Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression

Abstract

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize because of cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wild type, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1- 2, and 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wild-type chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate-derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

Fig. 1.. Structures of compounds (1 to 9).

The structures of isolated terpenes from PdFAC1.

Fig. 2.. The hypothesized scheme of A. nidulans–derived terpenes and prenylated metabolites pathways.

Fig. 3.. Squalene production is dependent on the presence of P. destructans–derived squalene synthase gene (sqsA).

(A) Squalene standard [100 parts per million (ppm)], (B) squalene detected from the control strain (TJW167), (C) squalene detected from the PdFAC1 transformed A. nidulans (TJW336), (D) squalene detected from the PdFAC1 transformed A. nidulans with deletion of sqsA gene (TJW337), and (E) relative intensities of squalene production from each strain.

Fig. 4.. Terpene production is dependent on the presence of P. destructans–derived squalene synthase gene (sqsA).

(A to C) Full-scan spectra of TJW167 (red), TJW336 (yellow), and TJW337 (blue), respectively. (D to G) Production of austinol decreased to 2% compared to the control strain and recovered after the deletion of sqsA. Austinol ([M + H]⁺ = 459.2006) was detected at 15.74 min. (H to K) Production of aspernidine A increased to 58-fold compared to the control strain and dropped to the same level with the control strain after the deletion of sqsA. Aspernidine A ([M + H]⁺ = 400.2482) was detected at 22.42 min.

Fig. 5.. Key correlations of COSY (bold) and HMBC (arrows) experiments for compounds 1 and 2.

Fig. 6.. Experimental CD spectrum of nidulene A (1) and calculated ECD spectra of (R) and (S) forms of nidulene A (1).

Fig. 7.. Neutrophil movement is affected by compounds 1, 3, 5, and 7.

(A) Nidulene E (3) is a chemoattractant for human neutrophils. Using a Boyden chamber, neutrophils stained with calcein AM were seeded in the upper chamber at 5 × 10⁴ cells per well and incubated at 37°C for 1 hour with the vehicle (DMSO; light gray bar), positive control (fMLP, 10 nM; dark gray bar), or nidulene E (3) at four different concentrations (0.1, 1.0, 10, or 100 μg/ml; blue bars) found in the lower chamber. The media control is indicated as a dotted line across the graph. (B to D) Inhibition of fMLP-driven neutrophil chemotaxis by nidulene A (1), nidulene C (4), and aspernidine F (7). As described above, neutrophils stained with calcein AM were seeded in the upper chamber at 5 × 10⁴ cells per well and incubated at 37°C for 1 hour with the vehicle (DMSO, light gray bar), the DMSO vehicle with fMLP (fMLP at 10 nM; medium gray bar), positive control (fMLP, 10 nM; dark gray bar), or the respective compounds at four different concentrations (0.1, 1.0, 10, or 100 μg/ml; red bars) found in the lower chamber. The media control is indicated as a dotted line across the graph. For all graphs (A to D), cells that migrated into the lower chamber were quantified using flow cytometry and the percent live cells that migrated were determined by calculating the percent live neutrophils from the loading control. Samples are listed as average ± SEM, and assays have been repeated to assure reproducibility. Statistical differences were assessed using one-way analysis of variance (ANOVA). Different letters denote statistical differences.