Cloning And Functional Analysis Of Aspergiolide A Related Polyketide Synthase Genes In Marine-derived Filamentous Fungus Aspergillus Glaucus

Aspergiolide A, a novel anthraquinone derivative, was produced by a marine-derived filamentous fungus Aspergillus glaucus HB1-19(CCTCC M206022). Aspergiolide A exhibited cytotoxicity against several cancer cell lines. It plays an important role for drug discovery and structure modifications. In our previous experiments, the aspergiolide A production from fermentation of the wild type strain (WT) was very low and thus limited the vivo activity analysis and pharmacological research. Using pathway inhibitor and precursor feeding and isotope labeling precursor feeding experiment, we demonstrated that12intact acetate units were incorporated into aspergiolide A by polyketide pathway. In this study, cloning and functional analysis of aspergiolide A related polyketide synthase genes in marine-derived Aspergillus glaucus HB1-19were investigated to set the basis for further regulation of aspergiolide A biosynthesis.According to the biosynthesis pathway of aspergiolide A, we suggest that aspergiolide A is related to type I NR-PKS. We first amplified NR-PKS gene fragment by LC1/LC2c degenerate primer pairs. Genome walking was then performed and a full length of DNA sequence of22.8kb including7complete ORFs, which contained a NR-PKS gene of8451bp, was finally obtained. The new obtained NR-PKS in A. gluacus was named as Agpksl. Afterwards, gene deletion of KS-AT domains of this NR-PKS was conducted, and a AAgKS-AT deficient strain was constructed by homologous integration of double crossover with the zeocin resistance gene Sh ble as a marker. The morphology and asexual reproduction of the AAgKS-AT strain significantly changed as compared to the WT. The ability of conidiospores of the AAgKS-AT was sligntly enhanced, while the ability of cleistothecia generation was significantly enhanced. Moreover, the color of△AgKS-AT colony presents faint yellow while the color of the WT is dark-green. The secondary metabolites, aspergiolide A production of the AAgKS-AT deduced from78mg/L to only9mg/L, which experienced a88%reduction compared with the WT, and production of Catenarin, the intermediate of aspergiolide A biosynthesis, decreased from240mg/L to140mg/L, which was41.7%lower than the WT. The results indicated that Agpksl was closely associated with the biosynthesis of aspergiolide A.