抄録
Polyketides are one of the largest and most important group of natural products. In spite of their structural diversity, the initial reactions of polyketide biosynthesis follow a common scheme that condensation of acyl primer with malonate units to form β-polyketomethylene intermediates and their cyclizations catalyzed by so called polyketide synthases (PKS). Although molecular genetic analysis of bacterial polyketide biosynthesis genes have been extensively carried out, only several PKS genes have been cloned from eukaryotic filamentous fungi, which are another rich source of polyketides, especially, aromatic compounds. All fungal PKS genes so far cloned code for iterative multifunctional type I PKS polypeptides. Though, little has been known how fungal PKSs control their reactions, that is, how to regulate the chain length of β-polyketomethylene intermediates and their cyclizations. Thus, we carried out functional analysis of Aspergillus nidulans wA gene which was assumed to code for PKS involved in conidial spore pigment biosynthesis. The wA gene was expressed in a heterologous fungus Aspergillus oryzae using fungal expression plasmid pTAex3. By starch induction, the A. oryzae transformant produced compounds of which structures were identified to be citreoisocoumarin (1) and its derivatives. In the course of these experiments, we found one base missing in the reported nucleotide sequence of wA gene that causes a frame shift in its C-terminus region. As a result, it was found that isocoumarins were produced by WA polypeptide with truncated C-terminus. The intact WA expression plasmid reconstructed was introduced into A. oryzae. The transformant produced a novel naphthopyrone compound YWA1 (4) by starch induction in place of isocoumarins. Both isocoumarins and naphthopyrone YWA1 (4) are heptaketides, but their cyclization patterns are different. That is, the former lack the second aromatic ring cyclization, in which the truncated C-terminus of WA polypeptide was suggested to be involved. By C-terminus modifications of WA polypeptide including further truncations and site-directed mutagenesis, C-terminus region, containing Ser^<1967> and His^<2129>, was identified to function as Claisen type cyclase. One of the characteristic feature of the WA polypeptide is that it contains two ACP motifs. Site-directed mutagenesis on Ser^<1682> and Ser^<1804> which are presumed phosphopantetheine modification sites revealed that both of two ACPs function independently in WA polyketide synthase reaction.
