Demonstration of the catalytic roles and evidence for the physical association of type I fatty acid synthases and a polyketide synthase in the biosynthesis of aflatoxin B-1

Background: Aflatoxin B-1 (compound 5) is a potent environmental carcinogen produced by certain Aspergillus species, Its first stable biosynthetic precursor is the anthraquinone norsolorinic acid (compound 3), which accumulates in the Aspergillus mutant strain NOR-1. Biochemical and genetic evidence suggest that this metabolite is synthesized in vivo by a specialized pair of fatty acid synthases (FAS-1 and FAS-2) and a separately transcribed polyketide synthase (PKS-A). Results: The N-acetylcysteamine (NAG) thioester of hexanoic acid was shown to efficiently support the biosynthesis of norsolorinic acid (compound 3) in the NOR-1 strain, In contrast, the mutants Dis-l and Dis-P, which are derived from NOR-1 by insertional inactivation of fas-1, produced unexpectedly low amounts of norsolorinic acid in the presence of hexanoylNAC. Controls eliminated defects in the parent strain or enhancement of degradative beta-oxidation activity as an explanation for the low level of production, Southern blots and restriction mapping of Dis-1 and Dis-2 suggested normal levels of expression of the PKS-A and FAS-2 proteins should be observed because the genes encoding these proteins are not physically altered by disruption of fas-1. Conclusions: The impaired ability of Dis-1 and Dis-2, harboring modified FAS-1 enzymes, to carry out norsolorinic acid synthesis implies the need for FAS-1 (and possibly also FAS-P) to physically associate with the PKS before biosynthesis can begin. The failure of the unaffected PKS alone to be efficiently primed by hexanoylNAC, and the presumed requirement for at least one of the FAS proteins to bind and transfer the C-6 unit to the PKS, is in contrast to behavior widely believed to occur for type I PKSs.