Fungal artificial chromosomes for mining of the fungal secondary metabolome.
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.


CONCLUSIONS
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.
Department of Medical Microbiology and Immunology and Bacteriology, University of Wisconsin at Madison, Madison, WI, USA. jwbok@facstaff.wisc.edu.
Jin Woo
JW
Bok
Intact Genomics, Inc., St Louis, MO, USA. rye@intactgenomics.com.|Lucigen Corporation, Middleton, WI, USA. rye@intactgenomics.com.
Rosa
R
Ye
Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA. kenneth.clevenger@northwestern.edu.
Kenneth D
KD
Clevenger
Lucigen Corporation, Middleton, WI, USA. dmead@lucigen.com.
David
D
Mead
Lucigen Corporation, Middleton, WI, USA. mniebauer@lucigen.com.
Megan
M
Wagner
Lucigen Corporation, Middleton, WI, USA. akrerowicz@lucigen.com.
Amanda
A
Krerowicz
Department of Chemistry, Northwestern University, Evanston, IL, USA. JessicaAlbright2014@u.northwestern.edu.
Jessica C
JC
Albright
Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA. AnthonyGoering2016@u.northwestern.edu.
Anthony W
AW
Goering
Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA. paul-thomas@northwestern.edu.|Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA. paul-thomas@northwestern.edu.
Paul M
PM
Thomas
Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA. n-kelleher@northwestern.edu.|Department of Chemistry, Northwestern University, Evanston, IL, USA. n-kelleher@northwestern.edu.|Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA. n-kelleher@northwestern.edu.
Neil L
NL
Kelleher
Department of Medical Microbiology and Immunology and Bacteriology, University of Wisconsin at Madison, Madison, WI, USA. npkeller@wisc.edu.
Nancy P
NP
Keller
Intact Genomics, Inc., St Louis, MO, USA. cwu@intactgenomics.com.|Lucigen Corporation, Middleton, WI, USA. cwu@intactgenomics.com.
Chengcang C
CC
Wu
D001233:Aspergillus nidulans / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D022201:Chromosomes, Artificial / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D016681:Genome, Fungal; D013058:Mass Spectrometry; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D005810:Multigene Family; D010879:Piperazines / Q000032:analysis / Q000378:metabolism
D010879:Piperazines; C093760:terezine D