Two prenyltransferases from Aspergillus ruber control the echinulin biosynthesis via exceptional sequential prenylations. EchPT1 catalyzes the first prenylation step, leading to preechinulin. The unique EchPT2 attaches, in a consecutive prenylation cascade, up to three dimethylallyl moieties to preechinulin and its dehydro forms neoechinulins A and B, resulting in the formation of at least 23 2- to 4-fold prenylated derivatives. Confirming these products in fungal extracts unravels the unprecedented catalytic relevance of EchPT2 for structural diversity.

The biosynthetic pathway of the prenylated salicylaldehyde flavoglaucin and congeners in Aspergillus ruber was elucidated by genome mining, heterologous expression, precursor feeding, and biochemical characterization. The polyketide skeleton was released as alkylated salicyl alcohols, which is a prerequisite for consecutive hydroxylation and prenylation, before reoxidation to the final aldehyde products. Our results provide an excellent example for a highly programmed machinery in natural product biosynthesis.

The prenyltransferases EchPT1 and EchPT2 from Aspergillus ruber are responsible for the consecutive prenylations of cyclo-L-Trp-L-Ala, leading to the formation of the triprenylated echinulin as the predominant product. In this study, we demonstrate that EchPT1 also accepts all stereoisomers of cyclo-Trp-Ala and cyclo-Trp-Pro and catalyses regiospecific reverse C2-prenylation at the indole nucleus. EchPT1 products were well accepted by EchPT2 for multiple consecutive prenylations, with conversion yields of 84 to 98% for six of the eight substrates. C2-, C5- and C7-triprenylated derivatives are identified as major enzyme products, with product yields of 40 to 86% in seven cases. High product yields of 25-36%, i.e. approximate 30% of the total enzyme products, were observed for tetraprenylated derivatives in the four reaction mixtures with one D- and one L-configured amino acid residues. To the best of our knowledge, enzymatic preparation of tetraprenylated cyclodipeptides with such high efficacy has not been reported prior to this study.

Prenylation of cyclodipeptides contributes largely to the structure diversification and biological activity. The prenylated products can be further metabolized by modifications like hydroxylation with cytochrome P450 enzymes or nonheme Fe/2-oxoglutarate-dependent oxygenases. Herein, we cloned and overexpressed NFIA_045530 from Neosartorya fischeri, which shares high sequence similarity with the nonheme Fe/2-oxoglutarate-dependent oxygenase FtmOx1 from Aspergillus fumigatus on the amino acid level. FtmOx1 is a member of the biosynthetic enzymes for fumitremorgin-type mycotoxins and catalyzes the conversion of fumitremorgin B to verruculogen by insertion of an oxygen molecule into the two prenyl moieties. The recombinant protein EAW25734 encoded by NFIA_045530 was purified to apparent homogeneity and then was used for incubation with intermediates of the fumitremorgin biosynthetic pathway. LC-MS analysis revealed no consumption of fumitremorgin B but good conversion with its biosynthetic precursor tryprostatin B in the presence of Fe and 2-oxoglutarate. Structure elucidation confirmed 22-hydroxylisotryprostatin B and 14α, 22-dihydroxylisotryprostatin B as the major enzyme products. Further detailed biochemical characterization led to the identification of a novel enzyme, which catalyzes a double bond migration within the dimethylallyl moiety of tryprostatin B with concomitant hydroxylation. Incubation with O-enriched atmosphere confirmed O as the major origin of the hydroxyl groups. Solvent exchange was also observed for that at C22. LC-MS analysis confirmed the presence of 22-hydroxylisotryprostatin B in a Neosartorya fischeri extract, highlighting the role of this enzyme in the metabolism of intermediates of the fumitremorgin/verruculogen pathway. A plausible reaction mechanism implementing a radical rearrangement prior to accepting a hydroxyl radical from Fe is discussed.