Polyketides are a group of extremely diverse natural products produced by polyketide synthases (PKSs). These compounds are isolated from bacteria, actinomycetes, fungi, and plants, and have a long and successful history as anticancer, antiviral, antibacterial, antifungal, antiparastic, and immunosuppressive agents. Numerous PKSs have been isolated from actinomycetes in the past decade. Their use in combinatorial biosynthesis has attracted considerable interest as a potential powerful new tool in natural product drug discovery. Only a few PKS systems have been investigated from filamentous fungi, and the mechanisms of fungal polyketide biosynthesis remain poorly understood. Brefeldin A, a macrolide polyketide produced by Eupenicilium brefeldianum, exhibits a wide range of antibiotic, antifungal and antiviral activities. The interest in brefeldin A has been greatly stimulated recently by the finding that it can selectively induce DNA fragmentation associated with apoptosis in certain types of tumor cells, and it is in pre-clinical development as an anticancer agent. Unfortunately, brefeldin A possesses a few undesirable pharmacokinetic properties, such as low aqueous solubility and poor bioavailability after oral administration. To address these issues, complicated chemical methods have been employed. On the other hand, so far almost no physiological and molecular genetic works have been performed in brefeldin A-producing E. brefeldianum. The proposed project will use the combination of gene cloning, gene disruption, over-expression and precursor-directed biosynthesis to generate brefeldin A analogs suitable for clinical applications, and to increase the fermentation titers of brefeldin A.
The specific aims of the project are: 1) to clone the brefeldin A PKS from E. brefeldianum, 2) to create fungal strains for the over-expression of brefeldin A production in both homologous and heterologous hosts, and 3) to generate novel analogs of brefeldin A by genetic engineering modification.