Isolation of a polyketide synthase gene from Dothistroma pini : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University, Palmerston North, New Zealand

Abstract

Dothistromin is a polyketide derived mycotoxin, produced by Dothistroma pini, which is structurally related to aflatoxins produced by Aspergillus parasiticus and Aspergillus flavus. Southern blot analysis of D. pini genomic DNA was carried out using a probe (KS-2) encoding the highly conserved β keto-acyl synthase domain from the polyketide synthase gene (pksLl) of A. parasiticus, which indicated the presence of a homologous gene in strain Dp 2 of D. pini. Subsequently, KS-2 hybridising lambda clones were isolated from a D. pini genomic library. A 2411 bp fragment was subcloned and sequenced. Sequence analysis recognised two functional protein domains, (β keto-acyl synthase (KS) and acyl transferase (AT), both of which are present in fatty acid and polyketide synthases. The sequence exhibited high homology with A. nidulans wA and A. parasiticus PKSL1 (62.3% and 59.9%) respectively, but only slight homology with the 6-MSA gene from Penicillium patulum and the atX gene from Aspergillus tereus. Additionally, a BLASTX search revealed some similarities with a number of FASs, although PKS genes had the highest scoring segment pairs. On the basis of these results, it is proposed that the 2.4 kb subcloned fragment encodes part of the D. pini PKS (pksDp) which synthesises the backbone polyketide and initiates dothistromin biosynthesis