RESUMO
Aspergillus westerdijkiae is the main producer of several biologically active polyketide metabolites including isoasperlactone and asperlactone. A 5298bp polyketide synthase gene "aomsas" has been cloned in Aspergillus westerdijkiae by using gene walking approach and RACE-PCR. The predicted amino acid sequence of aomsas shows an identity of 40-56% with different methylsalicylic acid synthase genes found in Byssochlamys nivea, P. patulum, A. terreus and Streptomyces viridochromogenes. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aomsas expression was found to be associated with the biosynthesis of isoasperlactone and asperlactone. Moreover an aomsas knockout mutant "aoDeltamsas" of A. westerdijkiae, not only lost the capacity to produce isoasperlactone and asperlactone, but also 6-methylsalicylic acid. The genetically complemented mutant ao+msas restored the biosynthesis of all the missing metabolites. Chemical complementation through the addition of 6-methylsalicylic acid, aspyrone and diepoxide to growing culture of aoDeltamsas mutant revealed that these compounds play intermediate roles in the biosynthesis of asperlactone and isoasperlactone.
Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Aspergillus/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ligases/genética , Ligases/metabolismo , Macrolídeos/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Sequência de Aminoácidos , Aspergillus/genética , Byssochlamys/enzimologia , Byssochlamys/genética , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Técnicas de Inativação de Genes , Teste de Complementação Genética , Dados de Sequência Molecular , Estrutura Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
The diversity of polyketide synthase (PKS) genes in Aspergillus ochraceus NRRL 3174 and Aspergillus carbonarius 2Mu134 has been investigated using different primer pairs previously developed for the ketosynthase (KS) domain of fungal PKSs. Nine different KS domain sequences in A. ochraceus NRRL 3174 as well as five different KS domain sequences in A. carbonarius 2Mu134 have been identified. The identified KS fragments were distributed in five different clusters on the phylogenetic tree, indicating that they most probably represent PKSs responsible for different functions.
Assuntos
Aspergillus ochraceus/enzimologia , Aspergillus/enzimologia , Policetídeo Sintases/química , Policetídeo Sintases/genética , Reação em Cadeia da Polimerase , Sequência de Aminoácidos , Aspergillus/genética , Aspergillus ochraceus/genética , Sequência de Bases , Cromatografia Líquida de Alta Pressão , DNA Bacteriano/análise , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Dados de Sequência Molecular , Ácido Penicílico/metabolismo , FilogeniaRESUMO
Fungi contaminating foods and feeds may produce many mycotoxins including ochratoxin A (OTA). Early and rapid detection of potential OTA producing fungi is important to reduce the negative impacts of OTA. In this study, two PCR specific primer pairs, AoLC35-12L/AoLC35-12R and AoOTAL/AoOTAR, were designed from a DNA sequence of a polyketide synthase gene in Aspergillus ochraceus NRRL 3174. On 14 different fungi tested by PCR, AoLC35-12L/AoLC35-12R amplified a unique band from either OTA or citrinin producers while AoOTAL/AoOTAR amplified one PCR product only from A. ochraceus. So these primers could be used to detect both OTA and citrinin producing fungi (AoLC35-12L/AoLC35-12R) or only A. ochraceus (AoOTAL/AoOTAR) from foodstuffs using PCR method.