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Nucleic Acids Research Advance Access published online on August 13, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn499
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© 2008 The Author(s)
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.

Methods Online

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

Jun Fu1,2, Silke C. Wenzel3, Olena Perlova3, Junping Wang1, Frank Gross1,2, Zhiru Tang2,4, Yulong Yin4, A. Francis Stewart2, Rolf Müller3 and Youming Zhang1,*

1Gene Bridges GmbH, BioInnovationsZentrum Dresden, 2Department of Genomics, Dresden University of Technology, BioInnovationsZentrum, Tatzberg 47-51, 01307 Dresden, 3Department of Pharmaceutical Biotechnology, Saarland University, PO Box 151150, 66041 Saarbrücken, Germany and 4Institute of Subtropical Agriculture, The Chinese Academy of Science, PO Box 10, Changsha, Hunan, Postcode 410125, P.R. China

*To whom correspondence should be addressed. Tel: +49 351 7965656; Fax: +49 351 7965655; Email: youming.zhang{at}genebridges.com

Correspondence may also be addressed to Rolf Müller. Tel: +49 681 302 70201; Fax: +49 681 302 70202; Email: rom{at}mx.uni-saarland.de

Received May 14, 2008. Revised July 10, 2008. Accepted July 18, 2008.

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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