RESUMO
The basidiomycete Hypholoma sublateritium produces the triterpenoid antitumor clavaric acid, an inhibitor of the human Ras-farnesyl transferase. The H. sublateritium squalene epoxidase gene (erg1) has been cloned and shown to encode a flavoprotein monooxygenase that requires FAD, NADPH, and P450 cofactors. Silencing of the erg1 gene in H. sublateritium using constructions expressed from the gdh promoter of Agaricus bisporus showed that the squalene epoxidase is involved in clavaric acid formation and in ergosterol biosynthesis; silenced expression of erg1 resulted in an ergosterol-dependent phenotype for full growth. Overexpression of erg1 gene resulted in up to 32% to 97% increment of clavaric acid production confirming its involvement in the biosynthesis of this antitumor product. Oxidosqualene (or dioxidosqualene) appears to be the branching point for primary metabolism (sterols) and secondary metabolites in basidiomycetes.
Assuntos
Agaricales/enzimologia , Lanosterol/análogos & derivados , Fitosteróis/biossíntese , Esqualeno Mono-Oxigenase/metabolismo , Agaricales/genética , Agaricales/metabolismo , Alquil e Aril Transferases/antagonistas & inibidores , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Ergosterol/biossíntese , Ergosterol/farmacologia , Expressão Gênica , Ordem dos Genes , Lanosterol/biossíntese , Lanosterol/farmacologia , Dados de Sequência Molecular , Naftalenos/farmacologia , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Compostos de Amônio Quaternário/farmacologia , RNA Antissenso/genética , Homologia de Sequência de Aminoácidos , Esqualeno Mono-Oxigenase/antagonistas & inibidores , Esqualeno Mono-Oxigenase/genética , Terbinafina , Transfecção , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismoRESUMO
The genetic organization of the left edge (tyIEDHFJ region) of the tylosin biosynthetic gene cluster from Streptomyces fradiae has been determined. Sequence analysis of a 12.9 kb region has revealed the presence of 11 ORFs, 10 of them belonging to the biosynthetic cluster. The putative functions of the proteins encoded by these genes are as follows: peptidase (ORF1, ddcA), tylosin resistance determinant (ORF2, tlrB), glycosyltransferase (ORF3, tylN), methyltransferase (ORF4, tylE), ketoreductase (ORF5, tylD), ferredoxin (ORF6, tylH2), cytochrome P450 (ORF7, tylH1), methyltransferase (ORF8, tylF), epimerase (ORF9, tylJ), acyl-CoA oxidase (ORF10, tylP) and receptor of regulatory factors (ORF11, tylQ). The functional identification of the genes in the proposed tylosin biosynthetic pathway has been deduced by database searches and previous genetic complementation studies performed with tylosin idiotrophic mutants blocked at various stages in tylosin biosynthesis. The tlrB gene has been shown to be useful as a tylosin resistance marker in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor and the effect of tylF on macrocin depletion has been confirmed. A pathway for the biosynthesis of 6-deoxy-D-allose, the unmethylated mycinose precursor, involving the genes tylD, tylJ and tylN is proposed.
