Fungal artificial chromosomes for mining of the fungal secondary metabolome

Abstract

BackgroundWith thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30–80 kb in size, breakthrough techniques are needed to characterize this SM wealth.ResultsHere we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery.ConclusionThe method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1561-x) contains supplementary material, which is available to authorized users.

Secondary metabolite arsenal of an opportunistic pathogenic fungus

“…This eight-gene BGC, or variations of it, is present sporadically in Aspergillus spp. including A. terreus [57], the dermatophytic genera Arthroderma and Trichophyton, the plant pathogenic Fusarium oxysporum and Fusarium avenaceum, and partially in the blackleg pathogen Leptosphaeria maculans. Over-production of this metabolite enhances virulence in a murine model of A. fumigatus infection [29], and its activity is associated with iron homeostasis and alterations in siderophore synthesis [58].…”

Modern mass spectrometry for synthetic biology and structure-based discovery of natural products

“…Through the use of fungal artificial chromosomes, Bok et al recombinantly expressed each of the 56 biosynthetic gene clusters from Aspergillus terreus in A. nidulans and identified the gene cluster for astechrome. 8 This technology has great potential when characterizing and exploring the biosynthetic gene clusters from unculturable or pathogenic fungi. Another platform that has disruptive potential was recently shown by Kang et al, where they developed a general CRISPR/Cas9 tool in yeast that combines high-throughput heterologous expression of microbial biosynthetic gene clusters with promoter replacement.…”

“…This can be done by manipulation of the native strain 6 or through heterologous expression of entire gene clusters in either targeted 7 or high-throughput fashions. 8, 9 Engineering efforts that seek to make ‘unnatural products’ also rely on the structure-based approach to screen and characterize the products made by engineering pre-existing gene clusters. 10 …”

Growth-Phase Sterigmatocystin Formation on Lactose Is Mediated via Low Specific Growth Rates in Aspergillus nidulans

“…In addition to being a model system for biochemical and genetic research in multi-cellular fungi [8], A. nidulans is also the industrial producer of the antifungal agent echinocandin B which inhibits the synthesis of glucan, a major component of the fungal cell wall [9]. It has also been used to investigate the biosynthesis of the beta-lactam antibiotic penicillin [10].…”