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Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition

Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to...

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Autores principales: Fu, Jun, Wenzel, Silke C., Perlova, Olena, Wang, Junping, Gross, Frank, Tang, Zhiru, Yin, Yulong, Stewart, A. Francis, Müller, Rolf, Zhang, Youming
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553598/
https://www.ncbi.nlm.nih.gov/pubmed/18701643
http://dx.doi.org/10.1093/nar/gkn499
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author Fu, Jun
Wenzel, Silke C.
Perlova, Olena
Wang, Junping
Gross, Frank
Tang, Zhiru
Yin, Yulong
Stewart, A. Francis
Müller, Rolf
Zhang, Youming
author_facet Fu, Jun
Wenzel, Silke C.
Perlova, Olena
Wang, Junping
Gross, Frank
Tang, Zhiru
Yin, Yulong
Stewart, A. Francis
Müller, Rolf
Zhang, Youming
author_sort Fu, Jun
collection PubMed
description Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes.
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spelling pubmed-25535982008-10-01 Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition Fu, Jun Wenzel, Silke C. Perlova, Olena Wang, Junping Gross, Frank Tang, Zhiru Yin, Yulong Stewart, A. Francis Müller, Rolf Zhang, Youming Nucleic Acids Res Methods Online Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes. Oxford University Press 2008-10 2008-08-13 /pmc/articles/PMC2553598/ /pubmed/18701643 http://dx.doi.org/10.1093/nar/gkn499 Text en © 2008 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Fu, Jun
Wenzel, Silke C.
Perlova, Olena
Wang, Junping
Gross, Frank
Tang, Zhiru
Yin, Yulong
Stewart, A. Francis
Müller, Rolf
Zhang, Youming
Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition
title Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition
title_full Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition
title_fullStr Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition
title_full_unstemmed Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition
title_short Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition
title_sort efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553598/
https://www.ncbi.nlm.nih.gov/pubmed/18701643
http://dx.doi.org/10.1093/nar/gkn499
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