A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Abstract

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of approximately 1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.

A fragmented aflatoxin-like gene cluster in the forest pathogen Dothistroma septosporum

“…Hybridizations were carried out at 42°C in buffer containing 50% deionized formamide, 5· SSC, 0.1% (w/ v) N-lauroylsarcosine, 0.02% (w/v) SDS, and 2% (w/v) Roche blocking reagent. Separation of D. septosporum chromosomes on a contour-clamped homogeneous electric field (CHEF) gel and Southern hybridization with [a-32 P]dCTP-labeled vbsA PCR fragment (amplified using primers vbs1aF and vbs-4R; Supplementary Table S1) were as described in Bradshaw et al (2006). DNA sequencing was carried out using an ABI Prism BigDyeä Terminator cycle sequencing ready reaction kit and an ABI3730 Genetic Analyzer (Applied Biosystems, Foster City, CA).…”

“…The dotA and pksA genes encode versicolorin reductase and polyketide synthase, respectively, which have 80% and 57% amino acid identity to their A. parasiticus orthologs, Ver-1 (AflM) and PksA (AflC). Targeted replacement of dotA and pksA confirmed they are both essential for dothistromin biosynthesis and they each have several other AF-like genes alongside (Bradshaw et al, 2002(Bradshaw et al, , 2006, suggesting they comprise two parts of a cluster of dothistromin biosynthetic genes. The aim of this work was to identify the remaining genes and to build up a complete picture of the dothistromin gene cluster, in order to provide further insight into the evolution of AF-like gene clusters.…”

The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism

“…4). Two functionally-characterized dothistromin biosynthetic genes, pksA and dotA (Bradshaw et al, 2002;Bradshaw et al, 2006) were analyzed, along with orthologs of two putative AF/ST/dothistromin regulatory genes (aflR and aflJ) that were identified in the D. septosporum genome (PID 75566 and 57214 respectively). Expression of Ds-veA was also assessed and, as expected, the DveA mutants KO1 and KO2 did not show Ds-veA expression above background.…”

“…Genes required for dothistromin biosynthesis appear to be orthologs of AF genes and a common biosynthetic pathway is proposed (Schwelm and Bradshaw, 2010;Shaw et al, 1978). However unlike AF genes, dothistromin genes are not tightly clustered, but are arranged in several mini-clusters on a 1.3 Mb chromosome (Bradshaw et al, 2006;Schwelm and Bradshaw, 2010). Furthermore, dothistromin is produced mainly during early exponential phase in culture, instead of during late exponential and stationary phases as is normally seen for secondary metabolites such as AF (Schwelm et al, 2008).…”

“…We examined whether the gene product regulates expression of dothistromin genes and other secondary metabolite genes in D. septosporum and whether veA-deficient mutants are able to cause disease in pine needles. Because dothistromin-deficient mutants also have impaired sporulation (Bradshaw et al, 2006) we also examined the possible effect of VeA on the development of this plant pathogen.…”