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Whole-genome and comparative genome analysis of Mucor racemosus C isolated from Yongchuan Douchi
Abstract
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.
Yongchuan douchi is a well-loved condiment. However, the aroma of rapid Yongchuan douchi is inferior to that of traditional Yongchuan douchi. The objective of this study was to improve the aroma quality of rapid Yongchuan douchi and evaluate the effect of aroma enhancement from the perspective of consumers. The aroma characteristics of samples were analyzed by consumers through flash profile (FP) ( n = 15) and rate-all-that-apply (RATA) ( n = 75). The results showed improvement in the aroma quality of rapid Yongchuan douchi with the two yeast strains, and consumers could perceive the modification of the aroma characteristics. It shows that the douchi aroma and sour aroma of rapid Yongchuan douchi increased significantly after aroma-enhancing fermentation, while the soy sauce aroma, soybean aroma, and musty aroma decreased. Similar results were obtained from FP and RATA. RATA showed that rapid Yongchuan douchi with aroma-enhancing fermentation aroused higher levels of positive emotions, which may increase the acceptance of consumers to rapid Yongchuan douchi. In summary, the aroma quality and consumer preference of rapid Yongchuan douchi were enhanced. The work provides insights into the quality improvement of rapid Yongchuan douchi from the perspective of consumers.
Aflatoxins (AFs) are the most important toxic, mutagenic, and carcinogenic fungal toxins that routinely contaminate food and feed. While more than 20 AFs have been identified to date, aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), and M1 (AFM1) are the most common. Over 25 species of Aspergillus have been shown to produce AFs, with Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius being the most important and well-known AF-producing fungi. These ubiquitous molds can propagate on agricultural commodities to produce AFs in fields and during harvesting, processing, transportation, and storage. Countries with warmer climates and that produce foods susceptible to AF contamination shoulder a substantial portion of the global AF burden. Pakistan’s warm climate promotes the growth of toxigenic fungi, resulting in frequent AF contamination of human foods and animal feeds. The potential for contamination in Pakistan is exacerbated by improper storage conditions and a lack of regulatory limits and enforcement mechanisms. High levels of AFs in common commodities produced in Pakistan are a major food safety problem, posing serious health risks to the population. Furthermore, aflatoxin contamination contributes to economic losses by limiting exports of these commodities. In this review, recent information regarding the fungal producers of AFs, prevalence of AF contamination of foods and feed, current regulations, and AF prevention and removal strategies are summarized, with a major focus on Pakistan.
The eggNOG (evolutionary gene genealogy Non-supervised Orthologous Groups) database is a bioinformatics resource providing orthology data and comprehensive functional information for organisms from all domains of life. Here, we present a major update of the database and website (version 6.0), which increases the number of covered organisms to 12 535 reference species, expands functional annotations, and implements new functionality. In total, eggNOG 6.0 provides a hierarchy of over 17M orthologous groups (OGs) computed at 1601 taxonomic levels, spanning 10 756 bacterial, 457 archaeal and 1322 eukaryotic organisms. OGs have been thoroughly annotated using recent knowledge from functional databases, including KEGG, Gene Ontology, UniProtKB, BiGG, CAZy, CARD, PFAM and SMART. eggNOG also offers phylogenetic trees for all OGs, maximising utility and versatility for end users while allowing researchers to investigate the evolutionary history of speciation and duplication events as well as the phylogenetic distribution of functional terms within each OG. Furthermore, the eggNOG 6.0 website contains new functionality to mine orthology and functional data with ease, including the possibility of generating phylogenetic profiles for multiple OGs across species or identifying single-copy OGs at custom taxonomic levels. eggNOG 6.0 is available at http://eggnog6.embl.de.
Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk‐clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk‐clotting activity under a solid‐state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25°C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45°C. The investigated enzyme was pronounced as thermal‐sensitive and lost activity completely after 10 min incubation at 55°C. The remarkable qualities of the studied enzyme, such as cost‐effective production, milk‐clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese‐making industry.
Practical Application
The milk‐clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers.
Pulmonary mucormycosis (PM) is a rare and life-threatening fungal infection. Here, we report a case of an acute T lymphoblastic leukemia patient with mixed infections of lethal invasive Mucormycosis and multi-drug resistant (MDR) bacteria. After receiving anti-infection drugs to control the patient's fever, he was treated with induction chemotherapy. However, the malignant hematological disease was poorly controlled by the chemotherapy and the patient developed more symptoms of infection. Although the results of multiple β-D-Glucan (G) and Galactomannan (GM) tests remained negative, several pathogens were detected using metagenomic next-generation sequencing (mNGS). In particular, mNGS identified Malassezia pachydermum, Mucor racemosus, and Lauteria mirabilis in the peripheral blood and local secretion samples. The Mucor and bacterial infections were further confirmed via multi-site and repeated fungal and bacterial cultures, respectively. Despite adjusting the anti-infection therapy according to the diagnostic results, the patient's blood disease and symptoms of infection were not alleviated. Additionally, the MDR Acinetobacter baumannii infection/colonization was not confirmed until the seventh culture of the peripheral venous catheter tip. Due to the patient's deteriorating conditions, his family decided to withdraw him from further treatment. Overall, mNGS can facilitate a diagnosis of Mucormycosis by providing clinical and therapeutic information to support conventional diagnostic approaches. For the early and timely diagnosis and treatment of PM, it is also necessary to consider the malignant hematological conditions and repeated tests through multiple detection methods.
The aim was to better understand the clinical characteristics of patients with mucormycosis in western China. We retrospectively investigated the clinical, laboratory, radiological and treatment profiles of mucormycosis patients during a 10-year period (2010–2019). As a result, 59 proven mucormycosis were enrolled in this study. It was found that 52.5% of patients had worse clinical outcomes. Pulmonary mucormycosis (PM) was the most common clinical manifestation. The most frequent risk factor was diabetes mellitus (38, 64.4%) for mucormycosis patients. Cough (43, 93.5%), fever (24, 52.2%) and hemoptysis/bloody phlegm (21, 45.7%) were the most common manifestations of PM. There were no differences in clinical manifestations, risk factors and laboratory tests between different clinical outcome groups (P>0.05). Lymph node enlargement (30, 65.2%), patchy shadows (28, 60.9%), cavitation (25, 53.3%) and bilateral lobe involvement (39, 84.8%) were the most common on chest CT. Nodule was more common in good outcome group (P <0.05). A total of 48 cases (81.4%) were confirmed by histopathological examination, 22 cases (37.3%) were confirmed by direct microscopy. PM patients were treated with amphotericin B/amphotericin B liposome or posaconazale had better clinical outcomes (P <0.05). In conclusion, PM was the most common clinical type of mucormycosis in China. Diabetes mellitus was the most common risk factor. PM has diverse imaging manifestations and was prone to bilateral lobes involvement. Early diagnosis and effective anti-mucor treatment contribute to successful treatment.
Aspergillus flavus is an opportunistic pathogen of crops, including peanuts and maize, and is the second leading cause of aspergillosis in immunocompromised patients. A. flavus is also a major producer of the mycotoxin, aflatoxin, a potent carcinogen, which results in significant crop losses annually. The A. flavus isolate NRRL 3357 was originally isolated from peanut and has been used as a model organism for understanding the regulation and production of secondary metabolites, such as aflatoxin. A draft genome of NRRL 3357 was previously constructed, enabling the development of molecular tools and for understanding population biology of this particular species. Here, we describe an updated, near complete, telomere-to-telomere assembly and re-annotation of the eight chromosomes of A. flavus NRRL 3357 genome, accomplished via long-read PacBio and Oxford Nanopore technologies combined with Illumina short-read sequencing. A total of 13,715 protein-coding genes were predicted. Using RNA-seq data, a significant improvement was achieved in predicted 5’ and 3’ untranslated regions, which were incorporated into the new gene models.
Cutaneous mucormycosis caused by Mucor irregularis (M. irregularis) is a rare condition that typically occurs in immunocompetent patients. Herein, we describe an immunocompromised patient with cutaneous M. irregularis infection who was successfully treated with debridement combined with vacuum assisted closure (VAC) negative pressure technique and split-thickness skin grafting. We present this case owing to its complexity and rarity and the successful treatment with surgical therapy. A 58-year-old man presented to our hospital with a history of skin ulcers and eschar on the right lower leg since two months. He had been receiving methylprednisolone therapy for bullous pemphigoid that occurred five months prior to the present lesions. Histopathological examination of a right leg lesion showed broad, branching hyphae in the dermis. Fungal culture and subsequent molecular cytogenetic analysis identified the pathogen as M. irregularis. After admission, methylprednisolone was gradually tapered and systemic treatment with amphotericin B (total dose 615 mg) initiated along with others supportive therapies. However, the ulcers showed no improvement, and amphotericin B had to be discontinued owing to development of renal dysfunction. After extensive surgical debridement combined with VAC and skin grafting, his skin ulcers were healed; subsequent fungal cultures of the lesions were negative. The patient exhibited no signs of recurrence at 36-month follow-up. Twenty-six cases with M. irregularis-associated cutaneous mucormycosis in literature were reviewed.
Bamboos are important non-timber forest plants widely distributed in the tropical and subtropical regions of Asia, Africa, America, and Pacific islands. They comprise the Bambusoideae in the grass family (Poaceae), including approximately 1,700 described species in 127 genera. In spite of the widespread uses of bamboo for food, construction and bioenergy, the gene repertoire of bamboo still remains largely unexplored. Raddia distichophylla (Schrad. ex Nees) Chase, belonging to the tribe Olyreae (Bambusoideae, Poaceae), is a diploid herbaceous bamboo with only slightly lignified stems. In this study, we report a draft genome assembly of the ∼589 Mb whole-genome sequence of R. distichophylla with a contig N50 length of 86.36 Kb. Repeat sequences account for ∼49.08% of the genome assembly, of which LTR retrotransposons occupy ∼35.99% of the whole genome. A total of 30,763 protein-coding genes were annotated in the R. distichophylla genome with an average transcript size of 2,887 bp. Access to this herbaceous bamboo genome sequence will provide novel insights into biochemistry, molecular marker-assisted breeding programs and germplasm conservation for bamboo species world-wide.
The Clusters of Orthologous Genes (COG) database, also referred to as the Clusters of Orthologous Groups of proteins, was created in 1997 and went through several rounds of updates, most recently, in 2014. The current update, available at https://www.ncbi.nlm.nih.gov/research/COG, substantially expands the scope of the database to include complete genomes of 1187 bacteria and 122 archaea, typically, with a single genome per genus. In addition, the current version of the COGs includes the following new features: (i) the recently deprecated NCBI’s gene index (gi) numbers for the encoded proteins are replaced with stable RefSeq or GenBank\ENA\DDBJ coding sequence (CDS) accession numbers; (ii) COG annotations are updated for >200 newly characterized protein families with corresponding references and PDB links, where available; (iii) lists of COGs grouped by pathways and functional systems are added; (iv) 266 new COGs for proteins involved in CRISPR-Cas immunity, sporulation in Firmicutes and photosynthesis in cyanobacteria are included; and (v) the database is made available as a web page, in addition to FTP. The current release includes 4877 COGs. Future plans include further expansion of the COG collection by adding archaeal COGs (arCOGs), splitting the COGs containing multiple paralogs, and continued refinement of COG annotations.
The safety of microbial cultures utilized for consumption is vital for public health and should be thoroughly assessed. Although general aspects on the safety assessment of microbial cultures have been suggested, no methodological detail nor procedural guideline have been published. Herein, we propose a detailed protocol on microbial strain safety assessment via whole-genome sequence analysis. A starter culture employed in traditional fermented pork production, nham, namely Lactobacillus plantarum BCC9546, was used as an example. The strain’s whole-genome was sequenced through several next-generation sequencing techniques. Incomplete plasmid information from the PacBio sequencing platform and shorter chromosome size from the hybrid Oxford Nanopore-Illumina platform were noted. The methods for 1) unambiguous species identification using 16S rRNA gene and average nucleotide identity, 2) determination of virulence factors and undesirable genes, 3) determination of antimicrobial resistance properties and their possibility of transfer, and 4) determination of antimicrobial drug production capability of the strain were provided in detail. Applicability of the search tools and limitations of databases were discussed. Finally, a procedural guideline for the safety assessment of microbial strains via whole-genome analysis was proposed.
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.
Background:
Despite a growing number of investigations on early diverging fungi, the corresponding lineages have not been as extensively characterized as Ascomycota or Basidiomycota ones. The Mucor genus, pertaining to one of these lineages is not an exception. To this date, a restricted number of Mucor annotated genomes is publicly available and mainly correspond to the reference species, Mucor circinelloides, and to medically relevant species. However, the Mucor genus is composed of a large number of ubiquitous species as well as few species that have been reported to specifically occur in certain habitats. The present study aimed to expand the range of Mucor genomes available and identify potential genomic imprints of adaptation to different environments and lifestyles in the Mucor genus.
Results:
In this study, we report four newly sequenced genomes of Mucor isolates collected from non-clinical environments pertaining to species with contrasted lifestyles, namely Mucor fuscus and Mucor lanceolatus, two species used in cheese production (during ripening), Mucor racemosus, a recurrent cheese spoiler sometimes described as an opportunistic animal and human pathogen, and Mucor endophyticus, a plant endophyte. Comparison of these new genomes with those previously available for six Mucor and two Rhizopus (formerly identified as M. racemosus) isolates allowed global structural and functional description such as their TE content, core and species-specific genes and specialized genes. We proposed gene candidates involved in iron metabolism; some of these genes being known to be involved in pathogenicity; and described patterns such as a reduced number of CAZymes in the species used for cheese ripening as well as in the endophytic isolate that might be related to adaptation to different environments and lifestyles within the Mucor genus.
Conclusions:
This study extended the descriptive data set for Mucor genomes, pointed out the complexity of obtaining a robust phylogeny even with multiple genes families and allowed identifying contrasting potentially lifestyle-associated gene repertoires. The obtained data will allow investigating further the link between genetic and its biological data, especially in terms of adaptation to a given habitat.
Douchi is a type of traditional Chinese flavoring food that has been used for thousands of years and is produced by multispecies solid-state fermentation. However, the correlation between the flavor, the microbiota, and the functional core microbiota in Aspergillus-type douchi fermentation remains unclear. In this study, Illumina MiSeq sequencing and chromatography were used to investigate the bacterial community and flavor components in Aspergillus-type douchi fermentation. The dominant phyla were Firmicutes, Proteobacteria, and Actinobacteria, and the dominant genera were Weissella, Bacillus, Anaerosalibacter, Lactobacillus, Staphylococcus, and Enterococcus. A total of 58 flavor components were detected during fermentation, including two alcohols, 14 esters, five pyrazines, three alkanes, four aldehydes, three phenols, six acids, and five other compounds. Bidirectional orthogonal partial least square modeling showed that Corynebacterium_1, Lactococcus, Atopostipes, Peptostreptococcus, norank_o__AKYG1722, Truepera, Gulosibacter, norank_f__Actinomycetaceae, and unclassified_f__Rhodobacteraceae are the functional core microbiota responsible for the formation of the flavor components during douchi fermentation. This is the first study to investigate the functional core microbiota in douchi fermentation using Illumina MiSeq sequencing and chromatographic techniques. Our findings extend our understanding of the relationships between flavor, the microbiota, and the functional core microbiota during Aspergillus-type douchi fermentation.
The mitochondrial citrate transport system, composed of citrate and malate transporters (MTs), can regulate the citrate efflux from mitochondria to cytosol, and then citrate is cleaved into OAA and acetyl-CoA which can be used for fatty acid (FA) biosynthesis. However, in the fungus Mucor circinelloides the molecular mechanism of citrate efflux from the mitochondria by this system and its role in FA synthesis is unclear. In the present study, we have analyzed the genome of high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 to find the potential genes involving in this system. Five potential genes are present in the genome of WJ11. These genes encode one citrate transport protein (CT), one tricarboxylate carrier (TCT), one MT, and two 2-oxoglutarate:malate antiporters (SoDIT-a and SoDIT-b). However, the genome of CBS 277.49 contains the same set of genes, except for the presence of just one SoDIT. The proteins from WJ11 had similar properties as their counterparts in CBS 277.49. Moreover, phylogenetic analyses revealed the evolutionary relationship of these proteins and illuminated their typical motifs related to potential functions. Additionally, the expression of these genes was analyzed to predict the possible functions in lipid metabolism in M. circinelloides. This is the first study to report the in silico analysis of structures and functions of the mitochondrial citrate transport system in M. circinelloides. This work showed a new strategy for research for the selection of candidate genes for further detailed functional investigation of the mitochondrial citrate transport system in lipid accumulation.
In this study, whole genome sequencing and comparative genomic analyses were performed for Mucilaginibacter polytrichastri RG4-7 and its carboxymethyl cellulose degradation potential was assessed. The results showed that the genome of strain RG4-7 was 5.84 Mb and contained 5019 predicted genes, in which a high proportion of strain-specific genes were related to carbohydrate metabolism. The carboxymethyl cellulose (CMC) degradation and cellulase activity tests revealed the strong cellulose degradation ability, CMCase and β-glucosidase activity in strain RG4-7. Real-time RT-PCR testing of most cellulose degradation related glycoside hydrolase (GH) families showed that GH9 (OKS85969), GH1 (OKS85832), GH3 (OKS89331 and OKS85615) were significantly up-regulated when strain RG4-7 was inoculated with CMC-Na, which suggested that GH9, GH1 and GH3 might determine its cellulose degradation ability. Certainly, further research need to be done to elucidate cellulose degradation mechanisms in strain RG4-7 in order to develop its industrial application value in lignocellulosic biomass degradation and waste management.
Alignment-based database search and sequence comparison are commonly used to detect horizontal gene transfer (HGT). However, with the rapid increase of sequencing depth, hundreds of thousands of contigs are routinely assembled from metagenomics studies, which challenges alignment-based HGT analysis by overwhelming the known reference sequences. Detecting HGT by k-mer statistics thus becomes an attractive alternative. These alignment-free statistics have been demonstrated in high performance and efficiency in whole-genome and transcriptome comparisons. To adapt k-mer statistics for HGT detection, we developed two aggregative statistics
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Intensive study of the metabolome during the Douchi fermentation can provide new knowledge for optimizing the fermentation process. In this work, the metabolic characterization throughout the fermentation of Mucor racemosus Douchi was investigated using gas chromatography with time‐of‐flight mass spectrometry. A total of 511 peaks were found, and 114 metabolites were identified. The fermentation process was clearly distinguished into two main phases by principal components analysis and orthogonal partial least squares‐discriminant analysis. All the samples in the score plots were within the 95% Hotelling T² ellipse. Two separated clusters can be seen clearly in the score plot, which represents the two stages of fermentation: koji‐making (within 48 hr) and postfermentation (after 48 hr). Besides, clear separation and discrimination by both methods were found among different fermentation time within 15 days, while the discrimination cannot be found with more than 15 days of fermentation, indicating that the fermentation of Douchi was finished in 15 days. Due to the synergistic effect of protease and hydrolase accumulated in the early stage, proteins and other big molecular substances are rapidly hydrolyzed into a large number of small molecule components. However, the activity of enzymes decreased with the further fermentation, and some free amino acids were consumed in Maillard reaction. Therefore, there was no significant change in the content of small molecular substances after 15 days of fermentation. Furthermore, the levels of some metabolites such as alanine and lysine involved in the fermentation varied significantly throughout the processes. This study provides new insights for the metabolomics characteristics of Douchi fermentation.
The increasing number of infections by species of Mucorales and their high mortality constitute an important concern for public health. This study aims to decipher the genetic basis of Mucor circinelloides pathogenicity, which displays virulence in a strain dependent manner. Assuming that genetic differences between strains may be linked to different pathotypes, we have conducted a study to explore genes responsible for virulence in M. circinelloides by whole genome sequencing of the avirulent strain NRRL3631 and comparison with the virulent strain CBS277.49. This genome analysis revealed 773 truncated, discontiguous and absent genes in the NRRL3631 strain. We also examined phenotypic traits resulting in reduced heat stress tolerance, chitosan content and lower susceptibility to toxic compounds (calcofluor white and sodium dodecyl sulphate) in the virulent strain, suggesting the influence of cell wall on pathogenesis. Based on these results, we focused on studying extracellular protein-coding genes by gene deletion and further pathotype characterization of mutants in murine models of pulmonary and systemic infection. Deletion of gene ID112092, which codes for a hypothetical extracellular protein of unknown function, resulted in significant reduction of virulence.
Although pathogenesis is a multifactorial process, these findings highlight the crucial role of surface and secreted proteins in M. circinelloides virulence and should promote further studies of other differential genes.
Many antibiotics, chemotherapeutics, crop protection agents and food preservatives originate from molecules produced by bacteria, fungi or plants. In recent years, genome mining methodologies have been widely adopted to identify and characterize the biosynthetic gene clusters encoding the production of such compounds. Since 2011, the 'antibiotics and secondary metabolite analysis shell-antiSMASH' has assisted researchers in efficiently performing this, both as a web server and a standalone tool. Here, we present the thoroughly updated antiSMASH version 4, which adds several novel features, including prediction of gene cluster boundaries using the ClusterFinder method or the newly integrated CASSIS algorithm, improved substrate specificity prediction for non-ribosomal peptide synthetase adenylation domains based on the new SANDPUMA algorithm, improved predictions for terpene and ribosomally synthesized and post-translationally modified peptides cluster products, reporting of sequence similarity to proteins encoded in experimentally characterized gene clusters on a per-protein basis and a domain-level alignment tool for comparative analysis of trans-AT polyketide synthase assembly line architectures. Additionally, several usability features have been updated and improved. Together, these improvements make antiSMASH up-to-date with the latest developments in natural product research and will further facilitate computational genome mining for the discovery of novel bioactive molecules.
The devastating infections that fungal pathogens cause in humans are underappreciated relative to viral, bacterial and parasitic diseases. In recent years, the contributions to virulence of reductive iron uptake, siderophore-mediated uptake and heme acquisition have been identified in the best studied and most life-threatening fungal pathogens: Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. In particular, exciting new work illustrates the importance of iron acquisition from heme and hemoglobin in the virulence of pathogenic yeasts. However, the challenge of establishing how these fungi gain access to hemoglobin in blood and to other sources of heme remains to be fully addressed. Recent studies are also expanding our knowledge of iron uptake in less-well studied fungal pathogens, including dimorphic fungi where new information reveals an integration of iron acquisition with morphogenesis and cell-surface properties for adhesion to host cells. Overall, the accumulating information provides opportunities to exploit iron acquisition for antifungal therapy, and new work highlights the development of specific inhibitors of siderophore biosynthesis and metal chelators for therapeutic use alone or in conjunction with existing antifungal drugs. It is clear that iron-related therapies will need to be customized for specific diseases because the emerging view is that fungal pathogens use different combinations of strategies for iron acquisition in the varied niches of vertebrate hosts.
Mucormycosis is a life-threatening infection caused by Mucorales fungi. Here we sequence 30 fungal genomes, and perform transcriptomics with three representative Rhizopus and Mucor strains and with human airway epithelial cells during fungal invasion, to reveal key host and fungal determinants contributing to pathogenesis. Analysis of the host transcriptional response to Mucorales reveals platelet-derived growth factor receptor B (PDGFRB) signaling as part of a core response to divergent pathogenic fungi; inhibition of PDGFRB reduces Mucorales-induced damage to host cells. The unique presence of CotH invasins in all invasive Mucorales, and the correlation between CotH gene copy number and clinical prevalence, are consistent with an important role for these proteins in mucormycosis pathogenesis. Our work provides insight into the evolution of this medically and economically important group of fungi, and identifies several molecular pathways that might be exploited as potential therapeutic targets.
Basal lineages of the kingdom of fungi comprise terrestrial and aquatic fungi, which are traditionally summarized as Chytridiomycota and Zygomycota in a colloquial sense. The zygomycetes build up the most basal lineage among the terrestrial fungi. As the earliest fungi to conquer terrestrial habitats, they facilitated early coevolution with other land-dwelling microorganisms with implications on the primary and secondary metabolism which is adaptive to the life in soil. Advances in next-generation genome sequencing allow us to gain a new layer of knowledge with respect to their metabolism and its regulation. Recent revisions of the metabolic potential of zygomycetes using genome mining suggested a hidden potential for the production of secondary metabolites. First insights suggest a revision of our understanding of the importance and mode of action of metabolic pathways revealing a fascinating potential to produce novel natural compounds. The aim of this review is to draw attention to the zygomycetes as a unique fungal group and to inspire the scientific community to explore its potential as participants in complex ecosystems for innovative metabolic regulatory frameworks and novel compounds.
Laccase is a key enzyme in the degradation of lignin by fungi. Reports indicate that the activity of this enzyme ranges from 3.5 to 484,000 U L-1. Our aim was to analyze how laccase activity is calculated in the literature, and to determine statistically whether variations in activity are due to biological properties or to inconsistencies in calculation.We found a general lack of consensus on the definition of enzyme activity, and enzymes are sometimes characterized in terms of reaction rate and specific activity.Moreover, enzymeactivity is calculated using at least seven different equations. Therefore, it is critical to standardize the calculation of laccase activity in order to compare results directly.
The genome of a high lipid-producing fungus Mucor circinelloides WJ11 (36% w/w lipid, cell dry weight, CDW) was sequenced and compared with that of the low lipid-producing strain, CBS 277.49 (15% w/w lipid, CDW), which had been sequenced by Joint Genome Institute. The WJ11 genome assembly size was 35.4 Mb with a G+C content of 39.7%. The general features of WJ11 and CBS 277.49 indicated that they have close similarity at the level of gene order and gene identity. Whole genome alignments with MAUVE revealed the presence of numerous blocks of homologous regions and MUMmer analysis showed that the genomes of these two strains were mostly co-linear. The central carbon and lipid metabolism pathways of these two strains were reconstructed and the numbers of genes encoding the enzymes related to lipid accumulation were compared. Many unique genes coding for proteins involved in cell growth, carbohydrate metabolism and lipid metabolism were identified for each strain. In conclusion, our study on the genome sequence of WJ11 and the comparative genomic analysis between WJ11 and CBS 277.49 elucidated the general features of the genome and the potential mechanism of high lipid accumulation in strain WJ11 at the genomic level. The different numbers of genes and unique genes involved in lipid accumulation may play a role in the high oleaginicity of strain WJ11.
Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.
The dynamic impact approach (DIA) represents an alternative to overrepresentation analysis (ORA) for functional analysis of time-course experiments or those involving multiple treatments. The DIA can be used to estimate the biological impact of the differentially expressed genes (DEGs) associated with particular biological functions, for example, as represented by the Kyoto encyclopedia of genes and genomes (KEGG) annotations. However, the DIA does not take into account the correlated dependence structure of the KEGG pathway hierarchy. We have developed herein a path analysis model (KEGG-PATH) to subdivide the total effect of each KEGG pathway into the direct effect and indirect effect by taking into account not only each KEGG pathway itself, but also the correlation with its related pathways. In addition, this work also attempts to preliminarily estimate the impact direction of each KEGG pathway by a gradient analysis method from principal component analysis (PCA). As a result, the advantage of the KEGG-PATH model is demonstrated through the functional analysis of the bovine mammary transcriptome during lactation.
Fungi cause opportunistic, nosocomial, and community-acquired infections. Among fungal infections (mycoses) zygomycoses are exceptionally severe, with a mortality rate exceeding 50 %. Immunocompromised hosts, transplant recipients, and diabetic patients with uncontrolled keto-acidosis and high iron serum levels are at risk. Zygomycota are capable of infecting hosts immune to other filamentous fungi. The infection often follows a progressive pattern, with angioinvasion and metastases. Moreover, current antifungal therapy frequently has an unfavorable outcome. Zygomycota are resistant to some of the routinely used antifungals, among them azoles (except posaconazole) and echinocandins. The typical treatment consists of surgical debridement of the infected tissues accompanied by amphotericin B administration. The latter has strong nephrotoxic side effects, which make it unsuitable for prophylaxis. Delayed administration of amphotericin and excision of mycelium-containing tissues worsens survival prognoses. More than 30 species of Zygomycota are involved in human infections, among them Mucorales is the most abundant. Prognosis and treatment suggestions differ for each species, which makes fast and reliable diagnosis essential. Serum sample PCR-based identification often gives false-negative results; culture-based identification is time-consuming and not always feasible. With the dawn of Zygomycota sequencing projects significant advancement is expected, as in the case of treatment of Ascomycota infections.
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The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system1 have facilitated the use of A. oryzae in modern biotechnology2, 3, 4. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7–9 Mb in comparison with the genomes of Aspergillus nidulans5 and Aspergillus fumigatus6. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.
Iron is an essential but, in excess, toxic nutrient. Therefore, fungi evolved fine-tuned mechanisms for uptake and storage of iron, such as the production of siderophores (low-molecular mass iron-specific chelators). In Aspergillus fumigatus, iron starvation causes extensive transcriptional remodeling involving two central transcription factors, which are interconnected in a negative transcriptional feed-back loop: the GATA-factor SreA and the bZip-factor HapX. During iron sufficiency, SreA represses iron uptake, including reductive iron assimilation and siderophore-mediated iron uptake, to avoid toxic effects. During iron starvation, HapX represses iron-consuming pathways, including heme biosynthesis and respiration, to spare iron and activates synthesis of ribotoxin AspF1 and siderophores, the latter partly by ensuring supply of the precursor, ornithine. In accordance with the expression pattern and mode of action, detrimental effects of inactivation of SreA and HapX are confined to growth during iron sufficiency and iron starvation, respectively. Deficiency in HapX, but not SreA, attenuates virulence of A. fumigatus in a murine model of aspergillosis, which underlines the crucial role of adaptation to iron limitation in virulence. Consistently, production of both extra and intracellular siderophores is crucial for virulence of A. fumigatus. Recently, the sterol regulatory element binding protein SrbA was found to be essential for adaptation to iron starvation, thereby linking regulation of iron metabolism, ergosterol biosynthesis, azole drug resistance, and hypoxia adaptation.
Mucormycosis is an emerging angioinvasive infection caused by the ubiquitous filamentous fungi of the Mucorales order of the
class of Zygomycetes. Mucormycosis has emerged as the third most common invasive mycosis in order of importance after candidiasis
and aspergillosis in patients with hematological and allogeneic stem cell transplantation. Mucormycosis also remains a threat
in patients with diabetes mellitus in the Western world. Furthermore, this disease is increasingly recognized in recently
developed countries, such as India, mainly in patients with uncontrolled diabetes or trauma. Epidemiological data on this
type of mycosis are scant. Therefore, our ability to determine the burden of disease is limited. Based on anatomic localization,
mucormycosis can be classified as one of 6 forms: (1) rhinocerebral, (2) pulmonary, (3) cutaneous, (4) gastrointestinal, (5)
disseminated, and (6) uncommon presentations. The underlying conditions can influence clinical presentation and outcome. This
review describes the emerging epidemiology and the clinical manifestations of mucormycosis.
Bacterial and fungal secondary metabolism is a rich source of novel bioactive compounds with potential pharmaceutical applications
as antibiotics, anti-tumor drugs or cholesterol-lowering drugs. To find new drug candidates, microbiologists are increasingly
relying on sequencing genomes of a wide variety of microbes. However, rapidly and reliably pinpointing all the potential gene
clusters for secondary metabolites in dozens of newly sequenced genomes has been extremely challenging, due to their biochemical
heterogeneity, the presence of unknown enzymes and the dispersed nature of the necessary specialized bioinformatics tools
and resources. Here, we present antiSMASH (antibiotics & Secondary Metabolite Analysis Shell), the first comprehensive pipeline
capable of identifying biosynthetic loci covering the whole range of known secondary metabolite compound classes (polyketides,
non-ribosomal peptides, terpenes, aminoglycosides, aminocoumarins, indolocarbazoles, lantibiotics, bacteriocins, nucleosides,
beta-lactams, butyrolactones, siderophores, melanins and others). It aligns the identified regions at the gene cluster level
to their nearest relatives from a database containing all other known gene clusters, and integrates or cross-links all previously
available secondary-metabolite specific gene analysis methods in one interactive view. antiSMASH is available at http://antismash.secondarymetabolites.org.
Enterococcus faecium ST20Kc and ST41Kc were isolated from kimchi, a traditional Korean fermented cabbage. Bacteriocins produced by both strains exhibited strong activity against Listeria monocytogenes and various Enterococcus spp., including 30 vancomycin-resistant enterococcal strains, but not against other lactic acid bacteria (LAB) on the evaluated test panel. The antimicrobials produced by the strains were found to be proteinaceous and stable even after exposure to varying pH, temperature, and chemicals used in the industry and laboratory processes. Antimicrobial activity of both strains was evaluated as bactericidal against exponentially growing cultures of L. monocytogenes ATCC® 15313™ and Enterococcus faecalis 200A. Based on tricine-SDS-PAGE, the molecular weights of the bacteriocins produced by the strains were between 4 and 6 kDa. Additionally, both strains were susceptible to antibiotics, including vancomycin, kanamycin, gentamycin, ampicillin, streptomycin, tylosin, chloramphenicol, clindamycin, and tetracycline. Adhesion genes, map, mub, and EF-Tu, were also detected in the genomes of both strains. With gastrointestinal stress induction, both strains showed high individual survival rates, and capability to reduce viable counts of L. monocytogenes ATCC® 15313™ and Enterococcus faecalis 200A in mixed cultures. Based on the metabolomics analysis, both strains were found to produce additional antimicrobial compounds, particularly, lactic acid, phenyllactic acid, and phenethylamine, which can be potentially involved in the antimicrobial interaction with pathogenic microorganisms.
The aroma compounds and the microbial community of oil furu, a specific fermented soybean curd, during fermentation were investigated using HS-SPME-GC/MS and high-throughput sequencing, respectively, and their correlations and the predicted functional roles of the microbiota in oil furu were analyzed. Twenty two volatile flavor compounds (relative odor activity value ≥1) were identified that contributed to the aroma profile, which were mainly associated with the aroma attributes. Lactobacillales, Trichosporon and Mucor racemosus were the predominant genera during pre-fermentation, while Candida and Tetragenococcus were predominant during ripening. Correlation analysis showed significant correlation between the microbiota and aroma profiles, and Candida, Empedobacter, Lactobacillus, Pseudomonas, Stenotrophomonas, Trichosporon and Mucor racemosus were significantly and strongly correlated with the characteristic volatile aroma compounds of oil furu (P <0.05, r >0.6). Functional analysis showed that metabolic pathways showed higher activity in oil furu, which mainly included amino acid, lipid and carbohydrate metabolism. The results allowed identification of the important aroma compounds and understanding the contribution of the microbiota, and would be useful for designing starter cultures to produce oil furu with desirable aroma properties and understanding its aroma formation pathways.
P. digitatum, the causative agent of green mold, is one of the most destructive pathogens in the citrus industry. To facilitate basal researches on this important plant pathogen, here we report a finished genome sequence for P. digitatum strain PDW03 using a combination of Illumina, PacBio, and Hi-C sequencing technologies. The assembly comprised 6 chromosomes from telomere to telomere and encodes approximately 9000 proteins. Genomic re-analyses identified 302 Carbohydrate-active enzymes, 420 secreted proteins, and 39 secondary metabolite (SM) gene clusters. Furthermore, we found 10 fragmentary SM clusters in the P. digitatum PDW03 genome. Pangenome analysis based on 5 P. digitatum genomes available showed that conserved orthogroups account for ~68% of the species pangenome. Taken together, this fully completed P. digitatum genome will provide an optimum resource for further researches to investigate the driving forces of fungal host switch and effectors functioning in plant-pathogen interaction.
Yongchuan douchi is a traditional fermented food in China. This study investigated the microbial community of two major types (Natural and artificial) of Yongchuan douchi, and their influences on quality. The results found that natural-type douchi had significantly better quality in its physicochemical properties such as higher amino nitrogen, darker color, and higher umami amino acids after post-fermentation (p < 0.05). A total of 58 volatile components were found in natural-type douchi compared to artificial-type with 42 identified. Microbial analysis found that natural-type douchi had the richness and diversity of the bacterial (fungus) community. However, artificial-type douchi was high in Candida genera may associated with its bitter after taste compared to natural-type. Following post-fermentation, natural-type douchi had higher abundance of Bacillus sp., Enterobacter sp., and Pseudomonas sp., while Bacillus sp., Weissella sp., and Aspergillus sp. compared to artificial-type douchi. Correlation analysis showed that physicochemical properties and sensory quality could be correlated positively or negatively with the microbial composition during the last stage of post-fermentation. In conclusion, the zhiqu process significantly affected the final quality of the two types of Yongchuan douchi as evidenced by the microbial composition in the post-fermentation process.
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.
A rapid dispersive liquid-liquid micro-extraction (DLLME) with in-situ derivatization method for extraction and purification of aflatoxins (AFs) in vegetable oils was developed and evaluated. Oil extract, dichloromethane and trifluoroacetic acid were mixed and injected into water to form a cloudy solution. AFs in the oil were extracted into the numerous liquid droplets (with diameters from a few microns to dozens of microns) of extractant, where derivatization was carried out in situ. The proposed sample preparation method was coupled with high performance liquid chromatography with fluorescence detection (HPLC-FLD) for determination of four AFs in vegetable oils. The method showed excellent linearity in three orders of magnitude, good relative recoveries, good repeatability and high sensitivity with limits of detection in range of 0.005–0.03 ng/mL. The accuracy of the method was also verified by certified reference sample. Finally, different kinds of vegetable oils from the local supermarket were analyzed.
In spring 2014, several wild passeriform garden birds were found severely ill or dead, all with severe periocular swellings. A blue tit (Cyanistes caeruleus) showing severe depression was humanely destroyed and sent for necropsy examination. In this bird, the lower eyelids were sagging, red and oedematous. Microscopical examination revealed marked infiltration of the eyelid tissue with fungal hyphae (stained by periodic acid–Schiff) without any inflammatory reaction. Polymerase chain reaction followed by sequencing identified Mucor racemosus and a so far unknown circovirus. The circovirus infection in this bird might have induced immunosuppression and thus facilitated the fungal infection. To our knowledge this is the first report of ocular M. racemosus infection associated with circovirus infection in a bird.
Mucor circinelloides is one of few oleaginous fungi that produce a useful oil rich in γ-linolenic acid but it usually only produces < 25% total lipid. Nevertheless, we isolated a new strain WJ11 that can produce up to 36% lipid of cell dry weight. In this study, we have systematically analyzed the global changes in protein levels between the high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 (15%, lipid/cell dry weight) at lipid accumulation phase through comparative proteome analysis. Proteome analysis demonstrated that the branched-chain amino acid and lysine metabolism, glycolytic pathway and pentose phosphate pathway in WJ11 were up-regulated, while the activity of tricarboxylic acid cycle and branch point enzyme for synthesis of isoprenoids were retarded compared with CBS 277.49. The coordinated regulation at proteome level indicates more acetyl-CoA and NADPH are provided for fatty acid biosynthesis in WJ11 compared with CBS 277.49.
The Mucor genus, a polyphyletic group pertaining to early diverging lineages of fungi, includes a high number of ubiquitous species. Some species have positive or negative impacts on human activities. Indeed, certain pathogenic Mucor species are a threat for animal and human health and identified more frequently as mycosis causative agents, especially in immunocompromised patients. On the contrary, a small number of Mucor species have been used for centuries in food manufacturing for cheese ripening or Asian fermented food production. Some species are also used as biotechnologically important microorganisms due to their high growth rates, dimorphism (for certain species) and their previously unsuspected potential for secondary metabolite production. Despite all these important roles played by Mucor spp., they have been less investigated than ascomycetous or basidiomycetous species and their taxonomy, metabolism and ecology are less documented when compared to their counterparts in the so-called higher fungi. Nevertheless, research focusing most often on the emblematic Mucor circinelloides species has led to increased knowledge on the biology of this genus, and overall on fungal biology. This is particularly documented for fungal dimorphism or light-induced gene regulation. The aim of this review is to give an overview of the current knowledge on Mucor morphology, taxonomy, ecology and genetics and of its importance regarding human health and industrial applications.
We previously identified the microbiota present during cheese ripening and observed high protease and lipase activity in Divle Cave cheese. To determine the contribution of individual isolates to enzyme activities, we investigated a range of species representing this microbiota for their proteolytic and lipolytic ability. In total, 17 fungal, 5 yeast and 18 bacterial strains, previously isolated from Divle Cave cheese, were assessed. Qualitative protease and lipase activities were performed on skim-milk agar and spirit-blue lipase agar, respectively, and resulted in a selection of strains for quantitative assays. For the quantitative assays, the strains were grown on minimal medium containing irradiated Divle Cave cheese, obtained from the first day of ripening. Out of 16 selected filamentous fungi, Penicillium brevicompactum, Penicillium cavernicola and Penicillium olsonii showed the highest protease activity, while Mucor racemosus was the best lipase producer. Yarrowia lipolytica was the best performing yeast with respect to protease and lipase activity. From the 18 bacterial strains, 14 and 11 strains, respectively showed protease and lipase activity in agar plates. Micrococcus luteus, Bacillus stratosphericus, Brevibacterium antiquum, Psychrobacter glacincola and Pseudomonas proteolytica displayed the highest protease and lipase activity. The proteases of yeast and filamentous fungi were identified as mainly aspartic protease by specific inhibition with Pepstatin A, whereas inhibition by PMSF (phenylmethylsulfonyl fluoride) indicated that most bacterial enzymes belong to serine type protease. Our results demonstrate that aspartic proteases, which usually have high milk clotting activity, are predominantly derived from fungal strains, and therefore fungal enzymes appear to be more suitable for use in the cheese industry. Microbial enzymes studied in this research might be alternatives for rennin (chymosin) from animal source because of their low cost and stable availability. Future studies will aim to purify these enzymes to test their suitability for use in similar artisanal cheeses or in large scale commercial cheeses.
In this work, Trichosporon fermentans CICC 1368, which has been shown to accumulate cellular lipids efficiently using industry-agricultural wastes, was subjected to preliminary genome analysis, yielding a genome size of 31.3 million bases and 12,702 predicted protein-coding genes. Our analysis also showed a high degree of gene duplications and unique genes compared with those observed in other oleaginous yeasts, with 3–4-fold more genes related to fatty acid elongation and degradation compared with those in Rhodosporidium toruloides NP11 and Yarrowia lipolytica CLIB122. Phylogenetic analysis with other oleaginous microbes suggested that the lipogenic capacity of T. fermentans was obtained during evolution after the divergence of genera. Thus, our study provided the first draft genome and comparative analysis of T. fermentans, laying the foundation for its genetic improvement to facilitate cost-effective lipid production.
The fungus Aspergillus flavus is known for its ability to produce the toxic and carcinogenic aflatoxins in food and feed. While aflatoxins are of most concern, A. flavus is predicted to be capable of producing many more metabolites based on a study of its complete genome sequence. Some of these metabolites could be of great importance in food and feed safety. Here we describe an analytical methodology based on Orbitrap HRMS technology that allows the untargeted determination of fungal metabolites, in support of the study of the function of genes involved in secondary metabolism in fungi. The applied strategy implies the detection and identification of differentially expressed metabolites in extracts of wild-type and mutant fungal strains, using Orbitrap high-resolution mass spectrometry (HRMS) accurate mass data. The suitability of this approach was demonstrated by the confirmation of previously characterised genes involved in the aflatoxin biosynthetic pathway, namely a polyketide synthase (pksA), an oxidoreductase (ordA) and a methyltransferase (omtA) gene. Subsequently, the proposed methodology was applied for the detection and identification of metabolites produced by a yet uncharacterised gene cluster in A. favus, cluster 23. Comparative Orbitrap HRMS analysis of extracts of A. flavus wild-type strain and an over-expression mutant for the transcription factor of gene cluster 23 (lepE) demonstrated that this gene cluster is responsible for the production a set of 2-pyridone derivatives, the leporins. Besides the known derivatives leporin B and leporin B precursor that could be identified by automatic de-replication of the accurate mass data, five other compounds belonging to this class of fungal secondary metabolites were detected and identified for the first time, combining MS and multiple-stage MS data.
A. oryzae 3.042 (China) and A. oryzae RIB40 (Japan) used for soy sauce fermentation show some regional differences. We sequenced the genome of A. oryzae 3.042, and compared it with A. oryzae RIB40 in an attempt to understand why different features are shown by these two A. oryzae strains. We predict 11,399 protein-coding genes in A. oryzae 3.042. The genomes of these two A. oryzae strains are collinear revealed by MUMmer analysis indicating that the differences are not obvious between them. Several strain-specific genes of two strains are identified by genome sequences' comparison, and they are classified into some groups which have the relationship with cell growth, cellular response and regulation, resistance, energy metabolism, salt tolerance and flavor formation. A. oryzae 3.042 showed stronger potential for mycelial growth and environmental stress resistance, such as the genes of chitinase and quinone reductase. Some genes unique to A. oryzae RIB40 were related to energy metabolism and salt tolerance, especially genes for Na+ and K+ transport, while others were associated with signal transduction and flavor formation. The genome sequence of A. oryzae 3.042 will facilitate the identification of the genetic basis of traits in A. oryzae 3.042, and accelerate our understanding of the different genetic traits of the two A. oryzae strains.
Douchi as a traditional fermentation soyfood with an acquired taste had been appreciated by consumers for thousands of years in China and few Asian countries. Unfortunately, few studies had been conducted on the changes of microbial community during the koji-making process, and whether the pathogenic microorganisms involving the fermentation process is still unclear. Therefore, the PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S and 18S rRNA genes was applied to characterise the dynamic changes of Bacteria, Bacilli and Fungi during koji-making process. The results of DGGE showed that eleven species of bacteria, nine species of bacilli and seven species of Fungi had been detected during the koji-making progress, of which the Bacillus subtilis and Aspergillus oryzae belonged to the dominant microorganisms. Also, eleven strains were isolated from Koji-making samples and were identified as B. subtilis, Bacillus amyloliquefaciens, A. oryzae, Brachybacterium sp., Aspergillus niger, Staphylococcus saprophyticus, Micrococcus sp., Staphylococcus gallinarum, Absidia corymbifera, Saccharomyces cerevisiae, Malassezia sp.; Our results revealed that pathogenic microorganisms were involved in the koji-making process, but the probiotic microorganisms occupied the dominant position of community in the koji-making process.
We review the literature on the enzymes, genes, and whole gene clusters underlying natural product biosyntheses and their regulation in filamentous fungi. We have included literature references from 1958, yet the majority of citations are between 1995 and the present. A total of 295 references are cited.
The advent of next-generation sequencing (NGS) has revolutionized genomic and transcriptomic approaches to biology. These new sequencing tools are also valuable for the discovery, validation and assessment of genetic markers in populations. Here we review and discuss best practices for several NGS methods for genome-wide genetic marker development and genotyping that use restriction enzyme digestion of target genomes to reduce the complexity of the target. These new methods -- which include reduced-representation sequencing using reduced-representation libraries (RRLs) or complexity reduction of polymorphic sequences (CRoPS), restriction-site-associated DNA sequencing (RAD-seq) and low coverage genotyping -- are applicable to both model organisms with high-quality reference genome sequences and, excitingly, to non-model species with no existing genomic data.
Fermentation process improvement of soybean residue cake, a Chinese traditional fermented food, and its physicochemical analysis during fermentation were studied. One of the dominant strains in the fermentation was isolated and identified as Mucor racemosus Fresenius. The fermentation process was improved by subsection fermentation. The crude protein content decreased from 19.95 ± 0.03% in the raw soybean residue to 16.85 ± 0.10% in the fermented products, and the formaldehyde nitrogen content increased from 0.068 ± 0.004% to 0.461 ± 0.022% in final fermented cakes. Hardness of samples significantly (P < 0.05) increased whereas springiness, cohesiveness, and resilience significantly (P < 0.05) decreased with increasing fermentation time, respectively. Microstructure observations showed obvious change of the surface of cake samples during the fermentation process.
Practical Application: During the soybean processing, it will produce plenty of by-products, and the most part of them is soybean residue. The discarded soybean residue causes economic loss. Fortunately, we can obtain nutritious and delicious fermented soybean residue cakes by fermenting soybean residue as raw material.