Genome-based characterization of two prenylation steps in the assembly of the stephacidin and notoamide anticancer agents in a marine-derived Aspergillus sp

Abstract

Stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a core bicyclo[2.2.2]diazaoctane ring system. These bioactive fungal secondary metabolites have a range of unusual structural and stereochemical features but their biosynthesis has remained uncharacterized. Herein, we report the first biosynthetic gene cluster for this class of fungal alkaloids based on whole genome sequencing of a marine-derived Aspergillus sp. Two central pathway enzymes catalyzing both normal and reverse prenyltransfer reactions were characterized in detail. Our results establish the early steps for creation of the prenylated indole alkaloid structure and suggest a scheme for the biosynthesis of stephacidin and notoamide metabolites. The work provides the first genetic and biochemical insights for understanding the structural diversity of this important family of fungal alkaloids.

Scheme 1

Biosynthetic subunits and putative route to paraherquamide (3) and malbrancheamide (4). Molecules in the boxes have been validated as the building blocks or biosynthetic intermediates based on precursor incorporation studies.

Scheme 2

The putative biosynthetic pathway for stephacidin A (14) and notoamide natural products. (a). The early stage in stephacidin and notaomide biosynthesis. 25 serves as the common precursor to 13 and 14. 13 is then converted into 11 and 12. Substrates used in NotC studies were labeled in blue. (b). Notoamide A (9) and B (10) are possibly derived from 14. The solid arrows represent reactions that have been confirmed with bioinformatic analysis, biochemical analysis, or precursor incorporation experiments, while the dashed arrows indicate proposed biosynthetic steps. The red symbol X indicates the reaction is not supported by the current study.

Figure 1

Genetic studies of fungal alkaloids produced in the marine-derived fungus Aspergillus sp. (a). Selected fungal alkaloids isolated from the marine-derived Aspergillus sp. Compound 16 was not reported in the fungal strain but was expected as the direct precursor of compound 15. (b) The notoamide biosynthetic gene cluster derived from complete sequencing and bioinformatic mining of Aspergillus sp. MF297-2 genome.

Figure 2

Determination of NotF and NotC prenyltransferase activities. (a). Identification of NotF product (18) by LC-MS/MS analysis as described in Methods. (b). Identification of NotC reaction product (25) by LC-MS/MS analysis as described in Methods. The product (25) was further characterized by ¹H and ¹³C NMR analysis. (c). Investigation of key residues in the reaction of the reverse prenyltransferase NotF by site-directed mutagenesis. Data shown are means ± s.d. from two independent experiments.