Whole-genome and comparative genome analysis of Mucor racemosus C isolated from Yongchuan Douchi.

Mucor racemosus is the predominant fungal in the zhiqu stage of the fermentation of Yongchuan Douchi (Mucor-type), which plays an important role in the fermentation process of Yongchuan Douchi. However, there is a lack of information on the genetic analysis of M. racemosus. In this study, we isolated and identified M. racemosus C (accession no JAPEHQ000000000) from Yongchuan Douchi and analyzed the physiological indicators, then genomic information of the strain to perform a comprehensive analysis of its fermentation capacity and safety. M. racemosus C had neutral protease activity up to 68.051 U/mL at 30 °C and alkaline protease activity up to 57.367 U/mL at 25 °C. In addition, comparing the genomic data with the COGs database (NCBI), it was predicted that M. racemosus C undergoes extensive amino acid metabolism, making C suitable for the production of fermented foods (e.g., Douchi, Syoyu, and sufu). Finally, we performed virulence genes and resistance genes analysis, hemolysis experiment, aflatoxins assay, antibiotic resistance assay to evaluate the safety of M. racemosus C, and the results showed that M. racemosus C was safe, non-toxin-producing and non-hemolytic.

Homologous Overexpression of Diacylglycerol Acyltransferase in Oleaginous Fungus Mucor circinelloides WJ11 Enhances Lipid Accumulation under Static Solid Cultivation.

Diacylglycerol acyltransferase (DGAT) catalyzes the binding of acyl-CoA to diacylglycerol to form triacylglycerol (TAG). Previous studies strongly indicate that DGAT2, rather than DGAT1, is crucial for TAG accumulation in the oleaginous fungus Mucor circinelloides. To increase the lipid content of M. circinelloides WJ11, McDGAT2 was overexpressed by homologous recombination; compared to the control strain Mc2075, transformants McDGAT2d showed a significant increase in biomass for both spores and mycelia (from 87.7 to 101.2 mg/g in spores and from 75.6 to 93.1 mg/g in mycelia). McDGAT2 overexpression under static solid fermentation gave a greater boost to lipid accumulation in mycelia than in spores. Total fatty acid content in mycelia increased by 68.0% (from 13.6 to 22.8%) and in spores by 26.3% (from 10.6 to 13.4%). However, under submerged fermentation, the lipid content of McDGAT2d was the same as the control, while biomass was slightly reduced. Transcriptomics showed that NADPH was derived mainly from the pentose phosphate pathway, acetyl-CoA was from multiple pathways, and leucine metabolism played an important role in substrate supply for fatty acid biosynthesis. Static solid fermentation may be the more suitable fermentation method for microbial oil production by filamentous fungi due to its lower fermentation costs.

Immobilization of fibrinolytic protease from Mucor subtilissimus UCP 1262 in magnetic nanoparticles.

This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.

Microbial Conjugation Studies of Licochalcones and Xanthohumol.

Microbial conjugation studies of licochalcones (1-4) and xanthohumol (5) were performed by using the fungi Mucor hiemalis and Absidia coerulea. As a result, one new glucosylated metabolite was produced by M. hiemalis whereas four new and three known sulfated metabolites were obtained by transformation with A. coerulea. Chemical structures of all the metabolites were elucidated on the basis of 1D-, 2D-NMR and mass spectroscopic data analyses. These results could contribute to a better understanding of the metabolic fates of licochalcones and xanthohumol in mammalian systems. Although licochalcone A 4'-sulfate (7) showed less cytotoxic activity against human cancer cell lines compared to its substrate licochalcone A, its activity was fairly retained with the IC50 values in the range of 27.35-43.07 µM.

Primary and secondary metabolites produced in Salvia miltiorrhiza hairy roots by an endophytic fungal elicitor from Mucor fragilis.

Salvia miltiorrhiza is one of the most commonly used medicinal materials in China. In recent years, the quality of S. miltiorrhiza has attracted much attention. Biotic and abiotic elicitors are widely used in cultivation to improve the quality of medicinal plants. We isolated an endophytic fungus, Mucor fragilis, from S. miltiorrhiza. We compared the effects of endophytic fungal elicitors with those of yeast extract together with silver ion, widely used together as effective elicitors, on S. miltiorrhiza hairy roots. Seventeen primary metabolites (amino acids and fatty acids) and five secondary metabolites (diterpenoids and phenolic acids) were analyzed after elicitor treatment. The mycelium extract promoted the accumulation of salvianolic acid B, rosmarinic acid, stearic acid, and oleic acid in S. miltiorrhiza hairy roots. Additionally, qPCR revealed that elicitors affect the accumulation of primary and secondary metabolites by regulating the expression of key genes (SmAACT, SmGGPPS, and SmPAL). This is the first detection of both the primary and secondary metabolites of S. miltiorrhiza hairy roots, and the results of this work should help guide the quality control of S. miltiorrhiza. In addition, the findings confirm that Mucor fragilis functions as an effective endophytic fungal elicitor with excellent application prospect for cultivation of medicinal plants.

Biogenic Gold Nanoparticles Decrease Methylene Blue Photobleaching and Enhance Antimicrobial Photodynamic Therapy.

Antibiotic resistance is a growing concern that is driving the exploration of alternative ways of killing bacteria. Here we show that gold nanoparticles synthesized by the mycelium of Mucor plumbeus are an effective medium for antimicrobial photodynamic therapy (PDT). These particles are spherical in shape, uniformly distributed without any significant agglomeration, and show a single plasmon band at 522-523 nm. The nanoparticle sizes range from 13 to 25 nm, and possess an average size of 17 ± 4 nm. In PDT, light (from a source consisting of nine LEDs with a peak wavelength of 640 nm and FWMH 20 nm arranged in a 3 × 3 array), a photosensitiser (methylene blue), and oxygen are used to kill undesired cells. We show that the biogenic nanoparticles enhance the effectiveness of the photosensitiser, methylene blue, and so can be used to kill both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The enhanced effectiveness means that we could kill these bacteria with a simple, small LED-based light source. We show that the biogenic gold nanoparticles prevent fast photobleaching, thereby enhancing the photoactivity of the methylene blue (MB) molecules and their bactericidal effect.

Anti-aging derivatives of cycloastragenol produced by biotransformation.

In this study, the microbial transformation of cycloastragenol (CA) by the fungi Mucor subtilissimus AS 3.2456 and Aspergillus oryzae AS 3.407 yielded 19 metabolites. Their structures were established based on extensive NMR and HR-MS data analyses, and six of them are new compounds. The two fungal strains exhibited distinct biocatalytic features. M. subtilissimus could catalyse hydroxylation and carbonylation reactions meanwhile the fragile 9,19-cyclopropane ring remained intact. A. oryzae preferred to catalyse hydroxylation, acetylation and ring expansion reactions. These highly specific reactions are difficult to achieve by chemical synthesis, particularly under mild conditions. Furthermore, we found that most of the metabolites could significantly extend the lifespan of Caenorhabditis elegans at 50 µM. These biotransformed derivatives of CA could be potential anti-aging agents.

Extraction and characterization of fungal chitin nanofibers from Mucor indicus cultured in optimized medium conditions.

Chitin nanofibers (ChNFs) were extracted from Mucor indicus by a purification method followed by a mechanical treatment, cultivated under obtained optimum culture medium conditions to improve fungal chitin production rate. A semi synthetic media containing 50 g/l glucose was used for cultivation of the fungus in shake flasks. The cell wall analysis showed that N-acetyl glucosamine (GlcNAc) content, which is an indication of chitin content, was maximum in presence of 2.5 g/l H3PO4, 2.5 g/l of NaOH, 1 g/l of yeast extract, 1 mg/l of plant hormones, and 1 ml/l of trace metals. The chemical characterizations demonstrated that the isolated fibers had a degree of deacetylation lower than of 10%, and were phosphate free. The FTIR results revealed successful removal of different materials during the purification step. The FE-SEM of fibrillated chitin illustrated an average diameter of 28 nm. Finally, X-ray diffraction results showed that the crystallinity index of nanofibers was 82%.

Comparative Analysis of Different Isolated Oleaginous Mucoromycota Fungi for Their γ-Linolenic Acid and Carotenoid Production.

γ-Linolenic acid (GLA) and carotenoids have attracted much interest due to their nutraceutical and pharmaceutical importance. Mucoromycota, typical oleaginous filamentous fungi, are known for their production of valuable essential fatty acids and carotenoids. In the present study, 81 fungal strains were isolated from different Egyptian localities, out of which 11 Mucoromycota were selected for further GLA and carotenoid investigation. Comparative analysis of total lipids by GC of selected isolates showed that GLA content was the highest in Rhizomucor pusillus AUMC 11616.A, Mucor circinelloides AUMC 6696.A, and M. hiemalis AUMC 6031 that represented 0.213, 0.211, and 0.20% of CDW, respectively. Carotenoid analysis of selected isolates by spectrophotometer demonstrated that the highest yield of total carotenoids (640 µg/g) was exhibited by M. hiemalis AUMC 6031 and M. hiemalis AUMC 6695, and these isolates were found to have a similar carotenoid profile with, ß-carotene (65%), zeaxanthin (34%), astaxanthin, and canthaxanthin (5%) of total carotenoids. The total fatty acids of all tested isolates showed moderate antimicrobial activity against Staphylococcus aureus and Salmonella Typhi, and Penicillium chrysogenum. To the best of our knowledge, this is the first report on the highest yield of total lipid accumulation (51.74% CDW) by a new oleaginous fungal isolate R. pusillus AUMC 11616.A. A new scope for a further study on this strain will be established to optimize and improve its total lipids with high GLA production. So, R. pusillus AUMC 11616.A might be a potential candidate for industrial application.

Soil fungi enable the control of gastrointestinal nematodes in wild bovidae captive in a zoological park: a 4-year trial.

The control of gastrointestinal nematodes among ruminants maintained in zoological parks remains difficult due to infective stages develop in the soil. For the purpose to improve the possibilities of the control of gastrointestinal nematodes (genera Trichostrongylus, Nematodirus, Chabertia and Haemonchus) affecting wild captive bovidae ruminants belonging to the subfamilies Antilopinae, Caprinae, Bovinae and Reduncinae, commercial pelleted feed enriched with a blend of 104-105 spores of both filamentous fungi Mucor circinelloides + Duddingtonia flagrans per kg meal was provided for a period of 3.5 years. All animals were dewormed at the beginning of the trial and also when exceeding a cut-off point of 300 eggs per gram of feces (EPG). The anthelmintic efficacy ranged between 96 and 100%. The need for repeating the administration of parasiticide treatment disappeared at the 24th month of study in the Antilopinae individuals, and at the 8th month in the Caprinae, Bovinae and Reduncinae. No side-effects were observed on the skin or in the digestive, respiratory or reproductive system. It was concluded that this strategy provides a sustainable tool for preventing the contamination of paddocks where captive ruminants are maintained, decreasing the risk of infection by gastrointestinal nematodes and consequently the need of frequent deworming.

Integrating the control of helminths in dairy cattle: Deworming, rotational grazing and nutritional pellets with parasiticide fungi.

Thirty-two Friesian cattle under a leaders/followers four-day rotation and passing eggs of trematodes and gastrointestinal nematodes (GIN) were studied in two trials for the integrated control of these helminths over two years. In the first trial, the effect of rotational pasturing was assessed on a group of leaders (milking cows, G-L1) and followers (dried-off cows and heifers, G-F1) supplemented daily with commercial nutritional pellets. In the second trial, leaders (G-L2) and followers (G-F2) were maintained under a rotational pasturing regime; the cows received daily commercial pelleted feed and heifers pellets manufactured with a blend of parasiticide fungi (3 × 105 chlamydospores of both Mucor circinelloides and Duddingtonia flagrans/kg pellet). Deworming via closantel and albendazole was performed in cows in each trial at the beginning of their drying periods, and fourteen days later, the fecal egg-count reductions (FECR) of Calicophoron daubneyi and GIN were from 94 to 100% (average 98 %), while the percentages of reduction of cattle shedding eggs (CPCR) were from 50 to 100% (average 77 % and 82 %, respectively). The heifers were dewormed one time only, at the beginning of each trial, and the values of FECR and CPCR were 100 % against C. daubneyi and 96 % and 83 %, respectively, against GIN. Over a period of 24 months, significantly higher numbers of helminth egg-output were observed in G-L1, with the lowest numbers in G-F2. C. daubneyi egg output was reduced by 5 % (G-L1) and 42 % (G-F1) at the end of trial 1 and by 83 % (G-L2) and 100 % (G-F2) at the end of trial 2; the numbers of GIN egg-output decreased by 13 % (G-L1) and 18 % (G-F1) at the end of trial 1, and by 72 % (G-L2) and 85 % (G-F2) at the end of trial 2. No adverse effects were detected in cattle taking pellets enriched with fungal spores (G-F2). It is concluded that long-term ingestion of spores of M. circinelloides and D. flagrans provides a valuable tool to improve the effect of rotational grazing and to lessen the risk of infection by C. daubneyi and GIN in dairy cattle, and accordingly, the performance of integrated control programs.

Microbial Transformation of Prenylquercetins by Mucor hiemalis.

Quercetin, one of the most widely distributed flavonoids, has been found to show various biological activities including antioxidant, anticancer, and anti-inflammatory effects. It has been reported that bioactivity enhancement of flavonoids has often been closely associated with nuclear prenylation, as shown in 8-prenylquercetin and 5'-prenylquercetin. It has also been revealed in many studies that the biological activities of flavonoids could be improved after glucosylation. Three prenylated quercetins were prepared in this study, and microbial transformation was carried out in order to identify derivatives of prenylquercetins with increased water solubility and improved bioavailability. The fungus M. hiemalis was proved to be capable of converting prenylquercetins into more polar metabolites and was selected for preparative fermentation. Six novel glucosylated metabolites were obtained and their chemical structures were elucidated by NMR and mass spectrometric analyses. All the microbial metabolites showed improvement in water solubility.

Novel Dual-Functional Enzyme Lip10 Catalyzes Lipase and Acyltransferase Activities in the Oleaginous Fungus Mucor circinelloides.

Lipases or triacylglycerol (TAG) lipases belong to the α/ß-hydrolases superfamily, which are enzymes capable of catalyzing the hydrolysis of the ester bond between fatty acids and glycerol. Interestingly, some lipases have been found to not only possess hydrolysis activity but also acyltransferase activity in yeasts and microalgae. Our present study reported a novel dual-functional Mucor circinelloides lipase Lip10 with a slight lipolysis activity but a noteworthy phospholipid/diacylglycerol acyltransferase (PDAT) activity. The purified Lip10 mutants prefer to utilize phosphatidyl serine to form TAG over phosphatidyl ethanolamine and phosphatidylcholine. Site-directed mutagenesis indicated that the histidine residue in the acyltransferase motif H-(X)4-D is indispensable for the PDAT activity of Lip10. Overexpression of the acyltransferase motif of Lip10 promoted cell growth by 12% and increased lipid production by 14% compared to the control, whilst overexpression of the lipase motif induced lipid degradation in M. circinelloides.

A Laboratory-Built Fully Automated Ultrasonication Robot for Filamentous Fungi Homogenization.

This article presents the design and development of a new hands-free ultrasonication robot for filamentous fungi homogenization. The platform was constructed with a modified inexpensive 3D printer, equipped with an upward-facing camera, a custom-designed wash station, and an add-on sonicator. While machine vision accomplished sample well screening based on image subtraction and color thresholding, it also determined the level of fungi homogeneity using color variance. Model fitting reveals that the process of filamentous fungi homogenization using ultrasonication included a period of significant exponential decay. Therefore, this procedure allowed for the rapid homogenization of the fungal samples during the initial stages of ultrasonication treatment followed by a deceleration in homogenization. Furthermore, a factorial experiment showed that higher sonicator power and temperature accelerated the homogenization process, while the cultivation time exhibited no effect on homogenization. In addition, the model parameters were varied between the wells, even when subjected to the same settings, meaning that the system cannot use the same asymptote of the homogeneity level to establish the termination time for different wells. Therefore, we used the standard deviation of the four most recent homogeneity level values to determine the termination time. This method was used for feedback control, forming a fully automated robot that did not require manual intervention during the experiment. A validation test on filamentous fungi demonstrated that the system was able to provide target quality of samples efficiently.

Difference in microbial community and taste compounds between Mucor-type and Aspergillus-type Douchi during koji-making.

Douchi has attracted people's attention because of its unique taste and rich health function. The microbes participated in the koji-making process contribute to taste compounds of Douchi. However, the majority of studies on Douchi focused on their functional components and the microbial community in single type of Douchi during koji-making so far. In the present study, the taste components of Mucor-type and Aspergillus-type Douchi were measured initially and the results showed that the amino acid and organic acid levels as well as the percentage of unsaturated fatty acids in Mucor-type Douchi were significantly higher than those in Aspergillus-type. The investigation of the microbial composition in two types of Douchi showed that Aspergillus, Candida, Meyerozyma and Lecanicillium were shared by >50% of samples during koji-making. Comparison of the microbial community between the two types of Douchi revealed that Meyerozyma and Lecanicillium were the main microbial community with significant difference during the initial stage of koji-making, while Candida was significantly different during the later stage of koji-making. When supplemented with Meyerozyma and Candida in Aspergillus-type Douchi, the level of all amino acid and organic acids as well as the percentage of unsaturated fatty acid was significant improved, which further validated the importance roles of the two microorganisms in enhancing the taste components of Douchi during koji-making. The results provide useful information on optimizing the microbial community structure of Douchi during the process of koji-making and improving the product quality.

Critical applications of Mucor circinelloides within a biorefinery context.

The establishment of an efficient and feasible biorefinery model depends on, among other factors, particularly the selection of the most appropriate microorganism. Mucor circinelloides is a dimorphic fungus species able to produce a wide variety of hydrolytic enzymes, lipids prone to biodiesel production, carotenoids, ethanol, and biomass with significant nutritional value. M. circinelloides also has been selected as a model species for genetic modification by being the first filamentous oleaginous species to have its genome fully characterized, as well as being a species characterized as a potential bioremediation agent. Considering the potential of replacing several nonrenewable feedstocks is widely dependent on fossil fuels, the exploitation of microbial processes and products is a desirable solution for promoting a green and sustainable future. Here, we introduce and thoroughly describe the recent and critical applications of this remarkable fungus within the context of developing a fungal-based biorefinery.

15N, 13C and 1H resonance assignments of FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus.

Invasive fungal infections are a leading cause of death in immunocompromised patients and remain difficult to treat since fungal pathogens, like mammals, are eukaryotes and share many orthologous proteins. As a result, current antifungal drugs have limited clinical value, are sometimes toxic, can adversely affect human reaction pathways and are increasingly ineffective due to emerging resistance. One potential antifungal drug, FK506, establishes a ternary complex between the phosphatase, calcineurin, and the 12-kDa peptidyl-prolyl isomerase FK506-binding protein, FKBP12. It has been well established that calcineurin, highly conserved from yeast to mammals, is necessary for invasive fungal disease and is inhibited when in complex with FK506/FKBP12. Unfortunately, FK506 is also immunosuppressive in humans, precluding its usage as an antifungal drug, especially in immunocompromised patients. Whereas the homology between human and fungal calcineurin proteins is > 80%, the human and fungal FKBP12s share 48-58% sequence identity, making them more amenable candidates for drug targeting efforts. Here we report the backbone and sidechain NMR assignments of recombinant FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus in the apo form and compare these to the backbone assignments of the FK506 bound form. In addition, we report the backbone assignments of the apo and FK506 bound forms of the Homo sapiens FKBP12 protein for evaluation against the fungal forms. These data are the first steps towards defining, at a residue specific level, the impacts of FK506 binding to fungal and mammalian FKBP12 proteins. Our data highlight differences between the human and fungal FKBP12s that could lead to the design of more selective anti-fungal drugs.

Hot off the Press.

A personal selection of 32 recent papers is presented covering various aspects of current developments in bioorganic chemistry and novel natural products such as huperphlegmine A from Huperzia phlegmaria.

Identification and Characterization of Diacylglycerol Acyltransferase from Oleaginous Fungus Mucor circinelloides.

Acyl-CoA:diacylglycerol acyltransferase (DGAT) is a pivotal regulator of triacylglycerol (TAG) synthesis. The oleaginous fungus Mucor circinelloides has four putative DGATs: McDGAT1A, McDGAT1B, McDGAT2A, and McDGAT2B, classified into the DGAT1 and DGAT2 subfamilies, respectively. To identify and characterize DGATs in M. circinelloides, these four genes were expressed in Saccharomyces cerevisiae H1246 (TAG-deficient quadruple mutant), individually. TAG biosynthesis was restored only by the expression of McDGAT2B, and TAG content was significantly higher in the mutants with McDGAT2B expression than in a S. cerevisiae mutant with endogenous DGA1 expression. McDGAT2B prefers saturated fatty acids to monounsaturated fatty acids and has an obvious preference for C18:3 (ω-6) according to the results of substrate preference experiments. Furthermore, only the mRNA expression pattern of McDGAT2B correlated with TAG biosynthesis during a fermentation process. Our experiments strongly indicate that McDGAT2B is crucial for TAG accumulation, suggesting that it may be an essential target for metabolic engineering aimed at increasing lipid content of M. circinelloides.

Assessing the potential of fatty acids produced by filamentous fungi as feedstock for biodiesel production.

Increased costs and limited availability of traditional lipid sources for biodiesel production encourage researchers to find more sustainable feedstock at low prices. Microbial lipid stands out as feedstock replacement for vegetable oil to convert fatty acid esters. In this study, the potential of three isolates of filamentous fungi (Mucor circinelloides URM 4140, M. hiemalis URM 4144, and Penicillium citrinum URM 4126) has been assessed as single-cell oil (SCO) producers. M. circinelloides 4140 had the highest biomass concentration with lipid accumulation of up to 28 wt% at 120 hr of cultivation. The profile of fatty acids revealed a high content of saturated (SFA) and monounsaturated fatty acids (MUFA), including palmitic (C16:0, 33.2-44.1 wt%) and oleic (C18:1, 20.7-31.2 wt%) acids, with the absence of polyunsaturated fatty acids (PUFA) having more than four double bonds. Furthermore, the predicted properties of biodiesel generated from synthesized SCOs have been estimated by using empirical models which were in accordance with the limits imposed by the USA (ASTM D6715), European Union (EN 14214), and Brazilian (ANP 45/2014) standards. These results suggest that the assessed filamentous fungus strains can be considered as alternative feedstock sources for high-quality biofuel production.