10/07/10

Zearalenone is a non-steroidal oestrogenic mycotoxin produced by F. graminearum that induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals. Zearalenone has also been shown to be an immunotoxin, mutagetic, haemotoxic and hepatotoxic, however the mechanisms of toxicity is not fully understood (Zinedine et al., 2007).

The compound zearalenone is synthesised by the combined action of two PKSs and an isoamyl alcohol oxidase (Kim et al., 2005b;Gaffoor and Trail, 2006;Lysoe et al., 2006). The involved genes are encoded by 39 kb large gene cluster consisting of four genes (Figure 10): FgPKS13, FgPKS4, ZEB1 and ZEB2. The ZEB1 protein contains a basic-region leucine zipper (bZIP) domain and functions as a cluster specific transcription factor that controls the expression of the cluster (Kim et al., 2005b).

The current model for zearalenone biosynthesis is shown in figure 11, based on (Kim et al., 2005b) and (Gaffoor et al., 2005). The biosynthetic pathway is initiated by FgPKS4, a reducing PKS, which catalyse the condensation of one acetyl-CoA and five malonyl-CoA units, resulting in a hexaketide. The three reducing domains of PKS4 are deployed to various degrees during the different cycles of synthesis: ketide no. 6 remains unreduced, ketide no. 1 and 3 are processed by the KR domain resulting in hydroxyl groups, ketide no. 5 is processed by the KR and DH domains resulting in an enoyl group, while ketide no. 2 and 4 are reduced completely to alkyls by the KR, DH and ER domains. The formed hexaketide is then passed onto the non-reducing FgPKS13, functioning as a starter unit for further extension of the polyketide chain. FgPKS13 completes three iterations extending the chain by three additional ketide units, resulting in a nonaketide. The nonaketide then undergo two rounds of intramolecular cyclication reactions, resulting in formation of an aromatic ring and a macrolide ring structure with a lactone bond. The final conversion of the formed zearalenol to zearalenone is catalyzed by the isoamyl alcohol oxidase, ZEB1, which converts the macrolide bound hydroxyl group to a ketone.

Targeted replacement of FgPKS4 results in down regulation of the PKS13 gene suggesting the existence of a regulatory feedback loop similar to that described for bikaverin biosynthesis in F. fujikuroi (Lysoe et al., 2006).

This zearalenone biosynthetic pathway is a good example of how multiple iPKSs can interact to form a common product. The two PKSs presumably interact directly, as FgPKS4 does not contain a thioesterase domain for releasing the produce following synthesis, instead the product is most likely transferred directly to the ACP domain of PKS13 where it functions as a starter unit. However the direct interaction and combined action of multiple iPKSs have not been proven experimentally yet. Another example of this situation is found in the aflatoxin biosynthesis in Aspergillus sp. where the starter unit is synthesised by a fatty acid synthase complex8 (FAS-1 alfa subunit and FAS-2 beta subunit) and then passed onto an iPKS (Bhatnagar et al., 2003). The key difference between these systems and single acting iPKSs is that the first acting PKS in the combined synthesis system lack a thioesterase type domain.

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