RESUMEN
BACKGROUND: Paclitaxel (Taxol™) is an important anticancer drug with a unique mode of action. The biosynthesis of paclitaxel had been considered restricted to the Taxus species until it was discovered in Taxomyces andreanae, an endophytic fungus of T. brevifolia. Subsequently, paclitaxel was found in hazel (Corylus avellana L.) and in several other endophytic fungi. The distribution of paclitaxel in plants and endophytic fungi and the reported sequence homology of key genes in paclitaxel biosynthesis between plant and fungi species raises the question about whether the origin of this pathway in these two physically associated groups could have been facilitated by horizontal gene transfer. RESULTS: The ability of the endophytic fungus of hazel Penicillium aurantiogriseum NRRL 62431 to independently synthesize paclitaxel was established by liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. The genome of Penicillium aurantiogriseum NRRL 62431 was sequenced and gene candidates that may be involved in paclitaxel biosynthesis were identified by comparison with the 13 known paclitaxel biosynthetic genes in Taxus. We found that paclitaxel biosynthetic gene candidates in P. aurantiogriseum NRRL 62431 have evolved independently and that horizontal gene transfer between this endophytic fungus and its plant host is unlikely. CONCLUSIONS: Our findings shed new light on how paclitaxel-producing endophytic fungi synthesize paclitaxel, and will facilitate metabolic engineering for the industrial production of paclitaxel from fungi.
Asunto(s)
Genoma Fúngico , Paclitaxel/biosíntesis , Penicillium/genética , Aciltransferasas/clasificación , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Farnesiltransferasa/clasificación , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hongos/genética , Transferencia de Gen Horizontal , Espectrometría de Masas , Oxigenasas de Función Mixta/clasificación , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Paclitaxel/análisis , Penicillium/clasificación , Filogenia , Análisis de Secuencia de ARNRESUMEN
Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Ginkgo biloba/citología , Ginkgo biloba/enzimología , Acetatos/química , Acetatos/metabolismo , Biocatálisis , Cromatografía Líquida de Alta Presión , Clonación Molecular , Sistema Enzimático del Citocromo P-450/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Electroforesis en Gel de Poliacrilamida , Ginkgo biloba/genética , Espectrometría de Masas , Datos de Secuencia Molecular , Análisis de Secuencia de ProteínaRESUMEN
Three new alkaloids, phomapyrrolidones A-C (1-3), bearing a cyclopenta[b]fluorene ring system were isolated from the mycelium extract of the endophytic fungal strain Phoma sp. NRRL 46751, inhabiting Saurauia scaberrinae. Methylation of 1 afforded its N-methyl derivative 4. The planar structures and relative configurations of 1-4 were elucidated by extensive spectroscopic analysis. Phomapyrrolidones B (2) and C (3) exhibited weak antitubercular activity at subcytotoxic concentrations.
Asunto(s)
Actinidiaceae/microbiología , Alcaloides/aislamiento & purificación , Alcaloides/farmacología , Antituberculosos/aislamiento & purificación , Antituberculosos/farmacología , Ascomicetos/química , Mycobacterium tuberculosis/efectos de los fármacos , Succinimidas/aislamiento & purificación , Succinimidas/farmacología , Alcaloides/química , Animales , Antituberculosos/química , Chlorocebus aethiops , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Estructura Molecular , Micelio/química , Resonancia Magnética Nuclear Biomolecular , Succinimidas/química , Células VeroRESUMEN
Phomodione, [(4aS(*),9bR(*))-2,6-diacetyl-7-hydroxy-4a,9-dimethoxy-8,9b-dimethyl-4a.9b-dihydrodibenzo[b,d]furan-1,3(2H,4H)-dione], an usnic acid derivative, was isolated from culture broth of a Phoma species, discovered as an endophyte on a Guinea plant (Saurauia scaberrinae). It was identified using NMR, X-ray crystallography, high resolution mass spectrometry, as well as infrared and Raman spectroscopy. In addition to phomodione, usnic acid and cercosporamide, known compounds with antibiotic activity, were also found in the culture medium. Phomodione exhibited a minimum inhibitory concentration of 1.6 microg/mL against Staphylococcus aureus using the disk diffusion assay, and was active against a representative oomycete, ascomycete and basidiomycete at between three and eight micro-grams per mL.
Asunto(s)
Actinidiaceae/microbiología , Ascomicetos/química , Benzofuranos/aislamiento & purificación , Ascomicetos/ultraestructura , Basidiomycota/efectos de los fármacos , Benzofuranos/química , Benzofuranos/farmacología , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Estructura Molecular , Oomicetos/efectos de los fármacos , Staphylococcus/efectos de los fármacosRESUMEN
Taxol (paclitaxel) a diterpenoid is one of the most effective anticancer drugs identified. Biosynthesis of taxol was considered restricted to the Taxus genera until Stierle et al. discovered that an endophytic fungus isolated from Taxus brevifolia could independently synthesize taxol. Little is known about the mechanism of taxol biosynthesis in microbes, but it has been speculated that its biosynthesis may differ from plants. The microbiome from the roots of Taxus chinensis have been extensively investigated with culture-dependent methods to identify taxol synthesizing microbes, but not using culture independent methods.,Using bar-coded high-throughput sequencing in combination with a metagenomics approach, we surveyed the microbial diversity and gene composition of the root-associated microbiomefrom Taxus chinensis (Pilger) Rehd. High-throughput amplicon sequencing revealed 187 fungal OTUs which is higher than any previously reported fungal number identified with the culture-dependent method, suggesting that T. chinensis roots harbor novel and diverse fungi. Some operational taxonomic units (OTU) identified were identical to reported microbe strains possessing the ability to synthesis taxol and several genes previously associated with taxol biosynthesis were identified through metagenomics analysis.
Asunto(s)
Hongos/metabolismo , Raíces de Plantas/microbiología , Taxus/microbiología , Antineoplásicos Fitogénicos/biosíntesis , ADN de Hongos/genética , Hongos/genética , Paclitaxel/biosíntesis , FilogeniaRESUMEN
The goal of this study was to evaluate the results and predictors of good recovery following involuntary hospitalization of violent substance abuse patients. Twenty patients (16 male, aged 13 to 53 years [mean+/-s.d.=32.9+/-10.2]) were admitted in 1997 with a median hospital time of 73.5 days (20 to 455) for exhibiting violent behavior following drug abuse and a loss of self-control. They were treated with psychiatric medication, a 12-step program (Minnesota), psychotherapy and family therapy, and, following hospitalization, counselling, psychotherapy, and participation in self-help groups. Follow-up ranged from 3 to 24 months (17.8+/-4.9). We studied the probability of maintenance of complete abstinence and social adaptation (professional-educational, family and legal parameters) using T and Fisher tests (significance level p< or =0.05). Of the twenty, thirteen patients (65%) achieved excellent social reintegration, and twelve maintained total abstinence. Two patients died (AIDS, hepatic cirrhosis). The chances of complete abstinence and social reintegration were increased by lower age at admission (p=0.02), some form of treatment following hospitalization (p=0.007), adherence to the entire period of treatment (p=0.05), and regular attendance at self-help groups (p=0.05). No significant differences were found in terms of other demographic parameters, drugs used (number or class), previous hospital admissions, length of hospitalization, or follow-up. Sixty percent of patients can expect an excellent outcome over a period of 18 months, according to strict clinical and social criteria. Early intervention and factors increasing adherence to prolonged treatment increase abstinence and social reintegration and thus should be further explored.