Identification and characterization of the asperthecin gene cluster of Aspergillus nidulans

Abstract

The sequencing of Aspergillus genomes has revealed that the products of a large number of secondary metabolism pathways have not yet been identified. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. It is, therefore, important to discover conditions or regulatory factors that can induce the expression of these genes. We report that the deletion of sumO, the gene that encodes the small ubiquitin-like protein SUMO in A. nidulans, caused a dramatic increase in the production of the secondary metabolite asperthecin and a decrease in the synthesis of austinol/dehydroaustinol and sterigmatocystin. The overproduction of asperthecin in the sumO deletion mutant has allowed us, through a series of targeted deletions, to identify the genes required for asperthecin synthesis. The asperthecin biosynthesis genes are clustered and include genes encoding an iterative type I polyketide synthase, a hydrolase, and a monooxygenase. The identification of these genes allows us to propose a biosynthetic pathway for asperthecin.

FIG. 1.

(A) HPLC profiles of extracts as detected by UV absorption at 254 nm. (B) Total ion chromatograms (TIC). Levels of production by the different strains were at the same order of magnitude. WT, sumO⁺ strain; sumOΔ, sumO deletion mutant; sumO_ki, sumOΔ strain complemented by the integration of a functional copy of sumO at the white (wA) locus; sumOΔ AN6000.3Δ, double deletion mutant.

FIG. 2.

Structures of austinol (1), dehydroaustinol (2), sterigmatocystin (3), asperthecin (4), emericellamide A (5), emericellamide C (6), emericellamide D (7), emericellamide E (8), and emericellamide F (9).

FIG. 3.

(A) Organization of the asperthecin synthase gene cluster in A. nidulans. Each arrow indicates the direction of transcription and the relative size of the open reading frame deduced from the analysis of the nucleotide sequence. Black arrows, representing the AN6000.3 (PKS), AN6001.3 (hydrolase), and AN6002.3 (monooxygenase) genes, are required for asperthecin synthesis. Gray arrows represent genes not involved in asperthecin biosynthesis according to our deletion analysis. (B) Analysis of the effects on asperthecin production of deletions of genes surrounding the asperthecin PKS gene. EIC were generated using the negative mode at m/z 317 for the sumOΔ AN5998.3Δ, sumOΔ AN5999.3Δ, sumOΔ AN6000.3Δ, sumOΔ AN6001.3Δ, sumOΔ AN6002.3Δ, sumOΔ AN6003.3Δ, and sumOΔ AN6004.3Δ strains. Levels of production by the different strains were at the same order of magnitude.

FIG. 4.

Proposed biosynthesis pathway for asperthecin (4) in A. nidulans. CoA, coenzyme A; compound 10, endocrocin-9-anthrone; compound 11, endocrocin; compound 12, emodin.