Small NRPS-like enzymes in Aspergillus sections Flavi and Circumdati selectively form substituted pyrazinone metabolites

Abstract

Aspergillus fungi produce mycotoxins that are detrimental to human and animal health. Two sections of aspergilli are of particular importance to cereal food crops such as corn and barley. Aspergillus section Flavi species like A. flavus and A. parasiticus produce aflatoxins, while section Circumdati species like A. ochraceus and A. sclerotiorum produce ochratoxin A. Mitigating these toxins in food and feed is a critical and ongoing worldwide effort. We have previously investigated biosynthetic gene clusters in Aspergillus flavus that are linked to fungal virulence in corn. We found that one such cluster, asa, is responsible for the production of aspergillic acid, an iron-binding, hydroxamic acid-containing pyrazinone metabolite. Furthermore, we found that the asa gene cluster is present in many other aflatoxin- and ochratoxin-producing aspergilli. The core gene in the asa cluster encodes the small nonribosomal peptide synthetase-like (NRPS-like) protein AsaC. We have swapped the asaC ortholog from A. sclerotiorum into A. flavus, replacing its native copy, and have also cloned both asaC orthologs into Saccharomyces cerevisiae. We show that AsaC orthologs in section Flavi and section Circumdati, while only containing adenylation-thiolation-reductase (ATR) domains, can selectively biosynthesize distinct pyrazinone natural products: deoxyaspergillic acid and flavacol, respectively. Because pyrazinone natural products and the gene clusters responsible for their production are implicated in a variety of important microbe-host interactions, uncovering the function and selectivity of the enzymes involved could lead to strategies that ultimately benefit human health.

Keywords: ATR; aflatoxin; aspergillic acid; biosynthesis; flavacol; mycotoxin; ochratoxin.

Figure 1

The pyrazinone precursors to aspergillic acids are formed by the NRPS-like enzyme AsaC in Aspergillus spp. (A) Schematic of the asa biosynthetic gene cluster responsible for aspergillic acid production contains the NRPS-like core gene asaC, which is comprised of Adenylation-Thiolation-Reductase (ATR) domains. Introns are shown as gaps in the genes. (B) Species in Aspergillus section Flavi such as A. flavus produce aspergillic acid from leucine and isoleucine through a deoxyaspergillic acid (1) intermediate. (C) Species in Aspergillus section Circumdati such as A. sclerotiorum produce neoaspergillic acid from two leucine residues from a flavacol (2) intermediate.

Figure 2

Sequence homology of AsaC in Aspergillus section Flavi is distinct from section Circumdati species. Phylogenetic tree of AsaC NRPS-like orthologs in Aspergillus section Flavi (blue) and Circumdati (orange) species. Chemical confirmation of the substituted pyrazinones and their derivatives produced by these species are in underlined bold font (Lebar et al., 2019). Blue: predominantly deoxyaspergillic acid-derived (1), including aspergillic acid and ferriaspergillin (3); orange: exclusively flavacol-derived (2), including neoaspergillic acid and ferrineoaspergillin (4). The node values represent the branch bootstrap values using the ultrafast bootstrap approximation.

Figure 3

A. flavus containing asaC from A. sclerotiorum produces an iron(III)-bound trimer found naturally only in Aspergillus section Circumdati species. (A–D) UV chromatograms of Aspergillus extracts grown on AFPA measured in relative absorbance units (AU) at λ = 310 nm.

Figure 4

AsaC forms aspergillic acid precursor pyrazinones. (A), left) Extracted ion chromatogram (EIC) of A. flavus ΔasaD. (A), right) High energy mass spectra of peak 1 from A. flavus ΔasaD extract. (B), left) EIC of A. sclerotiorum extract. (B), right) High energy mass spectra of peak 2 from A. sclerotiorum extract. (C), left) EIC of S. cerevisiae expressing asaC from A. flavus (asaC_AF) extract. (C), right) High energy mass spectra of peak 1 from S. cerevisiae asaC_AF extract. (D), left) EIC of S. cerevisiae expressing asaC from A. sclerotiorum (asaC_AS) extract. (D), right) High energy mass spectra of peak 2 from S. cerevisiae asaC_AS extract. 1: deoxyaspergillic acid; 2: flavacol.

Figure 5

The nonribosomal codes of AsaC in Aspergillus section Flavi and section Circumdati differ by only one residue. (A) Alignment of the 10 key amino acid residues in the nonribosomal code using GrsA-PheA as a reference. AsaC_AF represents the code from A. flavus as well as AsaC from all Aspergillus section Flavi species sequenced to date (see Figure 1 ). AsaC_AS represents the code from A. sclerotiorum and AsaC from all Aspergillus section Circumdati species sequenced to date (see Figure 1 ). Stick rendering model of the 10 key amino acids in AsaC_AF (B, C) and AsaC_AS (D) enzymes using the PheA crystal structure (pdb 1AMU) as a template. Key amino acid colors are consistent with the alignment in panel (A). Complexed amino acids leucine (B, D) or isoleucine (C) are colored yellow, and cAMP (in magenta) is orientated similarly near the top of each panel.

Figure 6

Proposed biosynthetic pathways of pyrazinone metabolites. (A) Schematic of the biosynthesis of aureusimine from valine and tyrosine by Staphylococcus aureus AusA. Adapted from (Wyatt et al., 2010; Zimmermann and Fischbach, 2010). (B) Schematic of the biosynthesis of flavacol (2) from two leucines by PvfC in Pseudomonas fluorescens and AsaC_AS in Aspergillus sclerotiorum. Adapted from (Morgan et al., 2021). (C) Schematic of the biosynthesis of deoxyaspergillic acid (1) from isoleucine and leucine by AsaC_AF in A flavus.