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Zygomycetous fungal infections pose an emerging medical threat among individuals with compromised immunity and metabolic abnormalities. Our pathophysiological understanding of these infections, particularly the role of fungal cell walls in growth and immune response, remains limited. Here we conducted multidimensional solid-state NMR analysis to examine cell walls in five Mucorales species, including key mucormycosis causative agents like Rhizopus and Mucor species. We show that the rigid core of the cell wall primarily comprises highly polymorphic chitin and chitosan, with minimal quantities of β-glucans linked to a specific chitin subtype. Chitosan emerges as a pivotal molecule preserving hydration and dynamics. Some proteins are entrapped within this semi-crystalline chitin/chitosan layer, stabilized by the sidechains of hydrophobic amino acid residues, and situated distantly from β-glucans. The mobile domain contains galactan- and mannan-based polysaccharides, along with polymeric α-fucoses. Treatment with the chitin synthase inhibitor nikkomycin removes the β-glucan-chitin/chitosan complex, leaving the other chitin and chitosan allomorphs untouched while simultaneously thickening and rigidifying the cell wall. These findings shed light on the organization of Mucorales cell walls and emphasize the necessity for a deeper understanding of the diverse families of chitin synthases and deacetylases as potential targets for novel antifungal therapies., (© 2024. The Author(s).)

DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF., (© 2024. The Author(s).)

During pulmonary mucormycosis, inhaled sporangiospores adhere to, germinate, and invade airway epithelial cells to establish infection. We provide evidence that HIF1α plays dual roles in airway epithelial cells during Mucorales infection. We observed an increase in HIF1α protein accumulation and increased expression of many known HIF1α-responsive genes during in vitro infection, indicating that HIF1α signaling is activated by Mucorales infection. Inhibition of HIF1α signaling led to a substantial decrease in the ability of R. delemar to invade cultured airway epithelial cells. Transcriptome analysis revealed that R. delemar infection induces the expression of many pro-inflammatory genes whose expression was significantly reduced by HIF1α inhibition. Importantly, pharmacological inhibition of HIF1α increased survival in a mouse model of pulmonary mucormycosis without reducing fungal burden. These results suggest that HIF1α plays two opposing roles during mucormycosis: one that facilitates the ability of Mucorales to invade the host cells and one that facilitates the ability of the host to mount an innate immune response., (© 2024. The Author(s).)

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO 4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO 4 and 5'-PO 4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO 4 terminus in the end-joining reaction, whereby the 2'-PO 4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO 4 installed by RNA ligase is removed by a dedicated NAD + -dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD + -dependent RNA 2'-phosphotransferase activity in vitro., (© 2024 Ghosh et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Some eukaryotic pre-tRNAs contain an intron that is removed by a dedicated set of enzymes. Intron-containing pre-tRNAs are cleaved by tRNA splicing endonuclease, followed by ligation of the two exons and release of the intron. Fungi use a "heal and seal" pathway that requires three distinct catalytic domains of the tRNA ligase enzyme, Trl1. In contrast, humans use a "direct ligation" pathway carried out by RTCB, an enzyme completely unrelated to Trl1. Because of these mechanistic differences, Trl1 has been proposed as a promising drug target for fungal infections. To validate Trl1 as a broad-spectrum drug target, we show that fungi from three different phyla contain Trl1 orthologs with all three domains. This includes the major invasive human fungal pathogens, and these proteins can each functionally replace yeast Trl1. In contrast, species from the order Mucorales, including the pathogens Rhizopus arrhizus and Mucor circinelloides , have an atypical Trl1 that contains the sealing domain but lacks both healing domains. Although these species contain fewer tRNA introns than other pathogenic fungi, they still require splicing to decode three of the 61 sense codons. These sealing-only Trl1 orthologs can functionally complement defects in the corresponding domain of yeast Trl1 and use a conserved catalytic lysine residue. We conclude that Mucorales use a sealing-only enzyme together with unidentified nonorthologous healing enzymes for their heal and seal pathway. This implies that drugs that target the sealing activity are more likely to be broader-spectrum antifungals than drugs that target the healing domains., (© 2024 Ahammed and van Hoof; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

CrgA has been shown to be a negative regulator of carotenogenesis in some filamentous fungi, while light irradiation is an inducible environmental factor for carotenoid biosynthesis. To clarify the relationship between CrgA and light-inducible carotenogenesis in Blakeslea trispora, the cis-acting elements of the btcrgA promoter region were investigated, followed by the analyses of correlation between the expression of btcrgA and carotenoid structural genes under different irradiation conditions. A variety of cis-acting elements associated with light response was observed in the promoter region of btcrgA, and transcription of btcrgA and carotenoid structural genes under different irradiation conditions was induced by white light with a clear correlation. Then, RNA interference and overexpression of btcrgA were performed to investigate their effects on carotenogenesis at different levels under irradiation and darkness. The analyses of transcription and enzyme activities of carotenoid structural gene, and accumulation of carotenoids among btcrgA-interfered, btcrgA-overexpressed, and wild-type strains under irradiation and darkness indicate that btcrgA negatively regulates the synthesis of carotenoid in darkness, while promotes the carotenogenesis under irradiation regardless of reduced or overexpression of btcrgA ., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Invasive fungal (IF) diseases are a leading global cause of mortality, particularly among immunocompromised individuals. The SARS-CoV-2 pandemic further exacerbated this scenario, intensifying comorbid IF infections such as mucormycoses of the nasopharynx. In the work reported here, it is shown that zygomycetes, significant contributors to mycoses, are sensitive to the natural product allicin. Inhibition of Mucorales fungi by allicin in solution and by allicin vapor was demonstrated. Mathematical modeling showed that the efficacy of allicin vapor is comparable to direct contact with the commercially available antifungal agent amphotericin B (ampB). Furthermore, the study revealed a synergistic interaction between allicin and the non-volatile ampB. The toxicity of allicin solution to human cell lines was evaluated and it was found that the half maximal effective concentration (EC 50 ) of allicin was 25-72 times higher in the cell lines as compared to the fungal spores. Fungal allicin sensitivity depends on the spore concentration, as demonstrated in a drop test. This study shows the potential of allicin, a sulfur-containing defense compound from garlic, to combat zygomycete fungi. The findings underscore allicin's promise for applications in infections of the nasopharynx via inhalation, suggesting a novel therapeutic avenue against challenging fungal infections.

Chromatin modifications play a fundamental role in controlling transcription and genome stability and yet despite their importance, are poorly understood in early-diverging fungi. We present a comprehensive study of histone lysine and DNA methyltransferases across the Mucoromycota, emphasizing heterochromatin formation pathways that rely on the Clr4 complex involved in H3K9-methylation, the Polycomb-repressive complex 2 driving H3K27-methylation, or DNMT1-like methyltransferases that catalyze 5mC DNA methylation. Our analysis uncovered H3K9-methylated heterochromatin as the major chromatin modification repressing transcription in these fungi, which lack both Polycomb silencing and cytosine methylation. Although small RNAs generated by RNA interference (RNAi) pathways facilitate the formation of heterochromatin in many eukaryotic organisms, we show that RNAi is not required to maintain either genomic or centromeric heterochromatin in Mucor . H3K9-methylation and RNAi act independently to control centromeric regions, suggesting a functional subspecialization. Whereas the H3K9 methyltransferase Clr4 and heterochromatin formation are essential for cell viability, RNAi is dispensable for viability yet acts as the main epigenetic, regulatory force repressing transposition of centromeric GremLINE1 elements. Mutations inactivating canonical RNAi lead to rampant transposition and insertional inactivation of targets resulting in antimicrobial drug resistance. This fine-tuned, Rdrp2-dependent RNAi activity is critical for genome stability, restricting GremLINE1 retroelements to the centromeres where they occupy long heterochromatic islands. Taken together, our results suggest that RNAi and heterochromatin formation are independent genome defense and regulatory mechanisms in the Mucorales, contributing to a paradigm shift from the cotranscriptional gene silencing observed in fission yeasts to models in which heterochromatin and RNAi operate independently in early-diverging fungi.

Background: Trisporic acids are considered to be key regulators of carotenoid biosynthesis and sexual reproduction in zygomycetes, but the mechanisms underlying this regulation have not been fully elucidated., Results: In this study, the relationships between trisporic acids and lycopene synthesis were investigated in Blakeslea trispora. The lycopene concentration in single fermentation by the (-) strain with the addition of 24 μg/L trisporic acids was slightly higher than that observed in mated fermentation. After transcriptomic analysis, a steroid 5α-reductase-like gene, known as SR5AL in B. trispora, was first reported. 5α-Reductase inhibitors reduced lycopene biosynthesis and downregulated the expression of sex determination and carotenoid biosynthesis genes. Overexpression of the SR5AL gene upregulated these genes, regardless of whether trisporic acids were added., Conclusion: These findings indicated that the SR5AL gene is a key gene associated with the response to trisporic acids., (© 2022. The Author(s).)

In this study, the biotransformation of ursolic acid by Circinella muscae CGMCC 3.2695 was investigated. Scaled-up biotransformation reactions yielded ten metabolites. Their structures were established based on extensive NMR and HR-ESI-MS data analyses, and four of them are new compounds. C . muscae could selectively catalyze hydroxylation, lactonisation, carbonylation and carboxyl reduction reactions. Furthermore, all the identified metabolites were evaluated for their anti-neuroinflammatory activities in LPS-induced BV-2 cells. Most metabolites displayed pronounced inhibitory effect on nitric oxide (NO) production. The results suggested that biotransformed derivatives of ursolic acid might be served as potential neuroinflammatory inhibitors.

The Mucorales fungal genus Rhizopus is used for the industrial production of organic acids, enzymes and fermented foods. The metabolic engineering efficiency of Rhizopus could be improved using gene manipulation; however, exogenous DNA rarely integrates into the host genome. Consequently, a genetic tool for Mucorales fungi needs to be developed. Recently, programmable nucleases that generate DNA double-strand breaks (DSBs) at specific genomic loci have been used for genome editing in various organisms. In this study, we examined gene disruption in Rhizopus oryzae using transcription activator-like effector nucleases (TALENs), with and without exonuclease overexpression. TALENs with an overexpressing exonuclease induced DSBs, followed by target site deletions. Although DSBs are repaired mainly by nonhomologous end joining in most organisms, our results suggested that in R. oryzae microhomology-mediated end joining was the major DSB repair system. Our gene manipulation method using TALENs coupled with exonuclease overexpression contributes to basic scientific knowledge and the metabolic engineering of Rhizopus., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N -substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica , and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates ( Aspergillus spp., Candida spp., Mucormycetes ) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl- N -dodecylpiperidin-4-amine and N -dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella . Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.

Mucormycosis is a deadly infection which is caused by fungi of the order Mucorales including species belonging to the genus Rhizopus, Mucor, Mycocladus, Rhizomucor, Cunninghamella, and Apophysomyces. Despite antifungal therapy and surgical procedures, the mortality rate of this disease is about 90-100% which is exceptionally high. The hypersensitivity of patients with raised available serum iron indicates that the Mucorales are able to use host iron as a critical factor of virulence. This is because iron happens to be a crucial element playing its role in the growth of cells and development. In this review, we have described Lactoferrin (Lf) as a potential iron-chelator. Lf is a naturally occurring glycoprotein which is expressed in most of the biological fluids. Moreover, Lf possesses exclusive anti-inflammatory effects along with several anti-fungal effects that could prove to be helpful to the pathological physiology of inexorable mucormycosis cases. This literature summarises the biological insights into the Lf being considered as a potential fungistatic agent and an immune regulator. The review also proposes that unique potential of Lf as an iron-chelator can be exploited as the adjunct treatment for mucormycosis infection., (Copyright © 2021. Published by Elsevier B.V.)

Fermented soybean products have attracted great attention due to their health benefits. In the present study, the hypoxia-injured PC12 cells induced by cobalt chloride (CoCl 2 ) were used to evaluate the neuroprotective potency of tofu fermented by Actinomucor elegans (FT). Results indicated that FT exhibited higher phenolic content and antioxidant activity than tofu. Moreover, most soybean isoflavone glycosides were hydrolyzed into their corresponding aglycones during fermentation. FT demonstrated a significant protective effect on PC12 cells against hypoxic injury by maintaining cell viability, reducing lactic dehydrogenase leakage, and inhibiting oxidative stress. The cell apoptosis was significantly attenuated by the FT through down-regulation of caspase-3, caspases-8, caspase-9, and Bax, and up-regulation of Bcl-2 and Bcl-xL. S-phase cell arrest was significantly inhibited by the FT through increasing cyclin A and decreasing the p21 protein level. Furthermore, treatment with the FT activated autophagy, indicating that autophagy possibly acted as a survival mechanism against CoCl 2 -induced injury. Overall, FT offered a potential protective effect on nerve cells in vitro against hypoxic damage.

Blakeslea trispora, a heterothallic Zygomycota with two mating types (termed "plus" and "minus"), is an ideal source of lycopene and β-carotene. The lycopene and β-carotene yields when the two type strains are used for fermentation separately are lower than those when they are joint together. To enhance the yield of lycopene and β-carotene in B. trispora, protoplast fusion technology was carried out between ATCC 14,271 (+) and ATCC 14,272 (-). After protoplast preparation, protoplast fusion, fusion sorting, fusion regeneration, and high-throughput screening, two fusions (Fu-1and Fu-2) with high lycopene and β-carotene yields were obtained. The lycopene yields of Fu-1 and Fu-2 were increased to 0.60 mg/gDW and 0.90 mg/gDW, which were respectively 3.62- and 5.44-fold those of 14,271 and 1.76- and 2.64-fold those of 14,272. The β-carotene yields of Fu-1 and Fu-2 were increased to 22.07 mg/gDW and 36.93 mg/gDW, which were respectively 1.72- and 2.89-fold those of 14,271 and 1.23- and 2.06-fold those of 14,272. In this study, the protoplast fusion technique was successfully used in Blakeslea trispora, providing new ideas for improving lycopene and β-carotene production.

Background: It has been reported that the genes coding for NADP-dependent glutamate dehydrogenases (NADP-GDHs) showed a cause-effect relationship with Yeast-Hypha (YH) reversible transition in a zygomycete Benjaminiella poitrasii. As YH transition is significant in human pathogenic fungi for their survival and proliferation in the host, the NADP-GDHs can be explored as antifungal drug targets., Methods: The yeast-form specific BpNADPGDH I and hyphal-form specific BpNADPGDH II of B. poitrasii were purified by heterologous expression in E. coli BL-21 cells and characterized. The structural analogs of L-glutamate, dimethyl esters of isophthalic acid (DMIP) and its derivatives were designed, synthesized and screened for inhibition of NADP-GDH activity as well as YH transition in B. poitrasii, and also in human pathogenic Candida albicans strains., Results: The BpNADPGDH I and BpNADPGDH II were found to be homo-hexameric proteins with native molecular mass of 282 kDa and 298 kDa, respectively and subunit molecular weights of 47 kDa and 49 kDa, respectively. Besides the distinct kinetic properties, BpNADPGDH I and BpNADPGDH II were found to be regulated by cAMP-dependent- and Calmodulin (CaM) dependent- protein kinases, respectively. The DMIP compounds showed a more pronounced effect on H-form specific BpNADPGDH II and inhibited YH transition as well as growth in B. poitrasii and C. albicans strains., Conclusion: The present study will be useful to design and develop antifungal drugs against dimorphic human pathogens using glutamate dehydrogenase as a target., Significance: Glutamate dehydrogenases can be explored as a target against human pathogenic fungi., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest with the content of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Iron is a key transition metal required by most microorganisms and is prominently utilised in the transfer of electrons during metabolic reactions. The acquisition of iron is essential and becomes a crucial pathogenic event for opportunistic fungi. Iron is not readily available in the natural environment as it exists in its insoluble ferric form, i.e., in oxides and hydroxides. During infection, the host iron is bound to proteins such as transferrin, ferritin, and haemoglobin. As such, access to iron is one of the major hurdles that fungal pathogens must overcome in an immunocompromised host. Thus, these opportunistic fungi utilise three major iron acquisition systems to overcome this limiting factor for growth and proliferation. To date, numerous iron acquisition pathways have been fully characterised, with key components of these systems having major roles in virulence. Most recently, proteins involved in these pathways have been linked to the development of antifungal resistance. Here, we provide a detailed review of our current knowledge of iron acquisition in opportunistic fungi, and the role iron may have on the development of resistance to antifungals with emphasis on species of the fungal basal lineage order Mucorales, the causative agents of mucormycosis.

The bacterial protease inhibitor domains known as Streptomyces subtilisin inhibitors (SSI) are rarely found in fungi. Genome analysis of a fungal pathogen, Choanephora cucurbitarum KUS-F28377, revealed 11 SSI-like domains that are horizontally transferred and sequentially diverged during evolution. We investigated the molecular function of fungal SSI-like domains of C. cucurbitarum , designated "choanepins." Among the proteins tested, only choanepin9 showed inhibitory activity against subtilisin as the target protease, accounting for 47% of the inhibitory activity of bacterial SSI. However, the binding affinity (expressed as the dissociation constant [ K d ]) of choanepin9 measured via microscale thermophoresis was 21 nM, whereas that for bacterial SSI is 34 nM. The trend of binding and inhibitory activity suggests that the two inhibitors exhibit different inhibitory mechanisms for subtilisin protease. Interestingly, choanepin9 was identified as a monomer in studies in vitro , whereas bacterial SSI is a homodimer. Based on these observations, we constructed a monomeric bacterial SSI protein with decreased binding affinity to abrogate its inhibitory activity. By altering the reactive sites of choanepin9 deduced from the P1 and P4 sites of bacterial SSI, we reestablished that these residues in choanepins are also crucial for modulating inhibitory activity. These findings suggest that the fungal SSI evolved to target specific cognate proteases by altering the residues involved in inhibitory reactivity (reactive sites) and binding affinity (structural integrity). The function of fungal SSI proteins identified in this study provides not only a clue to fungal pathogenesis via protease inhibition but also a template for the design of novel serine protease inhibitors. IMPORTANCE Until recently, Streptomyces subtilisin inhibitors (SSI) were reported and characterized only in bacteria. We found SSI-like domains in a plant-pathogenic fungus, Choanephora cucurbitarum KUS-F28377, which contains 11 sequentially diverged SSI-like domains. None of these fungal SSI-like domains were functionally characterized before. The active form of fungal SSI-like protein is a monomer, in contrast to the homodimeric bacterial SSI. We constructed a synthetic monomer of bacterial SSI to demonstrate the modulation of its activity based on structural integrity and not reactive sites. Our results suggest the duplication and divergence of SSI-like domains of C. cucurbitarum within the genome to inhibit various cognate proteases during evolution by modulating both binding and reactivity. The molecular functional characterization of fungal SSI-like domains will be useful in understanding their biological role and future biotechnological applications., (Copyright © 2020 American Society for Microbiology.)

Platelets are meanwhile recognized as versatile elements within the immune system and appear to play a key role in the innate immune response to pathogens including fungi. Previous experiments revealed platelet activation by direct contact with the hyphal-associated polysaccharide galactosaminogalactan (GAG). Since secreted fungal products may also be relevant and trigger immune reactions or thrombosis, we screened culture supernatants (SN) of human-pathogenic fungi for their capacity to activate platelets. For that purpose, platelets were incubated with SN from various fungal species; platelet activation and GAG deposition on the surface of platelets were detected by flow cytometry and electron and confocal microscopy, Culture supernatants of Aspergillus fumigatus and flavus isolates were potent platelet stimulators in a dose- and time-dependent manner, while SN of other Aspergillus species and all tested mucormycete species did not significantly induce platelet activation. The capacity of culture SN to activate platelets was dependent on fungal production of GAG and deposition of secreted GAG on the platelet surface; supernatants from mucormycetes or mutants of A. fumigatus lacking GAG secretion did not affect platelet activity. These results suggest that invading fungi can stimulate platelets not only locally through direct interactions with fungal hyphae, but can also act over a certain distance through secreted GAG., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Mucormycosis is an emergent, fatal fungal infection of humans and warm-blooded animals caused by species of the order Mucorales. Immune cells of the innate immune system serve as the first line of defence against inhaled spores. Alveolar macrophages were challenged with the mucoralean fungus Lichtheimia corymbifera and subjected to biotinylation and streptavidin enrichment procedures followed by LC-MS/MS analyses. A total of 28 host proteins enriched for binding to macrophage-L. corymbifera interaction. Among those, the HSP70-family protein Hspa8 was found to be predominantly responsive to living and heat-killed spores of a virulent and an attenuated strain of L. corymbifera. Confocal scanning laser microscopy of infected macrophages revealed colocalization of Hspa8 with phagocytosed spores of L. corymbifera. The amount of detectable Hspa8 was dependent on the multiplicity of infection. Incubation of alveolar macrophages with an anti-Hspa8 antibody prior to infection reduced their capability to phagocytose spores of L. corymbifera. In contrast, anti-Hspa8 antibodies did not abrogate the phagocytosis of Aspergillus fumigatus conidia by macrophages. These results suggest an important contribution of the heat-shock family protein Hspa8 in the recognition of spores of the mucoralean fungus L. corymbifera by host alveolar macrophages and define a potential immunomodulatory therapeutic target., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Microbial transformation of isorhodeasapogenin (1), the major steroidal sapogenin of Tupistra chinensis, was performed with the fungus Syncephalastrum racemosum (AS 3.264). As a result, nine new biotransformation metabolites (2-10) were isolated and their structures were elucidated by spectroscopic analysis. Hydroxylation, oxidation and glycosylation reactions were observed on the B, C, D and F rings of steroidal skeleton. Substrate (1) and its biotransformed metabolites 2-6, 8-10 were evaluated for their anti-neuroinflammatory effect on the NO accumulation induced by LPS in BV-2 cells. All the tested metabolites were found to have more potential anti-neuroinflammatory activity than the substrate. Especially, metabolites 2, 5 and 6 exhibited significant inhibition on NO production after hydroxylation at C-12 or C-15. Moreover, metabolite 2 dose-dependently reduced the LPS-induced protein expression of iNOS and COX-2., Competing Interests: Declaration of Competing Interest The authors have declared that there is no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

We investigated the influence of corn steep liquor (CSL) and cassava waste water (CWW) as carbon and nitrogen sources on the morphology and production of biomass and chitosan by Mucor subtilissimus UCP 1262 and Lichtheimia hyalospora UCP 1266. The highest biomass yields of 4.832 g/L ( M. subtilissimus UCP 1262) and 6.345 g/L ( L. hyalospora UCP 1266) were produced in assay 2 (6% CSL and 4% CWW), factorial design 2 2 , and also favored higher chitosan production (32.471 mg/g) for M. subtilissimus . The highest chitosan production (44.91 mg/g) by L. hyalospora (UCP 1266) was obtained at the central point (4% of CWW and 6% of CSL). The statistical analysis, the higher concentration of CSL, and lower concentration of CWW significantly contributed to the growth of the strains. The FTIR bands confirmed the deacetylation degree of 80.29% and 83.61% of the chitosan produced by M. subtilissimus (UCP 1262) and L. hyalospora (UCP 1266), respectively. M. subtilissimus (UCP 1262) showed dimorphism in assay 4-6% CSL and 8% CWW and central point. L. hyalospora (UCP 1266) was optimized using a central composite rotational design, and the highest yield of chitosan (63.18 mg/g) was obtained in medium containing 8.82% CSL and 7% CWW. The experimental data suggest that the use of CSL and CWW is a promising association to chitosan production.

Mycenarubin C, a previously unknown red pyrroloquinoline alkaloid, was isolated from fruiting bodies of the mushroom Mycena rosea and its structure was elucidated mainly by NMR spectroscopy and mass spectrometry. Unlike mycenarubin A, the major pyrroloquinoline alkaloid in fruiting bodies of M. rosea, mycenarubin C, contains an eight-membered ring with an additional C 1 unit that is hitherto unprecedented for pyrroloquinoline alkaloids known in nature. Incubation of mycenarubin A with an excess of formaldehyde revealed that mycenarubin C was generated nearly quantitatively from mycenarubin A. An investigation into the formaldehyde content of fresh fruiting bodies of M. rosea showed the presence of considerable amounts of formaldehyde, with values of 5 μg per gram of fresh weight in fresh fruiting bodies. Although mycenarubin C did not show bioactivity against selected bacteria and fungi, formaldehyde inhibits the growth of the mycoparasite Spinellus fusiger at concentrations present in fruiting bodies of M. rosea. Therefore, formaldehyde might play an ecological role in the chemical defence of M. rosea against S. fusiger. In turn, S. fusiger produces gallic acid-presumably to detoxify formaldehyde by reaction of this aldehyde with amino acids and gallic acid to Mannich adducts., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The effects of switching morphology and replacing supplementary nutrients with fungal extract (5 and 10 g/L) on the production of major metabolites and chitosan by Mucor rouxii were investigated. This approach was supposed to promote sustainability of the fermentation process and improve its economic feasibility. Different fungal morphologies, i.e., purely filamentous (PF), purely yeast-like (PY), mostly filamentous (MF), and mostly yeast-like (MY), were evaluated. The highest ethanol yields were obtained from the media supplemented with 10 g/L fungal extract for all morphologies, while adding nutrient salts did not make any improvements in these yields, except a slight decrease in the fermentation time. Except for PF morphology, the replacement of yeast extract favored the biomass production yields. Moreover, the alkali insoluble material (AIM) yields were higher as a result of the replacement for most cases. Furthermore, the replacement resulted in increased glucosamine and decreased N-acetyl-glucosamine content of AIM for almost all the morphologies. AIM yields of at least 0.25 g/g-glucose and maximum chitin/chitosan yield of 0.78 g/g-AIM were obtained from the solids remaining after autolysis process, which were higher than that obtained from the raw biomass. The maximum yield of 0.135 g/g-AIM purified chitosan with intact molecular weight was obtained from the biomass with PF morphology supplemented with 10 g/L fungal extract plus nutrients.

The effects of different mucor strains (Mucor racemosus, Actinomucor, and Mucor wutungkiao) on aroma and taste profiles based on proteolysis, lipolysis, and their catabolism in oil furu were studied. Gas chromatography-mass spectrometry and relative odor activity were used to monitor the changes of key volatile compounds and the differences in the characteristic aroma contents of oil furu. Using principal component analysis, the different fermentation strains had an effect on aroma profiles. The volatile compounds from metabolism of protein and fatty acid contributed to the aroma of oil furu with different contribution from the different strains, presumably due to their different enzymes. The electronic tongue and free amino acid profiles also showed strain differences of taste. Based on these results, optimization of the amount of each of the different mucor strains during cofermentation might achieve better flavor., (© 2020 Institute of Food Technologists®.)

The fungi in order Mortierellales are attractive producers for long-chain polyunsaturated fatty acids (PUFAs). Here, the genome sequencing and assembly of a novel strain of Mortierella sp. BCC40632 were done, yielding 65 contigs spanning of 49,964,116 total bases with predicted 12,149 protein-coding genes. We focused on the acetyl-CoA in relevant to its derived metabolic pathways for biosynthesis of macromolecules with biological functions, including PUFAs, eicosanoids and carotenoids. By comparative genome analysis between Mortierellales and Mucorales, the signature genetic characteristics of the arachidonic acid-producing strains, including Δ 5 -desaturase and GLELO-like elongase, were also identified in the strain BCC40632. Remarkably, this fungal strain contained only n-6 pathway of PUFA biosynthesis due to the absence of Δ 15 -desaturase or ω3-desaturase gene in contrast to other Mortierella species. Four putative enzyme sequences in the eicosanoid biosynthetic pathways were identified in the strain BCC40632 and others Mortierellale fungi, but were not detected in the Mucorales. Another unique metabolic trait of the Mortierellales was the inability in carotenoid synthesis as a result of the lack of phytoene synthase and phytoene desaturase genes. The findings provide a perspective in strain optimization for production of tailored-made products with industrial applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a , btwc-1b , and btwc-1c , in B. trispora Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a , btwc-1b , and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trispora IMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora , a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora ., (Copyright © 2020 American Society for Microbiology.)

The order Mucorales is a group of ancient fungi with limited tools for gene manipulation. The main consequence of this manipulation unwillingness is the limited knowledge about its biology compared to other fungal groups. However, the emerging of mucormycosis, a fungal infection caused by Mucorales, is attracting the medical spotlight in recent years because the treatments available are not efficient in reducing the high mortality associated with this disease. The result of this renewed interest in Mucorales and mucormycosis is an extraordinarily productive effort to unveil their secrets during the last decade. In this review, we describe the most compelling advances related to the genetic study of virulence factors, pathways, and molecular mechanisms developed in these years. The use of a few genetic study models has allowed the characterization of virulence factors in Mucorales that were previously described in other pathogens, such as the uptake iron systems, the mechanisms of dimorphism, and azole resistances. More importantly, recent studies are identifying new genes and mechanisms controlling the pathogenic potential of Mucorales and their interactions with the host, offering new alternatives to develop specific strategies against mucormycosis., Competing Interests: The authors declare no conflicts of interest.

As an ideal carotenoid producer, Blakeslea trispora has gained much attention due to its large biomass and high production of β-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified, as few investigations have been conducted at the molecular level in B. trispora In this study, a gene homologous to carotenogenesis regulatory gene ( crgA ) was cloned from the mating type (-) of B. trispora , and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the crgA -mediated regulation in B. trispora , we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora In contrast to the wild-type strain, the crgA null mutant (Δ crgA ) was defective in sporulation but accumulated much more β-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes ( hmgR , carB , and carRA ) for carotenoids throughout the culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids. IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids on a large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the present study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis, and other anabolic phenotypes. This will lead to a better understanding of the global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids., (Copyright © 2020 American Society for Microbiology.)

Blakeslea trispora is a natural source of carotenoids, including β-carotene and lycopene, which have industrial applications. Therefore, classical selective breeding techniques have been applied to generate strains with increased productivity, and microencapsulated β-carotene preparation has been used in food industry (Li et al., 2019). In B. trispora, lycopene is synthesized via the mevalonate pathway (Venkateshwaran et al., 2015). Lycopene cyclase, which is one of the key enzymes in this pathway, is a bifunctional enzyme that can catalyze the cyclization of lycopene to produce β-carotene and exhibit phytoene synthase activity (He et al., 2017).

In this study, the biotransformation of progesterone (1) and testosterone enanthate (5) using the whole cells of Circinella muscae was investigated for the first time. Microbial transformation of 1 with C. muscae afforded three known metabolites including 9α-hydroxyprogesterone (2), 14α-hydroxyprogesterone (3) and 6β,14α dihydroxyprogesterone (4) after 6 days of incubation at 26 °C. The biotransformation of 5 with C. muscae yielded a new metabolite; 8β,14α-dihydroxytestosterone (8), in addition to two known metabolites; 6β-hydroxytestosterone (6), and 9α-hydroxytestosterone (7). The structure of the metabolites were established on the basis of spectroscopic data., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Most mucoralean fungi are common soil saprotrophs and were probably among the first land colonisers. Although Mucoromycotina representatives grow well on simple sugar media and are thought to be unable to assimilate more complex organic compounds, they are often isolated from plant substrates. The main goal of the study was to explore the effects of isolation origin and phylogenetic placement on the carbon assimilation capacities of a large group of saprotrophic Mucoromycotina representatives (i.e. Umbelopsidales and Mucorales). Fifty two strains representing different Mucoromycotina families and isolated from different substrates were tested for their capacity to grow on 99 different carbon sources using the Biolog phenotypic microarray system and agar plates containing selected biopolymers (i.e. cellulose, xylan, pectin, and starch) as a sole carbon source. Although our results did not reveal a correlation between phylogenetic distance and carbon assimilation capacities, we observed 20 significant differences in growth capacity on specific carbon sources between representatives of different families. Our results also suggest that isolation origin cannot be considered as a main predictor of the carbon assimilation capacities of a particular strain. We conclude that saprotrophic Mucoromycotina representatives are, contrary to common belief, metabolically versatile and able to use a wide variety of carbon sources.

Biotransformation of oleanolic acid (OA) by Circinella muscae AS 3.2695 was investigated. Nine hydroxylated and glycosylated metabolites ( 1-9 ) were obtained. Their structures were elucidated as 3β,7β-dihydroxyolean-12-en-28-oic acid ( 1 ), 3β,7β,21β-trihydroxyolean-12-en-28-oic acid ( 2 ), 3β,7α,21β-trihydroxyolean-12-en- 28-oic acid ( 3 ), 3β,7β,15α-trihydroxyolean-12-en-28-oic acid ( 4 ), 7β,15α-dihydroxy- 3-oxo-olean-12-en-28-oic acid ( 5 ), 7β-hydroxy-3-oxo-olean-12-en-28-oic acid ( 6 ), oleanolic acid-28-O-β-D-glucopyranosyl ester ( 7 ), 3β,21β-dihydroxyolean-12-en-28- oic acid-28-O-β-D-glucopyranosyl ester ( 8 ), and 3β,7β,15α-trihydroxyolean-12-en- 28-oic acid-28-O-β-D-glucopyranosyl ester ( 9 ) by spectroscopic analysis. Among them, compounds 4 and 9 were new compounds. In addition, anti-inflammatory activities were assayed and evaluated for the isolated metabolites. Most of the metabolites exhibited significant inhibitory activities on lipopolysaccharides-induced NO production in RAW 264.7 cells.

In this study, the biocatalysis of 18 β -glycyrrhetinic acid by two strains of filamentous fungi, namely Rhizopus arrhizus AS 3.2893 and Circinella muscae AS 3.2695, was investigated. Scaled-up biotransformation reactions yielded 14 metabolites. Their structures were established based on extensive nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry data analyses, and seven of them are new compounds. The two fungal strains exhibited distinct biocatalytic features. R. arrhizus could catalyze hydroxylation and carbonylation reactions, whereas C. muscae preferred to catalyze hydroxylation and glycosidation reactions. These highly specific reactions are difficult to achieve by chemical synthesis, particularly under mild conditions. Furthermore, we found that most of the metabolites exhibited pronounced inhibitory activities on lipopolysaccharides-induced nitric oxide production in RAW264.7 cells. These biotransformed derivatives of 18 β -glycyrrhetinic acid could be potential anti-inflammatory agents., Competing Interests: The authors declare no conflicts of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

The effects of different fermentation strains on the flavor characteristics of fermented soybean curd (FSC) were investigated in this study. Fresh tofu was fermented by Actinomucor elegans, Rhizopus arrhizus, Mucor racemosus, and Rhizopus chinensis, either alone or in various combinations. The FSC manufacturing process included prefermentation by different strains at 28 °C for 60 hr, followed by salting at 16 °C for 7 days and finally proceeding postfermentation at 25 °C for 35 days. Subsequently, five tested samples were obtained, namely, sample A (fermented by A. elegans alone), R (fermented by R. arrhizus alone), AR (fermented by A. elegans and R. arrhizus at 5:1), AM (fermented by A. elegans and M. racemosus at 1:1), and RR (fermented by R. arrhizus and R. chinensis at 7:3). The flavors of the five samples were determined by E-nose, sensory evaluation, and GC-MS. E-nose system observed significant discriminations by principal component analysis and linear discriminant analysis analysis. Sensory evaluation ranked the overall sensory scores: AR>AM>A>RR>R. As shown in GC-MS results, sample AR also had, on average, the highest level of many volatiles. Out of 10 critical volatiles, the detected frequency of samples AR, AM, RR, A, and R was 10, 9, 9, 8, and 7, respectively. PLS2 regression model was used to explore the influence on flavor quality of different strains. All three analytic methods revealed similar results, with sample AR providing the best flavor quality, while the opposite was the case with sample R. Therefore, it could be concluded that A. elegans and R. arrhizus at 5:1 (v/v) was the optimal combination, and may likely promote the production of critical volatile compounds. PRACTICAL APPLICATION: The flavors of fermented soybean curds are influenced by various factors such as physicochemical and microorganism during the fermentation surroundings. The results of this work not only provide valuable information for FSC flavor studies, but can also guide the FSC industry to improve flavor quality by applying the most appropriate production strains., (© 2019 Institute of Food Technologists®.)

When sucrose was used as the carbon source, the Basidiomycete Coprinopsis cinerea showed poor growth and low laccase activity in pure culture, but greatly enhanced the level of laccase activity (>1800 U/L) during coculture with the Mucoromycete Gongronella sp. w5. As a result, the mechanism of laccase overproduction in coculture was investigated by starting from clarifying the function of sucrose. Results demonstrated that Gongronella sp. w5 in the coculture system hydrolyzed sucrose to glucose and fructose by an intracellular invertase. Fructose rather than glucose was supplied by Gongronella sp. w5  as the readily available carbon source for C. cinerea, and contributed to an alteration of its growth behavior and a basal laccase secretion of 110.6 ± 3.3 U/L. On the other hand, separating Gongronella sp. w5 of C. cinerea by transfer into dialysis tubes yielded the same level of laccase activity as without separation, indicating that enhanced laccase production probably resulted from the metabolites in the fermentation broth. Further investigation showed that the ethyl acetate-extracted metabolites generated by Gongronella sp. w5 induced C. cinerea laccase production. One of the laccase-inducing compounds namely p-hydroxybenzoic acid (HBA) was purified and identified from the extract. When using HBA as the inducer and fructose as the carbon source in monoculture, C. cinerea observed similar high laccase activity to that in coculture, and zymograms revealed the same expression of laccase Lcc9 as the main and Lcc1 and Lcc5 as the minor enzymes. Overall, our experiments verified that Gongronella sp. w5 elevates Coprinopsis cinerea laccase production by carbon source syntrophism and secondary metabolite induction.

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5-4.5 and at 70 °C. The enzyme exhibited high thermostability, for 1 h, up to 60 °C, and good tolerance to glucose (10 mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0 U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH 4 ) 2 SO 4 solution at pH 5.5-6.0 and fungus incubated at 40 °C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.

Introduction: Fungal infection burden related to Mucorales has been on the rise with significant associated morbidity and mortality. The major obstacle in the management has been lack of a non-invasive rapid and a reliable diagnostic test. Developing a culture-independent biomarker for the early diagnosis of mucormycosis is a major unmet need in modern mycology. Several approaches have been developed, such as immunohistochemistry (IHC) that can confirm the histopathologic diagnosis of the invasive mold infection, polymerase chain reaction (PCR) on formalin-fixed paraffin-embedded (FFPE) or fresh tissue, body fluids such as bronchoalveolar fluid (BAL), and detection directly from serum/blood. Serologic tests, matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), metabolomics and metagenomic shotgun sequencing are other evolving technologies. Area covered: In this review paper, we report the current status of the molecular diagnostics in the diagnosis of mucormycosis: serologic tests, IHC, PCR, protein-based with MALDI-TOF, metabolomics and metagenomic sequencing. Expert commentary: This review will conclude with an expert commentary on the potential uses/challenges of the currently available tests and the future of molecular diagnostics for mucormycosis.

Monitoring antifungal susceptibility patterns for new and established antifungal agents seems prudent given the increasing prevalence of uncommon species associated with higher antifungal resistance. We evaluated the activity of isavuconazole against 4,856 invasive yeasts and molds collected worldwide. The 4,856 clinical fungal isolates, including 2,351 Candida species isolates, 97 non- Candida yeasts, 1,972 Aspergillus species isolates, and 361 non- Aspergillus molds, including 292 Mucorales isolates collected in 2015 to 2016, were tested using CLSI methods. The MIC values for isavuconazole versus Aspergillus ranged from 0.06 to ≥16 μg/ml. The modal MIC for isavuconazole was 0.5 μg/ml (range, 0.25 [ A. nidulans and A. terreus species complex] to 4 μg/ml [ A. calidoustus and A. tubingensis ]). Eight A. fumigatus isolates had elevated isavuconazole MIC values at ≥8 μg/ml (non-wild type). Isavuconazole showed comparable activity to itraconazole against the Mucorales The lowest modal isavuconazole MIC values were seen for Rhizopus spp., R. arrhizus var. arrhizus , and R. microsporus (all 1 μg/ml). Candida species isolates were inhibited by ≤0.25 μg/ml of isavuconazole (range, 96.1% [ C. lusitaniae ] to 100.0% [ C. albicans , C. dubliniensis , C. kefyr , and C. orthopsilosis ]). MIC values were ≤1 μg/ml for 95.5% of C. glabrata isolates and 100.0% of C. krusei isolates. Isavuconazole was active against the non- Candida yeasts, including Cryptococcus neoformans (100.0% at ≤0.5 μg/ml). Isavuconazole exhibited excellent activity against most species of Candida and Aspergillus Isavuconazole was comparable to posaconazole and voriconazole against the less common yeasts and molds. Isavuconazole was generally less active than posaconazole and more active than voriconazole against the 292 Mucorales isolates. We confirm the potentially useful activity of isavuconazole against species of Rhizopus as determined by CLSI methods., (Copyright © 2018 Pfaller et al.)

Trachyloban-19-oic acid (1) is a diterpene very abundant in nature and its structural modification can furnish new bioactive compounds. Biotransformation of 1 by fungus Syncephalastrum racemosum provided three derivatives, two hydroxylated products (2-3) and one product of rearrangement (4). Products 3 and 4 have never been reported so far, to the best of our knowledge. Structure of 3 was formed after oxidation and rearrangement of compound 2. Compounds 1-4 were evaluated for inhibition of acetylcholinesterase, enzyme linked to the symptomatic control of Alzheimer's disease. All the compounds presented inhibitory activity higher than starting material 1, and product 3 presented IC 50  = 0.06 μM, which is about six times higher than activity found for galanthamine (IC 50  = 0.38 μM), the positive control used in this assay., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Horizontal gene transfer (HGT) can promote evolutionary adaptation by transforming a species' relationship to the environment. In most well-understood cases of HGT, acquired and donor functions appear to remain closely related. Thus, the degree to which HGT can lead to evolutionary novelties remains unclear. Mucorales fungi sense gravity through the sedimentation of vacuolar protein crystals. Here, we identify the octahedral crystal matrix protein (OCTIN). Phylogenetic analysis strongly supports acquisition of octin by HGT from bacteria. A bacterial OCTIN forms high-order periplasmic oligomers, and inter-molecular disulphide bonds are formed by both fungal and bacterial OCTINs, suggesting that they share elements of a conserved assembly mechanism. However, estimated sedimentation velocities preclude a gravity-sensing function for the bacterial structures. Together, our data suggest that HGT from bacteria into the Mucorales allowed a dramatic increase in assembly scale and emergence of the gravity-sensing function. We conclude that HGT can lead to evolutionary novelties that emerge depending on the physiological and cellular context of protein assembly.

This study evaluated corn steep liquor (CSL) and papaya peel juice (PPJ) in mixture as substrates for the cultivation (96h, 28°C, pH 5.6, 150rpm) of Mucorales fungi for chitosan production, and determined the growth-inhibitory effect of the fungal chitosan (FuCS) obtained under optimized conditions against phytopathogenic Colletotrichum species. All Mucorales fungi tested were capable of growing in CSL-PPJ medium, showing FuCS production in the range of 5.02 (Fennelomyces heterothalicus SIS 28) - 15.63mg/g (Cunninghamella elegans SIS 41). Highest FuCS production (37.25mg/g) was achieved when C. elegans was cultivated in medium containing 9.43% CSL and 42.5% PPJ. FuCS obtained under these conditions showed a deacetylation degree of 86%, viscosity of 120cP and molecular weight of 4.08×10 4 g/mol. FuCS at 5000, 7500 and 10,000ppm inhibited the growth of all Colletotrichum species tested. FuCS also induced alterations in the morphology of C. fructicola hyphae. CSL-PPJ mixtures are suitable substrates for the cultivation of Mucorales fungi for FuCS production. Chitosan from C. elegans cultivated in CSL-PPJ medium is effective in inhibiting phytopathogenic Colletotrichum species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mucoralean fungi are suitable microorganisms for the sustainable production of food, fodder, and fuels from inexpensive natural resources. Ethanol-producing Mucorales are particularly advantageous for second-generation ethanol production in comparison to the conventional ethanolic yeasts and bacteria. They are able to ferment a wide range of sugars to a range of valuable products, while they are typically resistance against the inhibitors available in different substrates, including untreated lignocellulosic hydrolysates. In addition to a high ethanol yield, the fungi produce several commercially valuable by-products, including chitosan, microbial oil (mainly polyunsaturated fatty acids), and protein. Moreover, the fungal extracts can replace the expensive nutrients required in fermentation. Besides, their morphologies can be altered from filamentous to yeast like and are adjustable based on the process requirement. The focus of this review is on applying Mucorales in producing ethanol and the biomass by-products thereof.

Lycopene is a carotenoid mainly present in red-colored fruits and vegetables. Its value in the pharmaceutical and food industry is linked to its benefits for the human health, including properties against cancer and cardiovascular diseases, and its use as a food colorant. Lycopene can be produced either by synthetic or natural means, but there is a preference for the second, since it is considered a more eco-friendly and less harmful process. Among natural methods for obtaining lycopene, microbial fermentation is a good alternative to extraction from plants that naturally contain lycopene, since it implies obtaining higher and more specific amounts of this carotenoid. This chapter describes lycopene production by fermentation of the fungus Blakeslea trispora, a naturally carotenoid producer, at 30 L scale. This procedure involves separated growth of the two sexual mating types of B. trispora during the vegetative stages and the use of a lycopene cyclase inhibitor to achieve lycopene accumulation during the production stage.

In this study, the degradation of metalaxyl was investigated in the presence of two Mucorales strains, previously isolated from soil subjected to repeated treatments with this fungicide and selected after enrichment technique. Fungal strains were characterised by a polyphasic approach using phylogenetic analysis of the Internal Transcribed Spacer (ITS) gene region, phenotypic characterisation by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) spectral analysis, and growth kinetics experiments. The strains were identified as Gongronella sp. and Rhizopus oryzae . The fungal growth kinetics in liquid cultures containing metalaxyl fits with Haldane model. Under laboratory conditions, the ability of Gongronella sp. and R. oryzae cultures to degrade metalaxyl was evaluated in liquid cultures and soil experiments. Both species were able to: (a) use metalaxyl as the main carbon and energy source; and (b) degrade metalaxyl in polluted soils, with rates around 1.0 mg kg - ¹ d - ¹. This suggests these strains could degrade metalaxyl in soils contaminated with this fungicide., Competing Interests: The authors declare no conflict of interest.

Context: Plants harbor endophytes with potential bioactivity. Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae) is reported to possess antioxidant, anti-inflammatory and anticancer activities., Objective: The antifungal and antiproliferative properties of endophytic fungi extracts and fractions from M. tomentosa were evaluated., Material and Methods: Endophytic fungi were isolated from the leaves of M. tomentosa and identified by ITS-rDNA sequence analysis. The antagonistic effect of the fungal strains was investigated against pathogenic fungi viz, Fusarium oxysporum, Sclerotinia sclerotiorium, Rhizoctonia solani, and Botrytis cinerea using the dual culture assay for 5-7 days. Antiproliferative effect of the fungal extracts and fractions (3.91-250 μg/mL) on HeLa cancer cell line was tested and IC 50 was calculated. Poisoning food assay and antifeedant activity against the pathogenic fungi and Spodoptera litura larvae, for 7 days and 2 h, respectively, was also tested at concentrations of 250, 500 and 1000 μg/mL., Results: Fungal endophytes Trichoderma longibrachiatum and Syncephalastrum racemosum were isolated from the leaves of M. tomentosa. Isolated endophytic fungal strains and solvent extracts showed MIC value of 1000 μg/mL against tested pathogenic fungi in the dual culture and poisoning food assays. Methanol fraction of S. racemosum isolate showed the most effective antiproliferative activity with IC 50 of 43.56 μg/mL. Minimal feeding deterrent activity against S. litura larvae was also observed., Discussion and Conclusion: These findings showed that the leaves of Markhamia tomentosa harbor strains of endophytic fungi with promising health benefits, and suggest their antifungal and antiproliferative effects against pathogenic fungi and HeLa cancer cell line.

Wastewater from textile industries contains azo dye residues that negatively affect most environmental systems. The biological treatment of these wastes is the best option due to safety and cost concerns. Here we isolated and identified 19 azo dye-degrading fungi and optimized conditions resulting in enhanced degradation. The fungi belonged to five species of Aspergillus and a single Lichtheimia sp. All fungi were evaluated for their ability to decolorize 20 azo dyes. While the most easily transformable azo dye was direct violet (decolorization ranged from 71.1 to 93.3%), the most resistant to decolorization was fast green azo dye. The greatest degradation potential of azo dyes (direct violet and methyl red) was optimized using the most promising four fungal strains and changing media glucose concentration, nitrogen source, and micronutrients. Biomass, lignin peroxidase, and laccases production were also determined in the optimization studies. The decolorization of both azo dyes by the four fungal strains was greatly enhanced by glucose supplementation. The fungal strains were not able to produce lignin peroxidases in the absence of organic nitrogen source. Both yeast extract and casamino acid supplementation enhanced decolorization of direct violet and methyl red dyes and production of lignin peroxidase by the fungal strains. In contrast, the laccases were absent in the similar medium enriched with the same organic nitrogen sources., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Most fungi are known to synthesize siderophores under iron limitation. However, arbuscular mycorrhizal fungi (AM fungi) have so far not been reported to produce siderophores, although their metabolism is iron-dependent. In an approach to isolate siderophores from AM fungi, we have grown plants of Tagetes patula nana in the presence of spores from AM fungi of the genus Glomus (G. etunicatum, G. mossae & unidentified Glomus sp.) symbiotically under iron limitation and sterile conditions. A siderophore was isolated from infected roots after 2-3 weeks of growth in pots containing low-iron sand with Hoagland solution. HPLC analysis of the root cell lysate revealed a peak at a retention time of 6.7 min which showed iron-binding properties in a chrome azurol S test. The compound was isolated by preparative HPLC and the structure was determined by high resolution electrospray FTICR-MS and GC/MS analysis of the hydrolysis products. From an observed absolute mass to charge ratio (m/z) of 401.11925 [M+H] + with a relative mass error of ∆ = 0.47 ppm an elemental composition of C 16 H 21 N 2 O 10 [M+H] + was derived, suggesting a molecular weight of 400 Da for glomuferrin. Corresponnding ion masses of m/z 423.10 and m/z 439.06 were asigned to the Na-adduct and K-adduct respectively. A mass of 455.03836 confirmed an Fe- complex with an elemental composition of C 16 H 19 N 2 O 10 Fe (∆ = 0.15 ppm). GC/MS analysis of the HCl lysate (6 N HCL, 12 h) revealed 1,4 butanediamine. Thus the proposed structure of the isolated siderophore from Glomus species consisted of 1,4 butanediamine amidically linked to two dehydrated citrate residues, similar to the previously identified bis-amidorhizoferrin. Thus, the isolated siderophore (glomuferrin) is a member of the rhizoferrin family previously isolated from fungi of the Mucorales (Zygomycetes).

Background: Oleaginous fungi can accumulate lipids by utilizing a wide range of waste substrates. They are an important source for the industrial production of omega-6 polyunsaturated fatty acids (gamma-linolenic and arachidonic acid) and have been suggested as an alternative route for biodiesel production. Initial research steps for various applications include the screening of fungi in order to find efficient fungal producers with desired fatty acid composition. Traditional cultivation methods (shake flask) and lipid analysis (extraction-gas chromatography) are not applicable for large-scale screening due to their low throughput and time-consuming analysis. Here we present a microcultivation system combined with high-throughput Fourier transform infrared (FTIR) spectroscopy for efficient screening of oleaginous fungi., Results: The microcultivation system enables highly reproducible fungal fermentations throughout 12 days of cultivation. Reproducibility was validated by FTIR and HPLC data. Analysis of FTIR spectral ester carbonyl peaks of fungal biomass offered a reliable high-throughput at-line method to monitor lipid accumulation. Partial least square regression between gas chromatography fatty acid data and corresponding FTIR spectral data was used to set up calibration models for the prediction of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, unsaturation index, total lipid content and main individual fatty acids. High coefficients of determination (R 2  = 0.86-0.96) and satisfactory residual predictive deviation of cross-validation (RPD CV  = 2.6-5.1) values demonstrated the goodness of these models., Conclusions: We have demonstrated in this study, that the presented microcultivation system combined with rapid, high-throughput FTIR spectroscopy is a suitable screening platform for oleaginous fungi. Sample preparation for FTIR measurements can be automated to further increase throughput of the system.