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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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Construction of the epo gene cluster and its expression in M. xanthus. (A), IR-Tps-bsd-oriT cassette (i) in pMycoMar-bsd-hyg plasmid was used as template to generate the PCR product of IR-Tps-bsd-oriT with homology arms. After recombineering, IR-Tps-bsd-oriT was inserted into the plasmid backbone of the stitched epo gene cluster (ii) to form p15A-epo-IR-Tps-bsd-oriT-zeo (iii). Background free template R6K-Tn5-kan was then used to generate IR-Tn5-kan PCR product (iv) with homology arms. The second round of recombineering was performed to build the final expression construct p15A-epo-IR-Tps-bsd-oriT-IR-kan (v). The verified and purified expression construct was electroporated into M. xanthus and the DNA fragment between the two IRs was integrated into the M. xanthus chromosome. (B) Extracts from the stable integrant M. xanthus DK1622-Mut8 were analyzed by HPLC-MS. Rows 1–3 show extracted ion chromatograms of HPLC–MS runs demonstrating that the different epothilones are biosynthesized. Epothilone B, C and D are the major compounds produced in this clone. Rows 4–6 show the same analysis for the wild-type M. xanthus DK1622 (WT) without the epo gene cluster. The fragmentation pattern of epothilone D produced in M. xanthus (row 7) is compared to authentic epothilone D reference (row 8).
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Figure 4: Construction of the epo gene cluster and its expression in M. xanthus. (A), IR-Tps-bsd-oriT cassette (i) in pMycoMar-bsd-hyg plasmid was used as template to generate the PCR product of IR-Tps-bsd-oriT with homology arms. After recombineering, IR-Tps-bsd-oriT was inserted into the plasmid backbone of the stitched epo gene cluster (ii) to form p15A-epo-IR-Tps-bsd-oriT-zeo (iii). Background free template R6K-Tn5-kan was then used to generate IR-Tn5-kan PCR product (iv) with homology arms. The second round of recombineering was performed to build the final expression construct p15A-epo-IR-Tps-bsd-oriT-IR-kan (v). The verified and purified expression construct was electroporated into M. xanthus and the DNA fragment between the two IRs was integrated into the M. xanthus chromosome. (B) Extracts from the stable integrant M. xanthus DK1622-Mut8 were analyzed by HPLC-MS. Rows 1–3 show extracted ion chromatograms of HPLC–MS runs demonstrating that the different epothilones are biosynthesized. Epothilone B, C and D are the major compounds produced in this clone. Rows 4–6 show the same analysis for the wild-type M. xanthus DK1622 (WT) without the epo gene cluster. The fragmentation pattern of epothilone D produced in M. xanthus (row 7) is compared to authentic epothilone D reference (row 8).

Mentions: To generate the conjugation/transposition cassette, by recombineering the blasticidin S-resistant gene (bsd) was inserted between the MycoMar transposase gene (Tps) and oriT in pMycoMar-hyg plasmid (32) to form the IR-Tps-bsd-oriT cassette (Figure 4A). The PCR product of this cassette with homology arms to the stitched p15A-epo-cm-zeo plasmid was used to replace the chloramphenicol (cm) gene in the backbone to form p15A-epo-IR-Tps-bsd-oriT-zeo. A PCR fragment containing the second IR plus Tn5-kan plus a ribosomal binding site (rbs) after the kan stop codon flanked with two homology arms to the p15A-epo-IR-Tps-bsd-oriT-zeo was used to replace zeo to form the final epothilone expression plasmid p15A-epo-IR-Tps-bsd-oriT-IR-kan for expressing in M. xanthus (Figure 4A). Since the Tn5 promoter is weak in P. putida (data not shown), after two rounds of recombineering the Pm promoter (toluic acid inducible) (29) plus its regulator gene (xylS) and the gentamycin-resistant gene (genta) were used to replace Tn5-kan to form p15A-epo-IR-Tps-bsd-oriT-IR-genta-xylS-Pm which is the final expression plasmid for P. putida expression. All of the engineering was done by recombineering.


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)

Construction of the epo gene cluster and its expression in M. xanthus. (A), IR-Tps-bsd-oriT cassette (i) in pMycoMar-bsd-hyg plasmid was used as template to generate the PCR product of IR-Tps-bsd-oriT with homology arms. After recombineering, IR-Tps-bsd-oriT was inserted into the plasmid backbone of the stitched epo gene cluster (ii) to form p15A-epo-IR-Tps-bsd-oriT-zeo (iii). Background free template R6K-Tn5-kan was then used to generate IR-Tn5-kan PCR product (iv) with homology arms. The second round of recombineering was performed to build the final expression construct p15A-epo-IR-Tps-bsd-oriT-IR-kan (v). The verified and purified expression construct was electroporated into M. xanthus and the DNA fragment between the two IRs was integrated into the M. xanthus chromosome. (B) Extracts from the stable integrant M. xanthus DK1622-Mut8 were analyzed by HPLC-MS. Rows 1–3 show extracted ion chromatograms of HPLC–MS runs demonstrating that the different epothilones are biosynthesized. Epothilone B, C and D are the major compounds produced in this clone. Rows 4–6 show the same analysis for the wild-type M. xanthus DK1622 (WT) without the epo gene cluster. The fragmentation pattern of epothilone D produced in M. xanthus (row 7) is compared to authentic epothilone D reference (row 8).
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Related In: Results  -  Collection

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Figure 4: Construction of the epo gene cluster and its expression in M. xanthus. (A), IR-Tps-bsd-oriT cassette (i) in pMycoMar-bsd-hyg plasmid was used as template to generate the PCR product of IR-Tps-bsd-oriT with homology arms. After recombineering, IR-Tps-bsd-oriT was inserted into the plasmid backbone of the stitched epo gene cluster (ii) to form p15A-epo-IR-Tps-bsd-oriT-zeo (iii). Background free template R6K-Tn5-kan was then used to generate IR-Tn5-kan PCR product (iv) with homology arms. The second round of recombineering was performed to build the final expression construct p15A-epo-IR-Tps-bsd-oriT-IR-kan (v). The verified and purified expression construct was electroporated into M. xanthus and the DNA fragment between the two IRs was integrated into the M. xanthus chromosome. (B) Extracts from the stable integrant M. xanthus DK1622-Mut8 were analyzed by HPLC-MS. Rows 1–3 show extracted ion chromatograms of HPLC–MS runs demonstrating that the different epothilones are biosynthesized. Epothilone B, C and D are the major compounds produced in this clone. Rows 4–6 show the same analysis for the wild-type M. xanthus DK1622 (WT) without the epo gene cluster. The fragmentation pattern of epothilone D produced in M. xanthus (row 7) is compared to authentic epothilone D reference (row 8).
Mentions: To generate the conjugation/transposition cassette, by recombineering the blasticidin S-resistant gene (bsd) was inserted between the MycoMar transposase gene (Tps) and oriT in pMycoMar-hyg plasmid (32) to form the IR-Tps-bsd-oriT cassette (Figure 4A). The PCR product of this cassette with homology arms to the stitched p15A-epo-cm-zeo plasmid was used to replace the chloramphenicol (cm) gene in the backbone to form p15A-epo-IR-Tps-bsd-oriT-zeo. A PCR fragment containing the second IR plus Tn5-kan plus a ribosomal binding site (rbs) after the kan stop codon flanked with two homology arms to the p15A-epo-IR-Tps-bsd-oriT-zeo was used to replace zeo to form the final epothilone expression plasmid p15A-epo-IR-Tps-bsd-oriT-IR-kan for expressing in M. xanthus (Figure 4A). Since the Tn5 promoter is weak in P. putida (data not shown), after two rounds of recombineering the Pm promoter (toluic acid inducible) (29) plus its regulator gene (xylS) and the gentamycin-resistant gene (genta) were used to replace Tn5-kan to form p15A-epo-IR-Tps-bsd-oriT-IR-genta-xylS-Pm which is the final expression plasmid for P. putida expression. All of the engineering was done by recombineering.

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