RESUMO
Clustering of biosynthetic genes for producing fungal secondary metabolites, which frequently consist of less than ten genes, has been recognized with numerous genomes. The heterologous expression of whole genes in the clusters will therefore produce various types of natural products when using a suitable fungal host. We introduced the whole gene cluster for the biosynthesis of diterpene aphidicolin into the fungal quadruple auxotrophic host, Aspergillus oryzae, by using four different vectors (pTAex3, pPTRI, pUSA and pAdeA) which harbor a starch-inducible promoter/terminator to examine the expression conditions. The resulting quadruple transformant carrying the genes of geranylgeranyl diphosphate synthase PbGGS, terpene synthase PbACS, and two monooxygenases (PbP450-1 and PbP450-2) produced aphidicolin. The double and triple transformants also respectively produced aphidicolan-16ß-ol and 3-deoxyaphidicolin. Alternative host Saccharomyces cerevisiae carrying the genes, PbGGS and PbACS, produced key intermediate aphidicolan-16ß-ol. This is the first example of a total biosynthesis of terpenoids using fungal hosts.
Assuntos
Alquil e Aril Transferases/metabolismo , Afidicolina/biossíntese , Aspergillus oryzae/genética , Inibidores Enzimáticos/metabolismo , Farnesiltranstransferase/metabolismo , Oxigenases de Função Mista/metabolismo , Saccharomyces cerevisiae/genética , Terpenos/metabolismo , Alquil e Aril Transferases/genética , Aspergillus oryzae/enzimologia , DNA Polimerase I/antagonistas & inibidores , Diterpenos/química , Diterpenos/metabolismo , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/genética , Cromatografia Gasosa-Espectrometria de Massas , Genes Fúngicos , Engenharia Genética/métodos , Vetores Genéticos , Oxigenases de Função Mista/genética , Família Multigênica , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/enzimologia , Terpenos/química , Transformação GenéticaRESUMO
Specific inhibitors of the MVA pathway (pravastatin) and the MEP pathway (fosmidomycin) were used to interfere with the biosynthetic flux which leads to the production of aphidicolin-like diterpene in leaf organ cultures of Scoparia dulcis. Treatment of leaf organs with fosmidomycin resulted in dose dependent inhibition of chlorophylls, carotenoids, scopadulcic acid B (SDB) and phytol production, and no effect on sterol production was observed. In response to the pravastatin treatment, a significant decrease in sterol and perturbation of SDB production was observed.
Assuntos
Afidicolina/biossíntese , Diterpenos/metabolismo , Eritritol/análogos & derivados , Scoparia/metabolismo , Fosfatos Açúcares/metabolismo , Antibacterianos/farmacologia , Antioxidantes/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Eritritol/metabolismo , Fosfomicina/análogos & derivados , Fosfomicina/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fotossíntese/efeitos dos fármacos , Folhas de Planta/metabolismo , Pravastatina/farmacologia , Scoparia/efeitos dos fármacos , Sementes/química , Espectrofotometria Ultravioleta , Esteróis/metabolismoRESUMO
The fungal diterpene, aphidicolin, is a well-known specific inhibitor of DNA polymerase alpha. Terpenoids are an important class of natural products. However, identification of the biosynthetic gene cluster in terpenoids is relatively rare compared with another important class of natural products, polyketides. To explore a reliable identification method for the biosynthetic gene cluster in fungal diterpenoids, cloning of the biosynthetic gene cluster of aphidicolin was employed. The application of a simple PCR method for genome walking based on the sequence of cDNA encoding aphidicolan-16beta-ol synthase (ACS) allowed us to analyze a 15.6-kb region of the Phoma betae genomic DNA. Six ORFs, PbGGS, ACS, PbP450-1, PbP450-2, PbTP, and PbTF were found in this region, and respectively expected to encode geranylgeranyl diphosphate synthase, diterpene synthase, two cytochrome P-450s, the transporter and transcription factor. Their amino acid sequences and introns were deduced by a corresponding cDNA analysis. This study shows that simple PCR-based genome walking without constructing a genomic DNA library is useful for identification of a small gene cluster. We propose a general strategy for the cloning the biosynthetic genes of fungal diterpenoids by using fungal GGS.
