natural antifungal agent against harmful filamentous fungi common in the food industry. Methods: Three filamentous fungi, including Aspergillus flavus, Aspergillus ochraceus, and Fusarium graminearum, were used in this study for the antifungal evaluation of ethanolic, hydroalcoholic, and two aqueous extracts of G. lucidum with different concentrations by the broth microdilution method. Results: The results showed that only the ethanolic and hydroalcoholic extracts completely inhibited the growth of A. flavus at 2 and 3.5 mg/mL, respectively. Also, no antifungal activity was observed for the aqueous extract for all the three studied fungi. In addition, A. flavus was found to be more sensitive to G. lucidum extracts compared to the two other studied fungi. Conclusions: The ethanolic extract of G. lucidum was effective on A. flavus and can be used as a natural antifungal agent to prevent the growth of this harmful filamentous fungus.

Aflatoxin and Disruption of Energy Metabolism

Aflatoxins constitute a cluster of mycotoxins that are derived from fungal metabolites and are produced from diverse fungi species, especially Aspergillus. They are a collection of closely linked heterocyclic compounds produced predominantly by two filamentous fungi, Aspergillus flavus and Aspergillus parasiticus. They are also known to cause severe health threats to humans and animals, thereby resulting to several complications like immunotoxicity, teratogenicity hepatotoxicity. Aflatoxins interfere with normal metabolic processes. This interference encompasses the regulatory processes that occur throughout the progression of energy metabolism. Thus, the effects of aflatoxins are seen in the inhibition of ATP generation, carbohydrate and lipid metabolism, mitochondrial structure and proteins synthesis. This chapter will focus on the mechanisms of aflatoxin-induced disruption of lipids, carbohydrates, and proteins metabolism, and how they affect the bioenergetic systems.