Depsipeptide Aspergillicins Revealed by Chromatin Reader Protein Deletion

Abstract

Expression of biosynthetic gene clusters (BGCs) in filamentous fungi is highly regulated by epigenetic remodeling of chromatin structure. Two classes of histone modifying proteins, writers (which place modifications on histone tails) and erasers (which remove the modifications), have been used extensively to activate cryptic BGCs in fungi. Here, for the first time, we present activation of a cryptic BGC by a third category of histone modifying proteins, reader proteins that recognize histone tail modifications and commonly mediate writer and eraser activity. Loss of the reader SntB (Δ sntB) resulted in the synthesis of two cryptic cyclic hexa-depsipeptides, aspergillicin A and aspergillicin F, in the fungus Aspergillus flavus. Liquid chromatography, high resolution mass spectrometry, and NMR analysis coupled with bioinformatic analysis and gene deletion experiments revealed that a six adenylation (A) domain nonribosomal peptide synthetase (NRPS, called AgiA) and O-methyltransferase (AgiB) were required for metabolite formation. A proposed biosynthetic scheme illustrates the requirement for unusual NRPS domains, such as a starting condensation domain and a thiolesterase domain proposed to cyclize the depsipeptides. This latter activity has only been found in bacterial but not fungal NRPS. The agi BGC-unique to A. flavus and some closely related species (e.g., A. oryzae, A. arachidicola)-is located next to a conserved Aspergillus siderophore BGC syntenic to other fungi.

Figure 1.

(A) UPLC-HRMS chromatograms (TIC, linked axis) of A.flavus wild-type (WT) and ΔsntB. Method 20–95% CH₃CN/H₂O gradient, 20 min. AF = aflatoxin B1; CA = cyclopiazonic acid. (B) Structures of aspergillicins A–G, siderophores fusarinine and TAFC, fumarylalanine, and 4′-methoxyviridicatin.

Figure 2.

(A) Proposed biosynthesis of aspergillicin A and F. Different colors highlight specific domains and peptide modifications. Domains: C_s, starter condensation domain; A, adenylation; PCP, peptidyl carrier protein; C, condensation; E, epimerase; NMeT, N-methyl transferase; TE, thiolesterase. (B) Putative aspergillicin BGC and flanking genes. Synteny analysis identifies that siderophore genes are conserved across different Aspergillus species. In A. arachidicola, the BGC is in two different contigs, indicated by the two brackets.

Figure 3.

UPLC-HRMS single ion chromatograms (linked axis), selecting the masses for aspergillicin A 1, aspergillicin F 2, aspergillicin C 4, and aspergillicin G 11. Method 20–95% CH₃CN/H₂O gradient, 20 min.