The work described in this application is aimed at developing in vitro methods for preparing polyketide-derived natural products. The polydetides comprise a class of clinically important compounds which include the tetracycline antibiotics, anthracycline antineoplastics, and antifungal agents. Polyketides are biosynthesized via the consectuve addition of acetate units, activated as malonyl coenzyme A, to a started unit of acetyl or propionyl CoA. Coenzyme A and its thioester derivatives cannot be used with isolated enzymes on a preparative scale because they are prohibitively expensive and are not available in larger quantities. This application describes work which will allow coenzyme A and its thioester derivatives to be recycled into their active forms using immobilized enzymes. Recycling of these materials will allow coenzyme A to be used for the production of fatty acids and polyketide natural products. Enzymes which can synthesize polyketide natural products have been isolated previously for biosynthetic studies. By employing coenzyme A recycling methodology it should be possible to produce useful quantities of these natural products. The first compounds that will be examined (orsellinic acid, alternariol) require only condensation reactions between acetyl and malonyl CoA thioesters. These reactions will be catalysed by immobolized enzymes isolated from the organisms which produce these substances. If these reactions are successful in producing these polyketide natural products, it will demonstrate the feasibility of using immobilized enzyme systems for producing secondary metabolite natural products. There are significant advantages of such technology. Intact microorganisms oftentimes are under strict regulatory control which limits the amount of product which can be formed. By removing the enzymes from the cell and stabilizing them through immobilization, continuous production of the secondary metabolites should be possible. Furthermore, by preparing analogs of the coenzyme A thioesters it should be possible to prepare analogs of natural products with different therapeutic properties. By employing the practical advantages of coenzyme recycling with the ability to preserve biosynthetic activity through immobilization, the proposed research should eventually lead to a new and practical method for preparing important pharameuticals.
