The potential role of fatty acid initiation in the biosynthesis of the fungal aromatic polyketide aflatoxin B1

Earlier work in this laboratory has shown the intact incorporation of [1-C-13]hexanoate into averufin (1), a key intermediate in aflatoxin B-1 biosynthesis. Parallel experiments with equimolar amounts of [1-C-13]butyrate, [1-C-13]-3-oxo-octanoate, and [1-C-13]-5-oxo-hexanoate gave no detectable specific incorporation of heavy isotope but low and equivalent background incorporation comparable to [1-C-13]acetate. Three of these potential intermediates in polyketide formation were reexamined as their corresponding N-acetylcysteamine (NAC) thioesters. The NAC thioester of [1-C-13]hexanoic acid gave a remarkably high 22% intact incorporation while the NAC thioester of [1-C-13]-3-oxo-octanoic acid afforded nearly 5% when an equimolar amount was administered to the producing organism Aspergillus parasiticus (ATCC 24551). In contrast, the NAC thioester of [1-C-13]butyric acid showed no selective enrichment of averufin. This negative result was tested further in a more sensitive experiment with the NAC thioester of [2,3-C-13(2)]butyric acid. No (1)J(CC) coupling was detectable, indicating an incorporation efficiency of <0.1%. [1-C-13,O-18(2)]Hexanoate was prepared and gave a 53% retention of O-18 relative to the C-13 internal standard in keeping with previous experiments with [1-C-13,O-18(2)]acetate. It is concluded from these data that the initial C-6 segment of polyketide biosynthesis is unlikely to arise by beta-oxidation of a higher fatty acid but more probably is generated by a specialized fatty acid synthase (FAS) that provides this unit either separately to the polyketide synthase (PKS) or as part of a larger FAS/PKS fusion. While these two physical arrangements cannot be distinguished by these experiments, both must accommodate comparatively efficient exchange of the NAC thioesters of both hexanoic and 3-oxo-octanoic acid, but not the NAC thioester of butyric acid.