RESUMO
The diterpene pleuromutilin is a ribosome-targeting antibiotic isolated from basidiomycete fungi, such as Clitopilus pseudo-pinsitus. The functional characterization of all biosynthetic enzymes involved in pleuromutilin biosynthesis is reported and a biosynthetic pathway proposed. In vitro enzymatic reactions and mutational analysis revealed that a labdane-related diterpene synthase, Ple3, catalyzed two rounds of cyclization from geranylgeranyl diphosphate to premutilin possessing a characteristic 5-6-8-tricyclic carbon skeleton. Biotransformation experiments utilizing Aspergillus oryzae transformants possessing modification enzyme genes allowed the biosynthetic pathway from premutilin to pleuromutilin to be proposed. The present study sets the stage for the enzymatic synthesis of natural products isolated from basidiomycete fungi, which are a prolific source of structurally diverse and biologically active terpenoids.
Assuntos
Basidiomycota/genética , Diterpenos/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Aspergillus oryzae/metabolismo , Basidiomycota/enzimologia , Vias Biossintéticas/genética , Ciclização , Diterpenos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Plasmídeos/genética , Plasmídeos/metabolismo , Compostos Policíclicos , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/metabolismo , PleuromutilinasRESUMO
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
Mutilin (4) and deoxy analogues 2 and 3 are biosynthetic precursors of pleuromutilin (1) in the later stage of biosynthesis. Precursors 2 and 3 are required for studies on the oxygenation steps in biosynthesis, and were synthesized from readily available 1 via 4 by deoxygenation of the hydroxy groups. Feeding experiments with the (2)H-labeled precursors confirmed their microbial conversion into 1.
Assuntos
Agaricales/metabolismo , Antibacterianos/metabolismo , Cetonas/metabolismo , Compostos Policíclicos/metabolismo , Deutério , Diterpenos/metabolismo , PleuromutilinasRESUMO
To obtain insight into how the cyclization pathway is controlled, the mechanism of diterpene synthase reactions (the putative phomactatriene synthase and taxadiene synthases) involving the same intermediate was investigated in detail. The mechanism of the initial transformation of GGDP to verticillen-12-yl cation (A+) was proposed based on the labelling pattern of phomactatriene (9a) obtained in the feeding experiments with 13C-labelled acetates. To obtain information on the reaction pathway of A+ to 9a and taxadiene, reactions of verticillol with various acids were conducted. Structural determination of products allowed us to propose a reaction pathway via cations A+, D+, E+, F+ and G+. Identification of hydrocarbons in mycelial extracts of phomactin-producing fungus supported the proposed reaction mechanism. Based on the results of ab initio calculations for highly flexible cation intermediates, a mechanism is proposed.
