RESUMO
Podophyllotoxin (PPT) is a plant natural product that serves as a precursor for the synthesis of many well-known chemotherapeutic drugs. The limited availability and high demand for the source plants of PPT have led to the exploration of alternative sources for this compound. In this study, we utilized the endophytic fungus Phialocephala podophylli (strain PPE7) that we isolated from the rhizomes of Podophyllum peltatum and is known to produce detectable amounts of PPT in broth culture. To date, the complete PPT biosynthetic pathway has yet to be determined in any species. Since fungi are well known for clustering genes that belong to secondary metabolite pathways, use of a fungal system for investigation of the PPT biosynthesis genes may ultimately lead to elucidation of the entire pathway. In this study, we investigated the secoisolariciresinol dehydrogenase (SD) gene that facilitates the dehydrogenation of secoisolariciresinol to form matairesinol, a mid-pathway intermediate product in PPT biosynthesis. We utilized PCR amplification to acquire the complete SD gene sequence in PPE7 and opted to synthesize the P. peltatum SD sequence for expression. Through western blotting, we confirmed the expression of the recombinant SD (PpSD) and verified protein functionality with a bioconversion assay followed by HPLC and LC-MS analyses. Here, we report the identification of the SD gene in PPE7; this is the first report of the SD gene in an endophytic fungus. Additionally, we established the groundwork for the future expression of the complete PPT biosynthetic pathway in the heterologous host Pichia pastoris.
Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/metabolismo , Álcool Oxidorredutases Dependentes de NAD(+) e NADP(+)/metabolismo , Podofilotoxina/biossíntese , Sequência de Aminoácidos , Ascomicetos/genética , Ascomicetos/isolamento & purificação , Sequência de Bases , Vias Biossintéticas , Clonagem Molecular , Meios de Cultura , DNA Fúngico/genética , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Álcool Oxidorredutases Dependentes de NAD(+) e NADP(+)/genética , Podophyllum peltatum/microbiologia , Conformação Proteica , Análise de Sequência de DNARESUMO
Mayapple rust is a common, disfiguring disease that is widespread in temperate eastern North America wherever the host, Podophyllum peltatum, occurs. Puccinia podophylli, the etiological agent of this rust, has been shown to be distantly related to both Puccinia and Uromyces as exemplified by their types. A systematic study was made to determine the generic classification of P. podophylli. Phylogenetic analyses of two rDNA loci from multiple specimens support the recognition of this taxon as a separate genus of Pucciniaceae. Based on historical literature and type material, P. podophylli was found to represent the type of the forgotten genus Allodus and it is correctly named Allodus podophylli. A neotype is designated for Puccinia podophylli Schwein. (≡ Allodus podophylli) and a lectotype is designated for Aecidium podophylli.