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 due to 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 new twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wildtype, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1-2, 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 wildtype 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.. Structure of compounds (1–9).

The structure 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 presence of P. destructans derived squalene synthase gene (sqsA).

(A) squalene standard (100 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) (E) relative intensities of squalene production from each strain.

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

(A–C) Full scan spectra of TJW167 (red), TJW336 (yellow), and TJW337 (blue), respectively. (D–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–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. 4.. Terpene production is dependent on presence of P. destructans derived squalene synthase gene (sqsA).

(A–C) Full scan spectra of TJW167 (red), TJW336 (yellow), and TJW337 (blue), respectively. (D–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–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 1–2.

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 impacted by compounds 1, 3, 5 and 7.

(A) Nidulene E (5) 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/well and incubated at 37 °C for 1 h with the vehicle (DMSO; light grey bar), positive control (fMLP, 10 nM; dark grey bar) or the nidulene E (5) at four different concentrations (0.1, 1, 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–D) Inhibition of fMLP-driven neutrophil chemotaxis by nidulene A (1), nidulene C (3), and aspernidine F (7). As described above, neutrophils stained with calcein AM were seeded in the upper chamber at 5 × 10⁴ cells/well and incubated at 37 °C for 1 h with the vehicle (DMSO, light grey bar), the DMSO vehicle with fMLP (fMLP at 10 nM; medium grey bar), positive control (fMLP, 10 nM; dark grey bar) or the respective compounds at four different concentrations (0.1, 1, 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–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 a one-way ANOVA. Different letters denote statistical differences.