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

Abstract

Earlier work in this laboratory has shown the intact incorporation of [1-'3~]hexanoate into averufin (I), a key intermediate in aflatoxin B1 biosynthesis. Parallel experiments with equimolar amounts of [l-13~]butyrate, [l-13~]-3-oxo-octanoate, and [l-13~]-5-oxo-hexanoate gave no detectable specific incorporation of heavy isotope but low and equivalent background incorporation comparable to [l-13~]acetate. Three of these potential intermediates in olyketide formation were reexamined as their P corresponding N-acetylcysteamine (NAC) thioesters. The NAC thioester of [I-' Clhexanoic acid gave a remarkably high 22% intact incorporation while the NAC thioester of [l-13~]-3-oxo-octanoic acid afforded nearly 5% when an equimolar amount was administered to the producing organism Aspergillusparasiticus (ATCC 2455 1). In contrast, the NAC thioester of [~-'~C ] b u t~r i c acid showed no selective enrichment of averufin. This negative result was tested further in a more sensitive experiment with the NAC thioester of [2,3-13~2]butyric acid. No l~c c coupling was detectable, indicatin an incorporation efficiency of <0.1%. F .[ 1 -1 3~, 1 s~2 ]~e x a n o a t e was pre ared and gave a 53% retention of "0 relative to the C lnternal standard in keeping with pre-vious experiments with [l-C, 02]acetate. It is concluded from these data that the initial C6 segment of polyketide biosynthesis is unlikely to arise by P-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 FASIPKS 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 3-0x0-octanoic acid, but not the NAC thioester of butyric acid. Ef 'tat est en accord avec les expkriences antCrieures avec du [I-13C,l 02]acCtate. Sur la base de ces donnCes on conclut que le segment initial en C6 de la synthkse du polycCtide ne provient probablement pas d'une oxydation P d'un acide gras de poids molCculaire plus ClevC, mais qu'il est vraisemblablement gCnCrC par une synthase d'acide gras spCcialiste (FAS) qui fournit cette unite soit d'une f a~o n sCparCe par rapport B la synthase de polycCtide (PKS) ou dans le cadre d'une fusion plus large FASIPKS. Alors que l'on ne peut pas distinguer entre ces deux arrangements physiques sur la base des expkriences rCalisCes, les deux doivent toutefois accommoder un Cchange efficace des thioesters de la NAC des acides tant hexanoi'que que 3-0x0-octanoi'que, mais pas de l'acide butyrique.[Traduit par la rCdaction]In 1982 we made the unexpected and intriguing observation that [1-13c]hexanoate incorporated label intact into averufin (I), a central intermediate in the biosynthesis of the potent mycotoxin aflatoxin B1 (2). The efficiency of linear fatty acid utilization in these whole-cell experiments was 3 4 % at C-1'. The specific incorporation was seen in a background of c...

Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase

“…The ability to bypass HexA͞HexB could be further demonstrated when [1-13 C]hexanoic acid was found to give a 3% specific incorporation into the aflatoxin precursor, averufin (7), in a blocked mutant of A. parasiticus (16). This acid, as the SNAC thioester to mimic the ACP͞CoA phosphopantetheine arm, dramatically increased whole-cell incorporation (Ϸ22% specific incorporation), and a remarkable selectivity was observed for hexanoyl over other short-chain SNAC thioesters (17). In a related set of experiments, intact incorporation of butanoate, heptanoate, or octanoate was not observed (18).…”

Characterization of the polyketide synthase gene (pksL1) required for aflatoxin biosynthesis in Aspergillus parasiticus

“…These toxic and carcinogenic compounds frequently contaminate food and feed commodities (3, 36). A typical AF, AFB 1 for example, is produced by the following generally accepted pathway: acetate building blocks (also hexanoate)3anthrone-derivative polyketide3norsolorinic acid (NA)3averanfin3averufin3hydroxyversicolorine3versiconal hemiacetal acetate3versicolorin B3versicolorin A3sterig-matocystin3o-methylsterigmatocystin3AFB 1 (13,15,27,58). NA is polyketide derived and represents the first stable intermediate in AF biosynthesis (34,37).…”

“…Genes involved in the initial steps of the pathway are still unknown; their identification and characterization are essential to understanding the initiation of AF biosynthesis (15). In the initial steps of AF biosynthesis, there is predicted to be a polyketide synthase (PKS), but the enzyme has not been isolated or characterized (15).…”

“…In the initial steps of AF biosynthesis, there is predicted to be a polyketide synthase (PKS), but the enzyme has not been isolated or characterized (15). Generally, a polyketide is synthesized by a PKS in a pattern resembling fatty acid biosynthesis (61).…”

“…on May 10, 2018 by guest http://jb.asm.org/ formation of the NA-derivative polyketide (15,58), there is no need for any reduction function from the PKS enzyme in synthesizing the NA-derivative polyketide (13,16). Similar observations were made for tetracenomycin biosynthesis and its PKS (39).…”