RESUMEN
Aspergillus species are among the most important filamentous fungi in terms of industrial use and because of their pathogenic or toxin-producing features. The genomes of several Aspergillus species have become publicly available in this decade, and genomic analyses have contributed to an integrated understanding of fungal biology. Stress responses and adaptation mechanisms have been intensively investigated using the accessible genome infrastructure. Mitogen-activated protein kinase (MAPK) cascades have been highlighted as being fundamentally important in fungal adaptation to a wide range of stress conditions. Reverse genetics analyses have uncovered the roles of MAPK pathways in osmotic stress, cell wall stress, development, secondary metabolite production, and conidia stress resistance. This review summarizes the current knowledge on the stress biology of Aspergillus species, illuminating what we have learned from the genomic data in this "post-genomic era."
Asunto(s)
Adaptación Fisiológica/genética , Aspergillus/genética , Sistema de Señalización de MAP Quinasas/genética , Estrés Fisiológico/genética , Aspergillus/fisiología , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Genómica , Presión Osmótica/fisiologíaRESUMEN
WYK-1 is a dipeptidyl peptidase IV inhibitor produced by Aspergillus oryzae strain AO-1. Because WYK-1 is an isoquinoline derivative consisting of three l-amino acids, we hypothesized that a nonribosomal peptide synthetase was involved in its biosynthesis. We identified 28 nonribosomal peptide synthetase genes in the sequenced genome of A. oryzae RIB40. These genes were also identified in AO-1. Among them, AO090001000009 (wykN) was specifically expressed under WYK-1-producing conditions in AO-1. Therefore, we constructed wykN gene disruptants of AO-1 after nonhomologous recombination was suppressed by RNA interference to promote homologous recombination. Our results demonstrated that the disruptants did not produce WYK-1. Furthermore, the expression patterns of 10 genes downstream of wykN were similar to the expression pattern of wykN under several conditions. Additionally, homology searches revealed that some of these genes were predicted to be involved in WYK-1 biosynthesis. Therefore, we propose that wykN and the 10 genes identified in this study constitute the WYK-1 biosynthetic gene cluster.
Asunto(s)
Aspergillus oryzae/enzimología , Aspergillus oryzae/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Inhibidores Enzimáticos/metabolismo , Isoquinolinas/metabolismo , Péptido Sintasas/metabolismo , Aspergillus oryzae/genética , Vías Biosintéticas/genética , ADN de Hongos/química , ADN de Hongos/genética , Eliminación de Gen , Datos de Secuencia Molecular , Familia de Multigenes , Péptido Sintasas/genética , Análisis de Secuencia de ADNRESUMEN
The gene manE, encoding a probable class I endoplasmic reticulum 1,2-α-mannosidases (ER-Man), was identified from the filamentous fungus Aspergillus oryzae due to similarity to orthologs. It removes a single mannose residue from Man(9)GlcNAc(2), generating Man(8)GlcNAc(2) isomer B. Disruption of manE caused drastic decreases in ER-Man activity in A. oryzae microsomes.
Asunto(s)
Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Retículo Endoplásmico/enzimología , Mananos/metabolismo , alfa-Manosidasa/genética , alfa-Manosidasa/metabolismo , Secuencia de Aminoácidos , Aspergillus oryzae/metabolismo , Isomerismo , Manosa/metabolismo , Microsomas/enzimología , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Aspergillus oryzae, although closely related to Aspergillus flavus, does not produce aflatoxin (AF). A. oryzae RIB strains can be classified into three groups (group 1-3) based on the structure of the AF biosynthesis gene homolog cluster (AFHC). In group 1 strains, where AFHC is present, the expression level of the aflR gene is extremely low and there is no expression of the other four AF homologue genes (avnA, verB, omtA and vbs). We conducted a detailed structural comparison of AFLR ORF and AFLJ ORF from A. oryzae and A. flavus and identified several amino-acid substitutions. If these substitutions induce inactivation of AFLR and AFLJ, AF biosynthesis of A. oryzae will be doubly inhibited at the transcriptional and translational level. In this study, we transferred aflR and aflJ to A. oryzae RIB67, a group 2 strain where more than half of AFHC is missing. Under control of the pgkA promoter, aflR and aflJ was expressed and avnA, verB, omtA and vbs gene expression were monitored by RT-PCR. We prepared six types of forced-expression vectors, including aflR (from A. oryzae RIB40 or its three mutants) or aflJ (from A. oryzae RIB40 or A. flavus RIB4011). RT-PCR analysis showed that transformants containing aflJ from A. oryzae displayed no expression of AF biosynthetic homologue genes, whereas aflR substitutions had no such effect. These results strongly suggest that the amino-acid substitutions in AFLJ of A. oryzae induce inactivation at the protein level.
Asunto(s)
Aflatoxinas/biosíntesis , Aspergillus oryzae/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Factores de Transcripción/metabolismo , Aflatoxinas/genética , Sustitución de Aminoácidos , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Aspergillus oryzae/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Familia de Multigenes , Mutación , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Transformación GenéticaRESUMEN
We screened actinomycetes capable of converting AS1387392 to AS1429716 and identified those strains capable of hydroxylation. Amycolatopsis azurea JCM 3275 was found to be a particularly efficient strain, capable of converting AS1387392 to AS1429716, with a yield of 44% after 9 h. This strain can metabolize not only the hydroxylation of phenylalanine at the meta and para positions but also the reduction of hydroxyketones, as shown by the isolation of bioconversion products. Examination of more suitable conversion conditions showed that pH 7.8 and 25 °C were the optimum pH and temperature for bioconversion, respectively. We also demonstrated the effect of carbon and nitrogen sources in the culture media on hydroxylation. Using this strain, we were able to efficiently produce AS1429716 as a chemical template. Further derivatization studies may provide more effective, safer immunosuppressants than those that are currently on-market.
Asunto(s)
Actinomycetales/metabolismo , Inmunosupresores/metabolismo , Péptidos Cíclicos/metabolismo , Carbono/química , Medios de Cultivo/química , Concentración de Iones de Hidrógeno , Hidroxilación , Nitrógeno/química , TemperaturaRESUMEN
AS1387392 was a novel and powerful histone deacetylase inhibitor with an excellent oral absorption profile, but this compound was lacking in active moieties, which are essential to synthesize more derivatives. In our screening program to identify actinomycetes capable of converting AS1387392 to AS1429716, which has an active moiety to synthesize more derivatives, we identified 12 strains capable of efficient hydroxylation. Results of phylogenetic analysis of 16S rDNA sequences suggested that these strains belonged to the genera Lentzea, Saccharopolyspora, Sphaerisporangium and Amycolatopsis. Morphological and chemical characteristics as well as results of phylogenetic analysis suggested that strain No. 7980 was a new species belonging to the genus Amycolatopsis, according to the FASTA search result of 16S rDNA gene sequence. Using these strains, we can easily produce AS1429716 as a chemical template for further chemical modifications, which may provide more effective and safer immunosuppressant.
Asunto(s)
Actinobacteria/metabolismo , ADN Ribosómico/química , Inmunosupresores/metabolismo , Péptidos Cíclicos/metabolismo , Actinobacteria/genética , Secuencia de Bases , ADN Bacteriano/química , Inhibidores de Histona Desacetilasas/metabolismo , Filogenia , Especificidad de la EspecieRESUMEN
The novel immunosuppressant AS1387392 has been isolated from Acremonium sp. No. 27082. This compound showed a strong inhibitory effect against mammalian histone deacetylase and T-cell proliferation. Further, AS1387392 showed a good oral absorption, and its plasma concentration was higher than that of FR235222, an analog of AS1387392 that inhibited histone deacetylase previously reported. Given these findings, AS1387392 may represent an important new lead in developing immunosuppressant.
Asunto(s)
Acremonium/química , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/efectos de los fármacos , Inmunosupresores/farmacología , Péptidos Cíclicos/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Femenino , Fermentación , Inhibidores de Histona Desacetilasas/aislamiento & purificación , Inhibidores de Histona Desacetilasas/farmacocinética , Histona Desacetilasas/metabolismo , Humanos , Inmunosupresores/aislamiento & purificación , Masculino , Ratones , Ratones Endogámicos BALB C , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/farmacocinética , Ratas , Ratas Endogámicas Lew , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
We compared atfA and atfB, the genes encoding the respective ATF/CREB-type transcription factors in Aspergillus oryzae. The germination ratio of DeltaatfA conidia was low without any stress, unlike that of DeltaatfB conidia. The DeltaatfA conidia were more sensitive to oxidative stress than the DeltaatfB conidia, which are also sensitive to oxidative stress. We compared the gene expressions of these strains by using a DNA microarray, GeneChip. Almost all the genes regulated by atfB were also regulated by atfA, but atfA also regulated many genes that were not regulated by atfB, including some genes putatively involved in oxidative stress resistance. The level of glutamate, the major amino acid in A. oryzae conidia, was significantly low only in the DeltaatfA conidia, and the glycerol accumulation during germination was not observed only in the DeltaatfA strain. We therefore concluded that atfA is involved in germination via carbon and nitrogen source metabolism.
Asunto(s)
Factores de Transcripción Activadores/genética , Aspergillus oryzae/fisiología , Proteínas Fúngicas/genética , Esporas Fúngicas/fisiología , Factores de Transcripción Activadores/química , Factores de Transcripción Activadores/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Aspergillus oryzae/genética , Catalasa/genética , Catalasa/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Glicerol/metabolismo , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Regiones Promotoras Genéticas , Alineación de Secuencia , Esporas Fúngicas/genética , Estrés Fisiológico , Trehalosa/metabolismoRESUMEN
In the course of our screening program for inhibitors of hepatic glucose production in rat hepatoma H4IIE-C3 cells, which were used as model liver cells, five naphtoquinone derivatives-javanicin, solaniol, 9-O-methylfusarubin, 5,10-dihydroxy-1,7-dimethoxy-3-methyl-1H-naphtho[2,3-c]pyran-6,9-dione, 9-O-methylbostrycoidin-and vanillin were selected from our natural product library. These naphtoquinone derivatives inhibited hepatic glucose production at IC(50) values of 3.8-29 microM, but showed cytotoxicity against hepatic cells after incubation for 48 h. However, vanillin showed an IC(50) value of 32 microM without exhibiting cytotoxicity at 50 microM. Therefore, we examined 12 vanillin derivatives to investigate their inhibitory activities against glucose production. Among these analogs, 4-hydro-3-methoxyacetophenone and 5-nitrosalicylaldehyde exhibited stronger inhibition than the other compounds at IC(50) values of 25 and 24 microM, respectively, with no cytotoxicity at a concentration of 50 microM. Hence, 4-hydro-3-methoxyacetophenone and 5-nitrosalicylaldehyde may be useful as a lead compound of anti-type 2 diabetic drugs.
Asunto(s)
Benzaldehídos/farmacología , Glucosa/biosíntesis , Hipoglucemiantes/farmacología , Hígado/metabolismo , Naftoquinonas/farmacología , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , RatasRESUMEN
FR901379 (WF11899A) is a novel echinocandin type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. Micafungin (FK463) is derived from the chemical modification of deacylated FR901379. In the present paper, we performed seven generation's strain-breeding, beginning with a wild type, was performed. Selection medium for screening and production medium for high FR901379 production were designed. Sodium chloride content in the selection plate was affected to FR901379 production and shrinkage of the colony size was observed in high producing strains. As selection markers, large colony-shrinking rate and large inhibition circle in the agar-piece method using C. albicans was selected. Using CMA medium with high sodium chloride, 3 mutants, M-1 to M-3, have achieved a high FR901379 production and M-3 showed 5.0 U/mL, while 1.0 U/mL of production was achieved in wild type strain. A-2 medium supplemented with 6% of soluble starch as a carbon source and 0.6% of ammonium sulfate as nitrogen source was also further effective for mutant screening. The FR901379 production of mutant M-4 (fourth generation) increased until 16.0 U/mL. The concentration of the phosphate salt in the medium seemed to inhibit the growth so as to extend the culture period. When the A-3 medium supplemented with low concentration of phosphate salt and magnesium sulfate as a sulfate source was designed and used, mutants with improved production were successively obtained. Finally, variant strain M-7 showed 30.0 U/mL of production, which was about 30 times higher than that of the wild strain.
Asunto(s)
Ascomicetos/genética , Ascomicetos/metabolismo , Reactores Biológicos/microbiología , Técnicas de Cultivo de Célula/métodos , Mejoramiento Genético/métodos , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/metabolismo , Ascomicetos/clasificación , Mutación , Especificidad de la EspecieRESUMEN
Using an Aspergillus oryzae EST database, we identified a gene encoding a transcription factor (atfB), which is a member of the ATF/CREB family. Expression of atfB was barely detectable during vegetative growth, but was readily detected during conidiation in solid-state culture. Microarray analyses showed that expression of many other genes, including catalase (catA), were downregulated in an atfB-disruptant. The expression of most of these genes was upregulated in the wild-type strain during the conidiation phase in solid-state culture, and the expression pattern was similar to that of atfB itself. In the absence of stress, e.g. heat-shock or hydrogen peroxide, the conidial germination ratios for the DeltaatfB strain and the wild-type strain were similar, but the stress tolerance of conidia carrying the DeltaatfB deletion was less than that of the wild-type conidia. CRE-like DNA motifs, which are bound by ATF/CREB proteins, were found in the promoters of most of the downregulated genes in the DeltaatfB strain. Thus, atfB appears to encode a transcription factor required for stress tolerance in conidia.
Asunto(s)
Aspergillus oryzae/genética , Regulación Fúngica de la Expresión Génica , Estrés Oxidativo , Esporas Fúngicas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Aspergillus oryzae/efectos de los fármacos , Aspergillus oryzae/crecimiento & desarrollo , Aspergillus oryzae/metabolismo , Secuencia de Consenso , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Respuesta al Choque Térmico , Peróxido de Hidrógeno/farmacología , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Alineación de Secuencia , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismo , Factores de Transcripción/químicaRESUMEN
We cloned the isovaleryl-CoA dehydrogenase (IVD)-encoding gene from Aspergillus oryzae. The promoter of ivdA was subjected to beta-glucuronidase (GUS) reporter assays in which certain amino acids were used as a major carbon source. L-leucine most strongly induced GUS-activity, while in the case of L-glutamate, significantly low activity was found, indicating that ivdA transcription was strongly repressed by glutamic acid.
Asunto(s)
Aminoácidos/farmacología , Aspergillus oryzae/enzimología , Isovaleril-CoA Deshidrogenasa/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Aspergillus oryzae/genética , Ácido Glutámico/farmacología , Leucina/farmacologíaRESUMEN
We found the orthologous genes required for RNA interference (RNAi) in the Aspergillus oryzae genome database, and constructed a set of tools for gene silencing using RNAi in A. oryzae. This system utilizes compatible restriction enzyme sites so that only a single target gene fragment is required to create the hairpin RNA cassette. For ease of handling, we also separated the construction of the hairpin RNA cassette for the target gene from its subsequent introduction into the expression vector. Using the brlA gene as a target for RNAi, we detected decreased mRNA levels and a delayed conidiation phenotype in the transformants. Furthermore, even though A. oryzae possesses three copies of the alpha-amylase gene, a single copy of an alpha-amylase RNAi construct was sufficient to downregulate the mRNA levels and decrease the enzymatic activity to 10% of control levels. Gene silencing by RNAi should provide a powerful genetic tool for post-genomic studies of the industrially important fungus A. oryzae.
Asunto(s)
Aspergillus oryzae/genética , Interferencia de ARN , Aspergillus oryzae/metabolismo , Vectores Genéticos/genética , Genoma Fúngico/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
Novel antifungal lipopeptides, FR209602, FR209603 and FR209604, were isolated from the fermentation broth of a fungal strain No. 738 which was identified as Coleophoma crateriformis from morphological and physiological characteristics. The antibiotics were purified by solvent extraction, HP-20, YMC-ODS and silica gel column chromatography and lyophilization. These compounds were structurally similar to FR901379 previously reported by ourselves which had a sulfate residue in the cyclic peptide portion.
Asunto(s)
Antifúngicos/aislamiento & purificación , Fermentación , Hongos/clasificación , Lipoproteínas/aislamiento & purificación , Péptidos Cíclicos/aislamiento & purificación , Antifúngicos/química , Hongos/metabolismo , Lipopéptidos , Lipoproteínas/química , Péptidos Cíclicos/químicaRESUMEN
When grown on solid agar medium, the mycelium of a filamentous fungus, Aspergillus oryzae, forms three morphologically distinct regions: the tip (T), white (W), and basal (B) regions. In this study, we developed the square-plate culture method, a novel culture method that enabled the extraction of mRNA samples from the three regions and analyzed the differential gene expression of the A. oryzae mycelium in concert with the microarray technique. Expression of genes involved in protein synthesis was predominant in the T region; relative expression was, at most, six times higher in the T region compared to the other regions. Genes encoding hypothetical proteins were expressed at high levels in the W and B regions. In addition, genes coding transporters/permeases were predominantly transcribed in the B region. By analyzing the expression patterns of genes in the three regions, we demonstrated the dynamic changes in the regulation of gene expression that occur along the mycelium of filamentous fungi. Consequently, our study established a method to analyze and screen for region-specific genes whose function may be essential for morphogenesis and differentiation in filamentous fungi and whose traits may be beneficial to the biotechnology industry.
Asunto(s)
Aspergillus oryzae/genética , Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Aspergillus oryzae/crecimiento & desarrollo , Western Blotting , Clonación Molecular/métodos , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
Recently we divided Aspergillus oryzae RIB strains into group 1, having seven aflatoxin biosynthesis homologous genes (aflT, nor-1, aflR, norA, avnA, verB, and vbs), and group 2, having three homologues (avnA, verB, and vbs). Here, partial aflatoxin homologous gene cluster of RIB62 from group 2 was sequenced and compared with that of RIB40 from group 1. RIB62 showed a large deletion upstream of ver-1 with more than half of the aflatoxin homologous gene cluster missing including aflR, a positive transcriptional regulatory gene. Adjacent to the deletion of the aflatoxin homologous gene cluster, RIB62 has a unique sequence of about 8 kb and a telomere. Southern analysis of A. oryzae RIB strains with four kinds of probe derived from the unique sequence of RIB62 showed that all group 2 strains have identical hybridizing signals. Polymerase chain reaction with specific primer set designed to amplify the junction between ver-1 and the unique sequence of RIB62 resulted in the same size of DNA fragment only from group 2 strains. Based on these results, we developed a useful genetic tool that distinguishes A. oryzae group 2 strains from the other groups' strains and propose that it might have differentiated from the ancestral strains due to chromosomal breakage.
Asunto(s)
Aflatoxinas/biosíntesis , Aspergillus oryzae/genética , Rotura Cromosómica , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Familia de Multigenes/genética , Aspergillus oryzae/metabolismo , Secuencia de Bases , Southern Blotting , Cromosomas Fúngicos/genética , ADN de Hongos/química , ADN de Hongos/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Orden Génico , Genes Fúngicos/genética , Modelos Genéticos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
To help assess the potential for aflatoxin production by Aspergillus oryzae, the structure of an aflatoxin biosynthesis gene homolog cluster in A. oryzae RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99% similarity to those of Aspergillus flavus, three genes shared 93% similarity or less. A 257-bp deletion in the aflT region, a frameshift mutation in norA, and a base pair substitution in verA were found in A. oryzae RIB 40. In the aflR promoter, two substitutions were found in one of the three putative AreA binding sites and in the FacB binding site. PCR primers were designed to amplify homologs of aflT, nor-1, aflR, norA, avnA, verB, and vbs and were used to detect these genes in 210 A. oryzae strains. Based on the PCR results, the A. oryzae RIB strains were classified into three groups, although most of them fell into two of the groups. Group 1, in which amplification of all seven genes was confirmed, contained 122 RIB strains (58.1% of examined strains), including RIB 40. Seventy-seven strains (36.7%) belonged to group 2, characterized by having only vbs, verB, and avnA in half of the cluster. Although slight expression of aflR was detected by reverse transcription-PCR in some group 1 strains, including RIB 40, other genes (avnA, vbs, verB, and omtA) related to aflatoxin production were not detected. aflR was not detected in group 2 strains by Southern analysis.
Asunto(s)
Aflatoxinas/biosíntesis , Aspergillus oryzae/metabolismo , Genes Fúngicos , Familia de Multigenes , Análisis de Secuencia de ADN , Aspergillus oryzae/clasificación , Aspergillus oryzae/genética , Southern Blotting , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Aspergillus oryzae/genética , Genoma Fúngico , Genómica , Ácido Aspártico Endopeptidasas/genética , Aspergillus oryzae/enzimología , Aspergillus oryzae/metabolismo , Cromosomas Fúngicos/genética , Sistema Enzimático del Citocromo P-450/genética , Genes Fúngicos/genética , Datos de Secuencia Molecular , Filogenia , SinteníaRESUMEN
We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.
Asunto(s)
Antibacterianos/metabolismo , Péptidos , Rhizopus/genética , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Recombinante , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.