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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 of epo gene cluster for P. putida FG2005 expression. PCR product of Cm resistance gene, Pm promoter and its regulator xylS gene (i) were inserted in front of epoA in the epo gene cluster (ii) to drive expression of the whole epo gene cluster (iii). PCR product of gentamycin resistance gene (iv) was used to replace Tn5-kan and cm to generate the final construct (v) for P. putida FG2005 expression. The verified final construct was integrated into the FG2005 genome by conjugation and transposition.
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Figure 5: Engineering of epo gene cluster for P. putida FG2005 expression. PCR product of Cm resistance gene, Pm promoter and its regulator xylS gene (i) were inserted in front of epoA in the epo gene cluster (ii) to drive expression of the whole epo gene cluster (iii). PCR product of gentamycin resistance gene (iv) was used to replace Tn5-kan and cm to generate the final construct (v) for P. putida FG2005 expression. The verified final construct was integrated into the FG2005 genome by conjugation and transposition.

Mentions: We next aimed to generate a derivative of the epo gene cluster for expression in P. putida by inserting the Pm promoter. To achieve this, a cm-xylS-Pm cassette with homology arms was amplified from the template plasmid pJB866-cm (in which cm was inserted behind the Pm regulator gene xylS in pJB866). The cm-xylS-Pm PCR product was inserted in front of the epoA gene in p15A-epo-IR-Tps-bsd-oriT-IR-kan to form the construct p15A-epo-IR-Tps-bsd-oriT-IR-kan-cm-xylS-Pm (Figure 5). Afterwards, genta with homology arms was used to remove Tn5-kan and cm to form the final epo construct p15A-epo-IR-Tps-bsd-oriT-IR-genta-xylS-Pm for P. putida FG2005 expression.Figure 5.


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 of epo gene cluster for P. putida FG2005 expression. PCR product of Cm resistance gene, Pm promoter and its regulator xylS gene (i) were inserted in front of epoA in the epo gene cluster (ii) to drive expression of the whole epo gene cluster (iii). PCR product of gentamycin resistance gene (iv) was used to replace Tn5-kan and cm to generate the final construct (v) for P. putida FG2005 expression. The verified final construct was integrated into the FG2005 genome by conjugation and transposition.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Engineering of epo gene cluster for P. putida FG2005 expression. PCR product of Cm resistance gene, Pm promoter and its regulator xylS gene (i) were inserted in front of epoA in the epo gene cluster (ii) to drive expression of the whole epo gene cluster (iii). PCR product of gentamycin resistance gene (iv) was used to replace Tn5-kan and cm to generate the final construct (v) for P. putida FG2005 expression. The verified final construct was integrated into the FG2005 genome by conjugation and transposition.
Mentions: We next aimed to generate a derivative of the epo gene cluster for expression in P. putida by inserting the Pm promoter. To achieve this, a cm-xylS-Pm cassette with homology arms was amplified from the template plasmid pJB866-cm (in which cm was inserted behind the Pm regulator gene xylS in pJB866). The cm-xylS-Pm PCR product was inserted in front of the epoA gene in p15A-epo-IR-Tps-bsd-oriT-IR-kan to form the construct p15A-epo-IR-Tps-bsd-oriT-IR-kan-cm-xylS-Pm (Figure 5). Afterwards, genta with homology arms was used to remove Tn5-kan and cm to form the final epo construct p15A-epo-IR-Tps-bsd-oriT-IR-genta-xylS-Pm for P. putida FG2005 expression.Figure 5.

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