Fungal artificial chromosomes for mining of the fungal secondary metabolome
| Autor: | David A. Mead, Amanda Krerowicz, Rosa Ye, Nancy P. Keller, Anthony W. Goering, Kenneth D. Clevenger, Neil L. Kelleher, Chengcang Wu, Paul M. Thomas, Megan Wagner, Jin Woo Bok, Jessica C. Albright |
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| Jazyk: | angličtina |
| Předmět: |
Autonomously replicating sequence
Human artificial chromosome Computational biology Genome Aspergillus nidulans Mass Spectrometry Piperazines Natural product discovery Escherichia coli Secondary metabolite (SM) gene clusters Genetics Metabolomics Chromosomes Artificial Aspergillus terreus Replicon Fungal artificial chromosome (FAC) Chromatography High Pressure Liquid biology Methodology Article Functional genomics biology.organism_classification Multigene Family Metabolome Heterologous expression Genome Fungal DNA microarray Biotechnology |
| Zdroj: | BMC Genomics |
| ISSN: | 1471-2164 |
| DOI: | 10.1186/s12864-015-1561-x |
| Popis: | 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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1561-x) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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