解析棘孢麴黴之倍半萜類-Aculenes的生合成途徑

Abstract

Aculenes are sesquiterpenes produced by Aspergillus aculeatus, a black filamentous fungus. Among the identified aculenes, aculenes A-D and asperaculene B contain an unprecedented norsesquiterpene scaffold that may derive from ent-daucane-type skeleton. As a cumulative number of aculenes that has been identified with chemical investigation, the genetic and enzymatic basis involved aculenes biosynthesis remains unknown. In this study, we have identified the biosynthetic gene cluster of aculenes and proposed the biosynthetic pathway based on gene inactivation, heterologous reconstitution, and biochemical characterization. From knockout strains and heterologous co-expression transformants of saccharomyces cerevisiae, we isolated thirteen aculene derivatives including seven new compounds. Based on the results, we discovered three P450 monooxygenases involving in multiple oxidative steps, one dioxygenase that introducing a double bond, and one non-ribosomal peptide synthase (NRPS) that transferring a proline moiety to the sesquiterpene skeleton. Furthermore, we characterized a terpene synthase that forms the ent-daucane-type skeleton of aculenes with heterologous expression. Biochemical characterization of the NRPS and hydrolase were also investigated. Interestingly, the deletion of a gene coding proline iminopeptidase on the aculene biosynthetic gene cluster did not influence the production of aculene A. The bioinformatic analysis on the A. aculeatus genome indicated that there are two proline iminopeptidase homologs within the genome, while most fungi only have one proline iminopeptidase. We hypothesized that the aculene-type metabolites may play a certain role in the interaction with the two proline iminopeptidase homologs. Further molecule-enzyme interaction will be investigated by enzymatic assay methods. We will continue to characterize the function of three P450s and to identify the key enzymes involved in the generation of norsesquiterpene skeleton. Furthermore, biological implication may be obtained based on the investigation of the interaction between aculene derivatives and proline iminopeptidase homologs.