Fungal artificial chromosomes for mining of the fungal secondary metabolome

Jin Woo Bok; Rosa Ye; Kenneth D. Clevenger; David Mead; Megan Wagner; Amanda Krerowicz; Jessica C. Albright; Anthony Whitney Goering; Paul Martin Thomas; Neil L Kelleher; Nancy P. Keller; Chengcang C. Wu

doi:10.1186/s12864-015-1561-x

Fungal artificial chromosomes for mining of the fungal secondary metabolome

Jin Woo Bok, Rosa Ye, Kenneth D. Clevenger, David Mead, Megan Wagner, Amanda Krerowicz, Jessica C. Albright, Anthony Whitney Goering, Paul Martin Thomas, Neil L Kelleher^*, Nancy P. Keller, Chengcang C. Wu

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

67 Scopus citations

Abstract

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.

Original language	English (US)
Article number	343
Journal	BMC Genomics
Volume	16
Issue number	1
DOIs	https://doi.org/10.1186/s12864-015-1561-x
State	Published - Dec 12 2015

Funding

We thank Berl Oakley for supplying us with strain LO4641. This work was supported in part by National Institutes of Health 1R43AI94885-1 to C.C.W. at Lucigen Corporation and to N.P.K, R01GM067725 to N.L.K., 5PO1GM084077 to N.P.K., and Innovation & Economic Development Research 101PRJ72KQ to N.P.K.

Keywords

Functional genomics
Fungal artificial chromosome (FAC)
Natural product discovery
Secondary metabolite (SM) gene clusters

ASJC Scopus subject areas

Genetics
Biotechnology

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10.1186/s12864-015-1561-x

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@article{0e16fb6991a14aa6a57805ae514bdc89,

title = "Fungal artificial chromosomes for mining of the fungal secondary metabolome",

abstract = "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.",

keywords = "Functional genomics, Fungal artificial chromosome (FAC), Natural product discovery, Secondary metabolite (SM) gene clusters",

author = "Bok, {Jin Woo} and Rosa Ye and Clevenger, {Kenneth D.} and David Mead and Megan Wagner and Amanda Krerowicz and Albright, {Jessica C.} and Goering, {Anthony Whitney} and Thomas, {Paul Martin} and Kelleher, {Neil L} and Keller, {Nancy P.} and Wu, {Chengcang C.}",

note = "Funding Information: We thank Berl Oakley for supplying us with strain LO4641. This work was supported in part by National Institutes of Health 1R43AI94885-1 to C.C.W. at Lucigen Corporation and to N.P.K, R01GM067725 to N.L.K., 5PO1GM084077 to N.P.K., and Innovation & Economic Development Research 101PRJ72KQ to N.P.K. Publisher Copyright: {\textcopyright} 2015 Bok et al.; licensee BioMed Central.",

year = "2015",

month = dec,

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doi = "10.1186/s12864-015-1561-x",

language = "English (US)",

volume = "16",

journal = "BMC Genomics",

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T1 - Fungal artificial chromosomes for mining of the fungal secondary metabolome

AU - Bok, Jin Woo

AU - Ye, Rosa

AU - Clevenger, Kenneth D.

AU - Mead, David

AU - Wagner, Megan

AU - Krerowicz, Amanda

AU - Albright, Jessica C.

AU - Goering, Anthony Whitney

AU - Thomas, Paul Martin

AU - Kelleher, Neil L

AU - Keller, Nancy P.

AU - Wu, Chengcang C.

N1 - Funding Information: We thank Berl Oakley for supplying us with strain LO4641. This work was supported in part by National Institutes of Health 1R43AI94885-1 to C.C.W. at Lucigen Corporation and to N.P.K, R01GM067725 to N.L.K., 5PO1GM084077 to N.P.K., and Innovation & Economic Development Research 101PRJ72KQ to N.P.K. Publisher Copyright: © 2015 Bok et al.; licensee BioMed Central.

PY - 2015/12/12

Y1 - 2015/12/12

N2 - 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.

AB - 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.

KW - Functional genomics

KW - Fungal artificial chromosome (FAC)

KW - Natural product discovery

KW - Secondary metabolite (SM) gene clusters

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ER -

Fungal artificial chromosomes for mining of the fungal secondary metabolome

Abstract

Funding

Keywords

ASJC Scopus subject areas

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