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

Fu J, Wenzel SC, Perlova O, Wang J, Gross F, Tang Z, Yin Y, Stewart AF, Müller R, Zhang Y - Nucleic Acids Res. (2008)

Bottom Line: However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range.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.

View Article: PubMed Central - PubMed

Affiliation: Gene Bridges GmbH, BioInnovationsZentrum Dresden, Department of Genomics, Dresden, Germany.

ABSTRACT
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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Engineering diagram for stitching of the epothilone gene cluster. pSuperCos-epo35 and pSuperCos-epo14 were the starting clones, which include an overlap in the epoD gene. pSuperCos-epo35 was retrofitted with the p15A origin and the chloramphenicol resistance gene from pACYC184 by subcloning to remove the pSuperCos backbone, introduce the short homology arm ‘(A)’ and create p15A-epo35. pSuperCos-epo14 was digested with ScaI and the epoA-D genes were recombined into p15A-epo35 by triple recombination using a bridging zeo. In (B), the NcoI digest reveals the correct product. (the NcoI sites in p15A-epo35 and p15A-epo are shown in Appendix 1 in Supplementary Material).
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Figure 3: Engineering diagram for stitching of the epothilone gene cluster. pSuperCos-epo35 and pSuperCos-epo14 were the starting clones, which include an overlap in the epoD gene. pSuperCos-epo35 was retrofitted with the p15A origin and the chloramphenicol resistance gene from pACYC184 by subcloning to remove the pSuperCos backbone, introduce the short homology arm ‘(A)’ and create p15A-epo35. pSuperCos-epo14 was digested with ScaI and the epoA-D genes were recombined into p15A-epo35 by triple recombination using a bridging zeo. In (B), the NcoI digest reveals the correct product. (the NcoI sites in p15A-epo35 and p15A-epo are shown in Appendix 1 in Supplementary Material).

Mentions: To stitch the epothilone gene cluster together (Figure 3A), epoD-K genes were subcloned into a p15A-Cm minimum vector that was generated by using linearized pACYC184 (41) as template to form p15A-epo35. The zeocin (zeo) resistance gene with a 5′ end homology arm to p15A-cm-epo35 and a 3′ end homology arm to a region in front of epoA in pSuperCos-epo14 was amplified by PCR. pSuperCos-epo14 was digested with ScaI to release a 26-kb fragment which carries the short homology arm to the zeo PCR product and a long homology arm (∼9.45 kb) to p15A-cm-epo35. After mixing the zeo PCR product with the digested pSuperCos-epo14, the mixture was transformed into a recombineering proficient host containing p15A-cm-epo35. The stitched full-length epothilone gene cluster was selected by zeo and analyzed by restriction digestion (Figure 3B). The junction regions of the stitched cluster were verified by sequencing.


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

Fu J, Wenzel SC, Perlova O, Wang J, Gross F, Tang Z, Yin Y, Stewart AF, Müller R, Zhang Y - Nucleic Acids Res. (2008)

Engineering diagram for stitching of the epothilone gene cluster. pSuperCos-epo35 and pSuperCos-epo14 were the starting clones, which include an overlap in the epoD gene. pSuperCos-epo35 was retrofitted with the p15A origin and the chloramphenicol resistance gene from pACYC184 by subcloning to remove the pSuperCos backbone, introduce the short homology arm ‘(A)’ and create p15A-epo35. pSuperCos-epo14 was digested with ScaI and the epoA-D genes were recombined into p15A-epo35 by triple recombination using a bridging zeo. In (B), the NcoI digest reveals the correct product. (the NcoI sites in p15A-epo35 and p15A-epo are shown in Appendix 1 in Supplementary Material).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553598&req=5

Figure 3: Engineering diagram for stitching of the epothilone gene cluster. pSuperCos-epo35 and pSuperCos-epo14 were the starting clones, which include an overlap in the epoD gene. pSuperCos-epo35 was retrofitted with the p15A origin and the chloramphenicol resistance gene from pACYC184 by subcloning to remove the pSuperCos backbone, introduce the short homology arm ‘(A)’ and create p15A-epo35. pSuperCos-epo14 was digested with ScaI and the epoA-D genes were recombined into p15A-epo35 by triple recombination using a bridging zeo. In (B), the NcoI digest reveals the correct product. (the NcoI sites in p15A-epo35 and p15A-epo are shown in Appendix 1 in Supplementary Material).
Mentions: To stitch the epothilone gene cluster together (Figure 3A), epoD-K genes were subcloned into a p15A-Cm minimum vector that was generated by using linearized pACYC184 (41) as template to form p15A-epo35. The zeocin (zeo) resistance gene with a 5′ end homology arm to p15A-cm-epo35 and a 3′ end homology arm to a region in front of epoA in pSuperCos-epo14 was amplified by PCR. pSuperCos-epo14 was digested with ScaI to release a 26-kb fragment which carries the short homology arm to the zeo PCR product and a long homology arm (∼9.45 kb) to p15A-cm-epo35. After mixing the zeo PCR product with the digested pSuperCos-epo14, the mixture was transformed into a recombineering proficient host containing p15A-cm-epo35. The stitched full-length epothilone gene cluster was selected by zeo and analyzed by restriction digestion (Figure 3B). The junction regions of the stitched cluster were verified by sequencing.

Bottom Line: However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range.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.

View Article: PubMed Central - PubMed

Affiliation: Gene Bridges GmbH, BioInnovationsZentrum Dresden, Department of Genomics, Dresden, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus