期刊
BMC GENOMICS
卷 16, 期 -, 页码 -出版社
BIOMED CENTRAL LTD
DOI: 10.1186/s12864-015-1561-x
关键词
Fungal artificial chromosome (FAC); Functional genomics; Secondary metabolite (SM) gene clusters; Natural product discovery
资金
- National Institutes of Health [1R43AI94885-1, R01GM067725, 5PO1GM084077]
- Innovation & Economic Development Research [101PRJ72KQ]
Background: With 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. Results: Here 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. Conclusion: The 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.
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