Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.

Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of soli...

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Autores principales:	Morten Thrane Nielsen, Jakob Blæsbjerg Nielsen, Diana Chinyere Anyaogu, Dorte Koefoed Holm, Kristian Fog Nielsen, Thomas Ostenfeld Larsen, Uffe Hasbro Mortensen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/52f4152da0a24051bb761a4448e348dc
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spelling	oai:doaj.org-article:52f4152da0a24051bb761a4448e348dc2021-11-18T08:58:22ZHeterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.1932-620310.1371/journal.pone.0072871https://doaj.org/article/52f4152da0a24051bb761a4448e348dc2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24009710/?tool=EBIhttps://doaj.org/toc/1932-6203Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway characterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to transformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL) encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects.Morten Thrane NielsenJakob Blæsbjerg NielsenDiana Chinyere AnyaoguDorte Koefoed HolmKristian Fog NielsenThomas Ostenfeld LarsenUffe Hasbro MortensenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 8, p e72871 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Morten Thrane Nielsen Jakob Blæsbjerg Nielsen Diana Chinyere Anyaogu Dorte Koefoed Holm Kristian Fog Nielsen Thomas Ostenfeld Larsen Uffe Hasbro Mortensen Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.
description	Fungal natural products are a rich resource for bioactive molecules. To fully exploit this potential it is necessary to link genes to metabolites. Genetic information for numerous putative biosynthetic pathways has become available in recent years through genome sequencing. However, the lack of solid methodology for genetic manipulation of most species severely hampers pathway characterization. Here we present a simple PCR based approach for heterologous reconstitution of intact gene clusters. Specifically, the putative gene cluster responsible for geodin production from Aspergillus terreus was transferred in a two step procedure to an expression platform in A. nidulans. The individual cluster fragments were generated by PCR and assembled via efficient USER fusion prior to transformation and integration via re-iterative gene targeting. A total of 13 open reading frames contained in 25 kb of DNA were successfully transferred between the two species enabling geodin synthesis in A. nidulans. Subsequently, functions of three genes in the cluster were validated by genetic and chemical analyses. Specifically, ATEG_08451 (gedC) encodes a polyketide synthase, ATEG_08453 (gedR) encodes a transcription factor responsible for activation of the geodin gene cluster and ATEG_08460 (gedL) encodes a halogenase that catalyzes conversion of sulochrin to dihydrogeodin. We expect that our approach for transferring intact biosynthetic pathways to a fungus with a well developed genetic toolbox will be instrumental in characterizing the many exciting pathways for secondary metabolite production that are currently being uncovered by the fungal genome sequencing projects.
format	article
author	Morten Thrane Nielsen Jakob Blæsbjerg Nielsen Diana Chinyere Anyaogu Dorte Koefoed Holm Kristian Fog Nielsen Thomas Ostenfeld Larsen Uffe Hasbro Mortensen
author_facet	Morten Thrane Nielsen Jakob Blæsbjerg Nielsen Diana Chinyere Anyaogu Dorte Koefoed Holm Kristian Fog Nielsen Thomas Ostenfeld Larsen Uffe Hasbro Mortensen
author_sort	Morten Thrane Nielsen
title	Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.
title_short	Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.
title_full	Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.
title_fullStr	Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.
title_full_unstemmed	Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.
title_sort	heterologous reconstitution of the intact geodin gene cluster in aspergillus nidulans through a simple and versatile pcr based approach.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/52f4152da0a24051bb761a4448e348dc
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Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach.

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