The validity of this putative biosynthetic pathway has been supported by a biomimetic total synthesis of alternariol, where 3,5,7,9,11,13-hexaoxotetradecanoic acid (corresponding to the penultimate product in ) could be cyclized to the natural product, albeit with low yield . A short and high-yielding total synthesis of alternariol with a biaryl coupling as the key step has been published quite recently .
Alternaria alternata produces more than 60 secondary metabolites, among which alternariol (AOH) and alternariol-9-methyl ether (AME) are important mycotoxins. Whereas the toxicology of these two polyketide-based compounds has been studied, nothing is known about the genetics of their biosynthesis. One of the postulated core enzymes in the biosynthesis of AOH and AME is polyketide synthase (PKS). In a draft genome sequence of A. alternata we identified 10 putative PKS-encoding genes. The timing of the expression of two PKS genes, pksJ and pksH, correlated with the production of AOH and AME. The PksJ and PksH proteins are predicted to be 2222 and 2821 amino acids in length, respectively. They are both iterative type I reducing polyketide synthases. PksJ harbors a peroxisomal targeting sequence at the C-terminus, suggesting that the biosynthesis occurs at least partly in these organelles. In the vicinity of pksJ we found a transcriptional regulator, altR, involved in pksJ induction and a putative methyl transferase, possibly responsible for AME formation. Downregulation of pksJ and altR caused a large decrease of alternariol formation, suggesting that PksJ is the polyketide synthase required for the postulated Claisen condensations during the biosynthesis. No other enzymes appeared to be required. PksH downregulation affected pksJ expression and thus caused an indirect effect on AOH production.
Mycotoxins - Their Biosynthesis in Alternaria
Here, we identified the polyketide synthase, PksJ, involved in alternariol biosynthesis in A. alternata. PksJ is predicted to be a 2225 amino acid long, multifunctional iterative type I RD-PKS with KS, AT, DH, ER, KR and ACP domains. The expression of the pksJ gene appeared to be highest from day seven onwards when compared to other PKS genes, which correlated well with AOH production. PksJ carries a peroxisomal targeting signal type 1 (PTS1) at the C-terminal end, which suggests that AOH biosynthesis takes place in peroxisomes. This is not the first example for a role of peroxisomes in secondary metabolite biosynthesis. Recently, this has been shown for an enzyme of the AK toxin biosynthesis in A. alternata and also for penicillin biosynthesis , , .
Two Separate Gene Clusters Encode the Biosynthetic …
Here we present the first report on a polyketide synthase involved in the biosynthesis of alternariol (AOH) and alternariol-methyl-ether (AME). We used both, gene deletion and RNA-silencing strategies to knock-down the function of PKS and other genes in A. alternata, which are involved in the biosynthetic pathway.
NMR Chemical Shifts of Common Laboratory Solvents …
Although much progress has been made on the molecular characterization of the genes involved in and the regulation of the biosynthesis of other mycotoxins like fumonisin or trichothecene, no report is yet available regarding alternariol biosynthesis. The genes involved in AOH biosynthesis have yet to be discovered, despite AOH being one of the major mycotoxins produced by Alternaria species such as A. alternata and despite the importance of AOH as contaminant of food and feed.
Constructing Polyketides: From Collie to Combinatorial Biosynthesis
Biosynthetic routes for AOH were first extensively studied by Thomas who suggested that this metabolite might be synthesized by head-to-tail condensations of acetate units. Later, Gatenbeck and Hermodsson determined that malonate formed by carboxylation of acetate was the polycondensing molecule. These authors also isolated an enzyme, alternariol-O-methytransferase from A. alternata that contained O-methyltransferase activity, which converted AOH to AME .