Acetylaszonalenin Biosynthesis in Neosartorya fischeri

Abstract

Based on the structural information of acetylaszonalenin iso-lated from Neosartorya fischeri, a putative biosynthetic gene cluster was identified in the genome sequence of this fungus by genomic mining. This cluster consists of three genes coding for a putative non-ribosomal peptide synthetase (AnaPS), a prenyl-transferase (AnaPT), and an acetyltransferase (AnaAT). The coding sequences of anaPT and anaAT were cloned in pQE70 and pQE60, respectively, and overexpressed in Escherichia coli. The soluble His 6 fusion proteins were purified to near homoge-neity and characterized biochemically. The structures of the enzymatic products were elucidated by NMR and mass spectros-copy analysis. AnaPT was found to catalyze the reverse prenyla-tion of (R)-benzodiazepinedione at position C3 of the indole moiety in the presence of dimethylallyl diphosphate, resulting in formation of aszonalenin. AnaAT was found to catalyze the acetylation of aszonalenin at position N1 of the indoline moiety in the presence of acetyl coenzyme A, resulting in formation of acetylaszonalenin. K m values of AnaPT were determined for di-methylallyl diphosphate at 156 ␮M and for (R)-benzodiaz-epinedione at 232 ␮M. K m values of AnaAT were determined for acetyl coenzyme A at 96 ␮M and for aszonalenin at 61 ␮M. The turnover numbers of the AnaPT and AnaAT reactions were determined at 1.5 and 0.14 s ؊1 , respectively.