RESUMEN
Strategies are needed for the robust production of cryptic, silenced, or engineered secondary metabolites in fungi. The filamentous fungus Fusarium heterosporum natively synthesizes the polyketide equisetin at >2 g L(-1) in a controllable manner. We hypothesized that this production level was achieved by regulatory elements in the equisetin pathway, leading to the prediction that the same regulatory elements would be useful in producing other secondary metabolites. This was tested by using the native eqxS promoter and eqxR regulator in F. heterosporum, synthesizing heterologous natural products in yields of â¼1 g L(-1). As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L(-1), leading to the practical synthesis of a selective antituberculosis agent. Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi. These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites.
Asunto(s)
Productos Biológicos/metabolismo , Fusarium/genética , Fusarium/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ingeniería Genética/métodos , Sintasas Poliquetidas/genética , Regiones Promotoras Genéticas/genética , Pirrolidinonas/metabolismo , Metabolismo Secundario , Tetrahidronaftalenos/metabolismoRESUMEN
Three new decalin-type tetramic acid analogues, pyrrolocins A (1), B (2), and C (3), were defined as products of a metabolic pathway from a fern endophyte, NRRL 50135, from Papua New Guinea. NRRL 50135 initially produced 1 but ceased its production before chemical or biological evaluation could be completed. Upon transfer of the biosynthetic pathway to a model host, 1-3 were produced. All three compounds are structurally related to equisetin-type compounds, with 1 and 3 having a trans-decalin ring system, while 2 has a cis-fused decalin. All were active against Mycobacterium tuberculosis, with the trans-decalin analogues 1 and 3 exhibiting lower MICs than the cis-decalin analogue 2. Here we report the isolation, structure elucidation, and antimycobacterial activities of 1-3 from the recombinant expression as well as the isolation of 1 from the wild-type fungus NRRL 50135.
Asunto(s)
Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Endófitos/química , Helechos/microbiología , Pirrolidinonas/aislamiento & purificación , Pirrolidinonas/farmacología , Antibacterianos/química , Bacillus subtilis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Naftalenos/química , Resonancia Magnética Nuclear Biomolecular , Papúa Nueva Guinea , Pirrolidinonas/química , Staphylococcus aureus/efectos de los fármacos , Estereoisomerismo , Streptococcus pneumoniae/efectos de los fármacos , Tetrahidronaftalenos/químicaRESUMEN
The methanol extract of Melochia odorata yielded three 4-quinolone alkaloids including waltherione A (1) and two new alkaloids, waltherione C (2) and waltherione D (3). Waltheriones A and C showed significant activities in an in vitro anti-HIV cytoprotection assay at concentrations of 56.2 and 0.84 µM and inhibition of HIV P24 formation of more than 50% at 1.7 and 0.95 µM, respectively. The structures of the alkaloids were established by spectroscopic data interpretation.
Asunto(s)
4-Quinolonas/aislamiento & purificación , Alcaloides/aislamiento & purificación , Fármacos Anti-VIH/aislamiento & purificación , Malvaceae/química , 4-Quinolonas/química , 4-Quinolonas/farmacología , Alcaloides/química , Alcaloides/farmacología , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Relación Dosis-Respuesta a Droga , Proteína p24 del Núcleo del VIH/antagonistas & inhibidores , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Papúa Nueva Guinea , Tallos de la Planta/química , QuinolinasRESUMEN
Two new triterpenoids were isolated from the leaves and twigs of Rhus taitensis. Their structures were elucidated by 1D and 2D NMR spectroscopic studies as 1,10,24,25,30-pentahydroxysqualene and dammar-20(22),24-diene-3 ß,26,27-triol. Both compounds exhibited moderate antimycobacterial activities with an MIC of 45 µg/mL.
Asunto(s)
Antibacterianos/farmacología , Extractos Vegetales/química , Rhus/química , Triterpenos/farmacología , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Supervivencia Celular , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Hojas de la Planta/química , Plantas Medicinales , Triterpenos/química , Triterpenos/aislamiento & purificaciónRESUMEN
Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/ß-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding ß-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrólidos/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Vacuolas/metabolismo , Proteínas Wnt/metabolismo , Acilación , Animales , Embrión no Mamífero , Desarrollo Embrionario/efectos de los fármacos , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Macrólidos/aislamiento & purificación , Unión Proteica , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Vacuolas/química , Proteína Wnt3 , Proteína Wnt3A , Xenopus , Proteínas de XenopusRESUMEN
Rapidly increasing experimental and clinical data provides evidence for the role of hypoxia inducible factor-1 (HIF-1) as a crucial mediator of tumor survival and progression. In our effort to identify inhibitors of the HIF-1 activation pathway, we screened fractions from marine invertebrates. Fractions from an extract of Petrosia (Strongylophora) strongylata potently inhibited the HIF-1 activation pathway. Strongylophorines 2, 3, and 8 isolated from the active fractions were found to be responsible for the HIF-1 inhibition with EC 50 values of 8, 13, and 6 microM, respectively.