In contrast to the toxic selectivity of some natural products that allow them to be used as antibiotics, fortuitous mainstays of human health and longevity, there are opposing, blunt toxicities by primitive mechanisms of action that overwhelm host defenses in preparation for systemic invasion. Fungi in particular can be pathogenic to animals and plants and use polyketide metabolites as virulence factors in these ways against their victims. Environmental toxins have detrimental effects on human health and must be closely monitored. That vigilance accounts for several $B/yr in losses to grain and nut crops and contaminated milk and meat destroyed. The aflatoxins are a paradigm among mycotoxins and occupy a central place in environmental toxicology. Hepatocarcinomas are the third most common cause of cancer death in the world. Hepatitis infections and dietary aflatoxin are major risk factors where clear epidemiological data correlate exposure to human disease. To the environmental carcinogen aflatoxin B1 can be added polyketide metabolites like T-2 toxin, zearalenone, fumonisin B, ochratoxin, patulin, citrinin and cercosporin. These secondary metabolites all derive from a family of fungal, polydomain enzymes called polyketide synthases (PKSs). They are fundamentally related to animal fatty acid synthases (FASs), but, unlike FASs, they synthesize reactive products that can undergo biosynthetic chemistry to a wide array of structural types. Many of the primitive toxins of pathogenic fungi arise from this versatile biosynthetic machinery. These enzymes have eluded investigation for years, especially compared to other types of PKSs, but the work of this lab in the last decade has made great inroads into this problem. These enzymes exhibit the unusual property of iterative catalysis where a small basis set of catalytic domains is reused multiple times but a fixed number of times to a specific, programmed end. This poorly understood catalytic behavior has been collectively called ¿programming¿ and is the goal of this research effort to understand. Great strides have been made in the current grant period with dramatic technical advances to dissect and reassemble functional domains for biochemical and structural analysis that we intend to drive the Research Plan proposed. A comprehensive approach is advanced to proceed on three fronts: protein X-ray crystallography of individual and multiple domains from the polyketide synthases, mechanistic enzymology of PKS function, and synthetic biology and combinatorial enzymology of a library of catalytic domains to elicit further understanding of function and to carry out syntheses of new products.