RESUMO
BACKGROUND: Aspergillus oryzae, a useful industrial filamentous fungus, produces limited varieties of secondary metabolites, such as kojic acid. Thus, for the production of valuable secondary metabolites by genetic engineering, the species is considered a clean host, enabling easy purification from cultured cells. A. oryzae has been evaluated for secondary metabolite production utilizing strong constitutive promoters of genes responsible for primary metabolism. However, secondary metabolites are typically produced by residual nutrition after microbial cells grow to the stationary phase and primary metabolism slows. We focused on a promoter of the secondary metabolism gene kojA, a component of the kojic acid biosynthetic gene cluster, for the production of other secondary metabolites by A. oryzae. RESULTS: A kojA disruptant that does not produce kojic acid was utilized as a host strain for production. Using this host strain, a mutant that expressed a polyketide synthase gene involved in polyketide secondary metabolite production under the kojA gene promoter was constructed. Then, polyketide production and polyketide synthase gene expression were observed every 24 h in liquid culture. From days 0 to 10 of culture, the polyketide was continuously produced, and the synthase gene expression was maintained. Therefore, the kojA promoter was activated, and it enabled the continuous production of polyketide for 10 days. CONCLUSIONS: The combined use of the kojA gene promoter and a kojA disruptant proved useful for the continuous production of a polyketide secondary metabolite in A. oryzae. These findings suggest that this combination can be applied to other secondary metabolites for long-term production.
Assuntos
Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Policetídeos/metabolismo , Regiões Promotoras Genéticas/genéticaRESUMO
In order to increase secondary metabolite production in filamentous fungi, a transcription factor gene in the biosynthetic gene cluster and global regulator genes such as laeA are considered plausible as targets for overexpression by genetic modification. In this study, we examined these overexpression effect in fungal sp. No. 14919 that produces FR901512, an HMG-CoA reductase inhibitor. Resultantly, the productivity was improved at 1.7-1.8 fold by overexpressing frlE, a transcription factor gene in the biosynthetic gene cluster, whereas productivity did not change by overexpression of laeA and veA. Furthermore, we searched for extra transcription factors affecting the productivity by transcriptome analysis between wild-type strain and highly productive UV mutants. After verifying productivity decrease by overexpression, Drf1, a novel transcription factor encoded by drf1 was identified as the negative regulator. Because each frlE product (FrlE) and Drf1 worked on the same cluster in positive and negative regulatory manners, their network was analyzed.
Assuntos
Fungos/metabolismo , Genes Fúngicos , Família Multigênica , Policetídeos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fungos/genética , Análise de Sequência de RNARESUMO
In the production of useful microbial secondary metabolites, the breeding of strains is generally performed by random mutagenesis. However, because random mutagenesis introduces many mutations into genomic DNA, the causative mutations leading to increased productivity are mostly unknown. Therefore, although gene targeting is more efficient for breeding than random mutagenesis, it is difficult to apply. In this study, a wild-type strain and randomly mutagenized strains of fungal sp. No. 14919, a filamentous fungus producing the HMG-CoA reductase inhibitor polyketide FR901512, were subjected to point mutation analysis based on whole genome sequencing. Among the mutated genes found, mutation of the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) had a positive effect on increasing FR901512 productivity. By complementing the SCAP gene in the SCAP-mutated strain, productivity was decreased to the level of the SCAP-intact strain. Conversely, when either the SCAP or SREBP gene was deleted, the productivity was significantly increased. By genomic transcriptional analysis, the expression levels of three enzymes in the ergosterol biosynthesis pathway were shown to be decreased by SCAP mutation. These findings led to the hypothesis that raw materials of polyketides, such as acetyl-CoA and malonyl-CoA, became more available for FR901512 biosynthesis due to depression in sterol biosynthesis caused by knockout of the SREBP system. This mechanism was confirmed in Aspergillus terreus producing the polyketide lovastatin, which is structurally similar to FR901512. Thus, knockout of the SREBP system should be considered significant for increasing the productivities of polyketides, such as HMG-CoA reductase inhibitors, by filamentous fungi.
Assuntos
Aspergillus/metabolismo , Fungos/metabolismo , Técnicas de Inativação de Genes , Lovastatina/biossíntese , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Tetra-Hidronaftalenos/metabolismo , Aspergillus/genética , Proteínas de Ligação a DNA/genética , Fungos/genética , Proteínas de Membrana/genética , Mutagênese , Mutação Puntual , Policetídeo Sintases/metabolismo , Sequências Reguladoras de Ácido Nucleico , Metabolismo Secundário , Fatores de Transcrição/genética , Sequenciamento Completo do GenomaRESUMO
Ustiloxins were found recently to be the first example of cyclic peptidyl secondary metabolites that are ribosomally synthesized in filamentous fungi. In this work, two function-unknown genes (ustYa/ustYb) in the gene cluster for ustiloxins from Aspergillus flavus were found experimentally to be involved in cyclization of the peptide. Their homologous genes are observed mainly in filamentous fungi and mushrooms. They have two "HXXHC" motifs that might form active sites. Computational genome analyses showed that these genes are frequently located near candidate genes for ribosomal peptide precursors, which have signal peptides at the N-termini and repeated sequences with core peptides for the cyclic portions, in the genomes of filamentous fungi, particularly Aspergilli, as observed in the ustiloxin gene cluster. Based on the combination of the ustYa/ustYb homologous genes and the nearby ribosomal peptide precursor candidate genes, 94 ribosomal peptide precursor candidates that were identified computationally from Aspergilli genome sequences were classified into more than 40 types including a wide variety of core peptide sequences. A set of the predicted ribosomal peptide biosynthetic genes was experimentally verified to synthesize a new cyclic peptide compound, designated as asperipin-2a, which comprises the amino acid sequence in the corresponding precursor gene, distinct from the ustiloxin precursors.
Assuntos
Aspergillus flavus/genética , Genes Fúngicos , Genes Sintéticos , Peptídeos Cíclicos/genética , Sequência de Aminoácidos , Genoma Fúngico , Dados de Sequência Molecular , Família Multigênica , Peptídeos Cíclicos/química , Ribossomos/metabolismoRESUMO
MOTIVATION: Ustiloxins A and B are toxic cyclic tetrapeptides, Tyr-Val/Ala-Ile-Gly (Y-V/A-I-G), that were originally identified from Ustilaginoidea virens, a pathogenic fungus affecting rice plants. Contrary to our report that ustiloxin B is ribosomally synthesized in Aspergillus flavus, a recent report suggested that ustiloxins are synthesized by a non-ribosomal peptide synthetase in U.virens. Thus, we analyzed the U.virens genome, to identify the responsible gene cluster. RESULTS: The biosynthetic gene cluster was identified from the genome of U.virens based on homologies to the ribosomal peptide biosynthetic gene cluster for ustiloxin B identified from A.flavus. It contains a gene encoding precursor protein having five Tyr-Val-Ile-Gly and three Tyr-Ala-Ile-Gly motifs for ustiloxins A and B, respectively, strongly indicating that ustiloxins A and B from U.virens are ribosomally synthesized. AVAILABILITY AND IMPLEMENTATION: Accession codes of the U.virens and A.flavus gene clusters in NCBI are BR001221 and BR001206, respectively. Supplementary data are available at Bioinformatics online.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Família Multigênica , Peptídeos Cíclicos/genética , Ribossomos/metabolismo , Ustilago/genética , Sequência de Aminoácidos , Vias Biossintéticas , Proteínas Fúngicas/biossíntese , Genoma Fúngico , Dados de Sequência Molecular , Peptídeos Cíclicos/biossíntese , Análise de Sequência de DNA/métodos , Homologia de Sequência de Aminoácidos , Ustilago/crescimento & desenvolvimento , Ustilago/metabolismoRESUMO
The biosynthetic machinery of the first fungal ribosomally synthesized and post-translationally modified peptide (RiPP) ustiloxinâ B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side-chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5'-phosphate (PLP)-dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi.
Assuntos
Vias Biossintéticas , Fungos/metabolismo , Peptídeos Cíclicos/metabolismo , Fungos/enzimologia , Fungos/genética , Família Multigênica , Oxirredutases/genética , Oxirredutases/metabolismo , Peptídeos Cíclicos/genética , Processamento de Proteína Pós-Traducional , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Fatty acids are attractive molecules as source materials for the production of biodiesel fuel. Previously, we attained a 2.4-fold increase in fatty acid production by increasing the expression of fatty acid synthesis-related genes in Aspergillus oryzae. In this study, we achieved an additional increase in the production of fatty acids by disrupting a predicted acyl-CoA synthetase gene in A. oryzae. The A. oryzae genome is predicted to encode six acyl-CoA synthetase genes and disruption of AO090011000642, one of the six genes, resulted in a 9.2-fold higher accumulation (corresponding to an increased production of 0.23 mmol/g dry cell weight) of intracellular fatty acid in comparison to the wild-type strain. Furthermore, by introducing a niaD marker from Aspergillus nidulans to the disruptant, as well as changing the concentration of nitrogen in the culture medium from 10 to 350 mM, fatty acid productivity reached 0.54 mmol/g dry cell weight. Analysis of the relative composition of the major intracellular free fatty acids caused by disruption of AO090011000642 in comparison to the wild-type strain showed an increase in stearic acid (7 to 26 %), decrease in linoleic acid (50 to 27 %), and no significant changes in palmitic or oleic acid (each around 20-25 %).
Assuntos
Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Coenzima A Ligases/genética , Ácidos Graxos/metabolismo , Cromatografia Líquida de Alta Pressão , Coenzima A Ligases/metabolismo , Ácidos Graxos/análise , Teste de Complementação Genética , Engenharia Genética/métodos , Filogenia , Triglicerídeos/análiseRESUMO
Ustiloxin B is a secondary metabolite known to be produced by Ustilaginoidea virens. In our previous paper, we observed the production of this compound by Aspergillus flavus, and identified two A. flavus genes responsible for ustiloxin B biosynthesis (Umemura et al., 2013). The compound is a cyclic tetrapeptide of Tyr-Ala-Ile-Gly, whose tyrosine is modified with a non-protein coding amino acid, norvaline. Although its chemical structure strongly suggested that ustiloxin B is biosynthesized by a non-ribosomal peptide synthetase, in the present study, we observed its synthesis through a ribosomal peptide synthetic (RiPS) pathway by precise sequence analyses after experimental validation of the cluster. The cluster possessed a gene (AFLA_094980), termed ustA, whose translated product, UstA, contains a 16-fold repeated peptide embedding a tetrapeptide, Tyr-Ala-Ile-Gly, that is converted into the cyclic moiety of ustiloxin B. This result strongly suggests that ustiloxin B is biosynthesized through a RiPS pathway and that UstA provides the precursor peptide of the compound. The present work is the first characterization of RiPS in Ascomycetes and the entire RiPS gene cluster in fungi. Based on the sequence analyses, we also proposed a biosynthetic mechanism involving the entire gene cluster. Our finding indicates the possibility that a number of unidentified RiPSs exist in Ascomycetes as the biosynthetic genes of secondary metabolites, and that the feature of a highly repeated peptide sequence in UstA will greatly contribute to the discovery of additional RiPS.
Assuntos
Aspergillus flavus/genética , Família Multigênica , Peptídeos Cíclicos/genética , Ribossomos/metabolismo , Vias Biossintéticas , Genes Fúngicos/fisiologia , Peptídeos Cíclicos/biossíntese , Análise de Sequência de DNARESUMO
Mammalian circadian clocks consist of complexly integrated regulatory loops, making it difficult to elucidate them without both the accurate measurement of system dynamics and the comprehensive identification of network circuits. Toward a system-level understanding of this transcriptional circuitry, we identified clock-controlled elements on 16 clock and clock-controlled genes in a comprehensive surveillance of evolutionarily conserved cis elements and measurement of their transcriptional dynamics. Here we report the roles of E/E' boxes, DBP/E4BP4 binding elements and RevErbA/ROR binding elements in nine, seven and six genes, respectively. Our results indicate that circadian transcriptional circuits are governed by two design principles: regulation of E/E' boxes and RevErbA/ROR binding elements follows a repressor-precedes-activator pattern, resulting in delayed transcriptional activity, whereas regulation of DBP/E4BP4 binding elements follows a repressor-antiphasic-to-activator mechanism, which generates high-amplitude transcriptional activity. Our analysis further suggests that regulation of E/E' boxes is a topological vulnerability in mammalian circadian clocks, a concept that has been functionally verified using in vitro phenotype assay systems.
Assuntos
Ritmo Circadiano/genética , Transcrição Gênica , Animais , Células Cultivadas , Biologia Computacional , Proteínas de Ligação a DNA/genética , Fatores de Ligação G-Box , Regulação da Expressão Gênica , Genes Reguladores , Genes erbA , Genes rev , Ratos , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
The Aspergillus oryzae glucoamylase-encoding gene glaB is expressed specifically and strongly only during solid-state cultivation (SSC). To elucidate the basis for the specificity, the glaB promoter was analyzed by electrophoretic gel mobility shift assay (EMSA) which indicated two protein-binding elements from -382 to -353 and from -332 to -313. To confirm that these regions contained cis-elements, deletion analysis of the promoter was undertaken using ß-glucuronidase as a reporter. The results of the deletion analysis were consistent with the EMSA results. The promoter missing the -332 to -313 element was not induced by low water activity stress during SSC.
Assuntos
Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Regulação Fúngica da Expressão Gênica , Glucana 1,4-alfa-Glucosidase/biossíntese , Regiões Promotoras Genéticas , Sítios de Ligação , Análise Mutacional de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Ligação Proteica , Deleção de SequênciaRESUMO
Microbial production of fats and oils is being developed as a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillus oryzae. Examination of the A. oryzae genome demonstrates that it contains two fatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhanced the expression of fatty acid synthesis-related genes by replacing their promoters with the promoter from the constitutively highly expressed gene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthase genes we successfully increased the production of fatty acids and triglycerides by more than two-fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesterase increased productivity to a lesser extent. Increasing expression of acetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored using quantitative real-time reverse transcription polymerase chain reaction. Our data demonstrate that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.
Assuntos
Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Vias Biossintéticas/genética , Ácidos Graxos/metabolismo , Engenharia Metabólica/métodos , Triglicerídeos/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library by in vitro peptide selection using the evolutionary molecular engineering method "cDNA display". In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.
Assuntos
Aptâmeros de Peptídeos/farmacologia , Proliferação de Células/efeitos dos fármacos , Interleucina-6/antagonistas & inibidores , Aptâmeros de Peptídeos/química , Aptâmeros de Peptídeos/genética , Linhagem Celular Tumoral , Cisteína/química , Cistina/química , DNA Complementar/genética , Ensaios de Triagem em Larga Escala , Humanos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Seleção GenéticaRESUMO
KK-1, a cyclic depsipeptide with 10 residues produced by a filamentous fungus Curvularia clavata BAUA-2787, is a promising pesticide active compound with high activity against many plant pathogens, especially Botrytis cinerea. As a first step toward the future mass production of KK-1 through synthetic biological approaches, we aimed to identify the genes responsible for the KK-1 biosynthesis. To achieve this, we conducted whole genome sequencing and transcriptome analysis of C. clavata BAUA-2787 to predict the KK-1 biosynthetic gene cluster. We then generated the overexpression and deletion mutants for each cluster gene using our originally developed transformation system for this fungus, and analyzed the KK-1 production and the cluster gene expression levels to confirm their involvement in KK-1 biosynthesis. As a result of these, a region of approximately 71 kb was found, containing 10 open reading frames, which were co-induced during KK-1 production, as a biosynthetic gene cluster. These include kk1B, which encodes nonribosomal peptide synthetase with a domain structure that is consistent with the structural features of KK-1, and kk1F, which encodes a transcription factor. The overexpression of kk1F increased the expression of the entire cluster genes and, consequently, improved KK-1 production, whereas its deletion decreased the expression of the entire cluster genes and almost eliminated KK-1 production, demonstrating that the protein encoded by kk1F regulates the expressions of the other nine cluster genes cooperatively as the pathway-specific transcription factor. Furthermore, the deletion of each cluster gene caused a reduction in KK-1 productivity, indicating that each gene is involved in KK-1 production. The genes kk1A, kk1D, kk1H, and kk1I, which showed a significant decrease in KK-1 productivity due to deletion, were presumed to be directly involved in KK-1 structure formation, including the biosynthesis of the constituent residues. kk1C, kk1E, kk1G, and kk1J, which maintained a certain level of KK-1 productivity despite deletion, were possibly involved in promoting or assisting KK-1 production, such as extracellular transportation and the removal of aberrant units incorporated into the peptide chain.
RESUMO
The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.
Assuntos
Aspergillus fumigatus/genética , Aspergillus nidulans/genética , Aspergillus oryzae/genética , Genoma Fúngico/genética , Genômica , Aspergillus fumigatus/fisiologia , Aspergillus nidulans/fisiologia , Aspergillus oryzae/fisiologia , Sequência de Bases , Sequência Consenso/genética , Sequência Conservada/genética , Evolução Molecular , Genes Fúngicos Tipo Acasalamento/genética , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Proteoma/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA , Sintenia/genéticaRESUMO
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 system have facilitated the use of A. oryzae in modern biotechnology. 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 nidulans and Aspergillus fumigatus. 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.
Assuntos
Aspergillus oryzae/genética , Genoma Fúngico , Genômica , Ácido Aspártico Endopeptidases/genética , Aspergillus oryzae/enzimologia , Aspergillus oryzae/metabolismo , Cromossomos Fúngicos/genética , Sistema Enzimático do Citocromo P-450/genética , Genes Fúngicos/genética , Dados de Sequência Molecular , Filogenia , SinteniaRESUMO
We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA-protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a 'ligation site' for T4 RNA ligase, a 'biotin site' for solid-phase handling, a 'reverse transcription primer site' for the efficient and rapid conversion from an unstable mRNA-protein fusion (mRNA display) to a stable mRNA/cDNA-protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a 'restriction enzyme site' for the release of a complex from the solid support. This enables not only stabilizing mRNA-protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.
Assuntos
DNA Complementar/biossíntese , Biblioteca de Peptídeos , Peptídeos/química , Proteínas/química , RNA Mensageiro/química , Sistema Livre de Células , DNA/química , Dissulfetos/química , Técnicas Genéticas , Biossíntese de Proteínas , Puromicina/química , Receptores de Interleucina-6/química , Transcrição ReversaRESUMO
Kojic acid is produced in large amounts by Aspergillus oryzae as a secondary metabolite and is widely used in the cosmetic industry. Glucose can be converted to kojic acid, perhaps by only a few steps, but no genes for the conversion have thus far been revealed. Using a DNA microarray, gene expression profiles under three pairs of conditions significantly affecting kojic acid production were compared. All genes were ranked using an index parameter reflecting both high amounts of transcription and a high induction ratio under producing conditions. After disruption of nine candidate genes selected from the top of the list, two genes of unknown function were found to be responsible for kojic acid biosynthesis, one having an oxidoreductase motif and the other a transporter motif. These two genes are closely associated in the genome, showing typical characteristics of genes involved in secondary metabolism.
Assuntos
Aspergillus oryzae/genética , Proteínas Fúngicas/genética , Microbiologia Industrial , Pironas/metabolismo , Aspergillus oryzae/metabolismo , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Glucose/metabolismo , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Demand for novel antifungal drugs for medical and agricultural uses has been increasing because of the diversity of pathogenic fungi and the emergence of drug-resistant strains. Genomic resources for various living species, including pathogenic fungi, can be utilized to develop novel and effective antifungal compounds. We used Aspergillus oryzae as a model to construct a reporter system for exploring novel antifungal compounds and their target genes. The comprehensive gene expression analysis showed that the actin-encoding actB gene was transcriptionally highly induced by benomyl treatment. We therefore used the actB gene to construct a novel reporter system for monitoring responses to cytoskeletal stress in A. oryzae by introducing the actB promoter::EGFP fusion gene. Distinct fluorescence was observed in the reporter strain with minimum background noise in response to not only benomyl but also compounds inhibiting lipid metabolism that is closely related to cell membrane integrity. The fluorescent responses indicated that the reporter strain can be used to screen for lead compounds affecting fungal microtubule and cell membrane integrity, both of which are attractive antifungal targets. Furthermore, the reporter strain was shown to be technically applicable for identifying novel target genes of antifungal drugs triggering perturbation of fungal microtubules or membrane integrity.
Assuntos
Actinas/genética , Antifúngicos/farmacologia , Aspergillus oryzae/efeitos dos fármacos , Aspergillus oryzae/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Regiões Promotoras Genéticas , Fusão Gênica Artificial , Fluorescência , Proteínas Fúngicas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismoRESUMO
Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.
Assuntos
Aspergillus flavus/genética , Aspergillus oryzae/genética , Genoma Fúngico/genética , Genômica , Aspergillus flavus/classificação , Aspergillus flavus/enzimologia , Aspergillus oryzae/classificação , Aspergillus oryzae/enzimologia , Reatores Biológicos , Metabolismo dos Carboidratos/genética , Produtos Agrícolas/microbiologia , DNA Fúngico/genética , Fermentação , Alimentos Fermentados , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Redes e Vias Metabólicas/genética , Família Multigênica , Fenótipo , Filogenia , Doenças das Plantas/prevenção & controle , Metabolismo Secundário/genéticaRESUMO
We previously developed two methods (loop-out and replacement-type recombination) for generating large-scale chromosomal deletions that can be applied to more effective chromosomal engineering in Aspergillus oryzae. In this study, the replacement-type method is used to systematically delete large chromosomal DNA segments to identify essential and nonessential regions in chromosome 7 (2.93 Mb), which is the smallest A. oryzae chromosome and contains a large number of nonsyntenic blocks. We constructed 12 mutants harboring deletions that spanned 16- to 150-kb segments of chromosome 7 and scored phenotypic changes in the resulting mutants. Among the deletion mutants, strains designated Delta5 and Delta7 displayed clear phenotypic changes involving growth and conidiation. In particular, the Delta5 mutant exhibited vigorous growth and conidiation, potentially beneficial characteristics for certain industrial applications. Further deletion analysis allowed identification of the AO090011000215 gene as the gene responsible for the Delta5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loop-helix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes.