RESUMO
Fungal highly reducing polyketide synthases (HRPKSs) are an enigmatic group of multidomain enzymes that catalyze the biosynthesis of structurally diverse compounds. This variety stems from their intrinsic programming rules, which permutate the use of tailoring domains and determine the overall number of iterative cycles. From genome sequencing and mining of the producing strain Eupenicillium brefeldianum ATCC 58665, we identified an HRPKS involved in the biosynthesis of an important protein transport-inhibitor Brefeldin A (BFA), followed by reconstitution of its activity in Saccharomyces cerevisiae and in vitro. Bref-PKS demonstrated an NADPH-dependent reductive tailoring specificity that led to the synthesis of four different octaketide products with varying degrees of reduction. Furthermore, contrary to what is expected from the structure of BFA, Bref-PKS is found to be a nonaketide synthase in the absence of an associated thiohydrolase Bref-TH. Such chain-length control by the partner thiohydrolase was found to be present in other HRPKS systems and highlights the importance of including tailoring enzyme activities in predicting fungal HRPKS functions and their products.
Assuntos
Anti-Infecciosos/metabolismo , Brefeldina A/metabolismo , Eupenicillium/enzimologia , Hidrolases/metabolismo , Policetídeo Sintases/metabolismo , Anti-Infecciosos/química , Brefeldina A/química , Clonagem Molecular , Eupenicillium/química , Eupenicillium/genética , Eupenicillium/metabolismo , Genoma Fúngico , Dados de Sequência Molecular , Família Multigênica , Policetídeo Sintases/genética , Saccharomyces cerevisiae/genética , TranscriptomaRESUMO
Azaphilones are a class of fungal metabolites characterized by a highly oxygenated pyrano-quinone bicyclic core and exhibiting a broad range of bioactivities. Although widespread among various fungi, their biosynthesis has not been thoroughly elucidated. By activation of a silent (aza) gene cluster in Aspergillus niger ATCC 1015, we discovered six azaphilone compounds, azanigerones A-F (1, 3-7). Transcriptional analysis and deletion of a key polyketide synthase (PKS) gene further confirmed the involvement of the aza gene cluster. The biosynthetic pathway was shown to involve the convergent actions of a highly reducing PKS and a non-reducing PKS. Most significantly, in vitro reaction of a key flavin-dependent monooxygenase encoded in the cluster with an early benzaldehyde intermediate revealed its roles in hydroxylation and pyran-ring formation to afford the characteristic bicylic core shared by azaphilones.
Assuntos
Aspergillus niger/genética , Proteínas Fúngicas/genética , Pigmentos Biológicos/biossíntese , Piranos/metabolismo , Aspergillus niger/química , Aspergillus niger/enzimologia , Benzopiranos/química , Proteínas Fúngicas/metabolismo , Técnicas de Inativação de Genes , Hidroxilação , Família Multigênica , Pigmentos Biológicos/química , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Piranos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
A dazzling array of enzymes is used by nature in making structurally complex natural products. These enzymes constitute a molecular toolbox that may be used in the construction and fine-tuning of pharmaceutically active molecules. Aided by technological advancements in protein engineering, it is now possible to tailor the activities and specificities of these enzymes as biocatalysts in the production of both natural products and their unnatural derivatives. These efforts are crucial in drug discovery and development, where there is a continuous quest for more potent agents. Both rational and random evolution techniques have been utilized in engineering these enzymes. This review will highlight some examples from several large families of natural products.
