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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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Diagram of myxochromide S gene cluster engineering. In the first step, the inverted repeat and MycoMar transposase gene (IR-Tps, i) plus amp (ii) were inserted into the mchS expression plasmid (iii) backbone by triple recombination to delete zeo and kan (iv). The MycoMar Tps plus IR fragment (i) was generated by PCR from the original MycoMar transposon (12). In the second step, the oriT-tet-trpE-cm-xylS section (13) in the backbone was replaced with a right IR and Tn5-kan cassette (v), which included a ribosomal binding site for the mchS gene cluster, by selection for kanamycin resistance. In the third step, the modified plasmid (vi) was electroporated into M. xanthus and kanamycin-resistant colonies were selected. To introduce the gene cluster into P. putida, an additional cassette containing oriT and the tetracycline-resistant gene (oriT-tetR-tet, vii) was inserted between amp and the pUC origin (pUC) to form the final construct (viii) for P. putida expression. See remarks in the Materials and Methods section.
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Figure 1: Diagram of myxochromide S gene cluster engineering. In the first step, the inverted repeat and MycoMar transposase gene (IR-Tps, i) plus amp (ii) were inserted into the mchS expression plasmid (iii) backbone by triple recombination to delete zeo and kan (iv). The MycoMar Tps plus IR fragment (i) was generated by PCR from the original MycoMar transposon (12). In the second step, the oriT-tet-trpE-cm-xylS section (13) in the backbone was replaced with a right IR and Tn5-kan cassette (v), which included a ribosomal binding site for the mchS gene cluster, by selection for kanamycin resistance. In the third step, the modified plasmid (vi) was electroporated into M. xanthus and kanamycin-resistant colonies were selected. To introduce the gene cluster into P. putida, an additional cassette containing oriT and the tetracycline-resistant gene (oriT-tetR-tet, vii) was inserted between amp and the pUC origin (pUC) to form the final construct (viii) for P. putida expression. See remarks in the Materials and Methods section.

Mentions: The pUC-mchS was derived from a SuperCos 1 vector that contains most of the mchS pathway. The missing gene at the 3′ end was added, along with a cassette for conjugation as described (29). Two rounds of recombineering were used for engineering of the pUC-mchS plasmid. We used our recently developed technology named triple recombination for the first round. By electroporation, PCR products of IR-Tps cassette (IR, inverted repeat; Tps, MycoMar transposase gene) and the ampicillin resistance gene (0.3 μg each in 2 μl) were cotransformed into recombineering proficient competent cells in which the pUC-mchS is resident. Recombinants were selected on LB plates containing 100 μg/ml of Amp. For second round recombineering, the IR-Tn5-kan cassette (Tn5, Tn5 promoter; kan, kanamycin resistance gene) flanked with homology arms was generated by PCR. The PCR product (0.3 μg) were used for recombineering and the recombinants were selected on LB plates with 15 μg/ml of Km (Figure 1).Figure 1.


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)

Diagram of myxochromide S gene cluster engineering. In the first step, the inverted repeat and MycoMar transposase gene (IR-Tps, i) plus amp (ii) were inserted into the mchS expression plasmid (iii) backbone by triple recombination to delete zeo and kan (iv). The MycoMar Tps plus IR fragment (i) was generated by PCR from the original MycoMar transposon (12). In the second step, the oriT-tet-trpE-cm-xylS section (13) in the backbone was replaced with a right IR and Tn5-kan cassette (v), which included a ribosomal binding site for the mchS gene cluster, by selection for kanamycin resistance. In the third step, the modified plasmid (vi) was electroporated into M. xanthus and kanamycin-resistant colonies were selected. To introduce the gene cluster into P. putida, an additional cassette containing oriT and the tetracycline-resistant gene (oriT-tetR-tet, vii) was inserted between amp and the pUC origin (pUC) to form the final construct (viii) for P. putida expression. See remarks in the Materials and Methods section.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Diagram of myxochromide S gene cluster engineering. In the first step, the inverted repeat and MycoMar transposase gene (IR-Tps, i) plus amp (ii) were inserted into the mchS expression plasmid (iii) backbone by triple recombination to delete zeo and kan (iv). The MycoMar Tps plus IR fragment (i) was generated by PCR from the original MycoMar transposon (12). In the second step, the oriT-tet-trpE-cm-xylS section (13) in the backbone was replaced with a right IR and Tn5-kan cassette (v), which included a ribosomal binding site for the mchS gene cluster, by selection for kanamycin resistance. In the third step, the modified plasmid (vi) was electroporated into M. xanthus and kanamycin-resistant colonies were selected. To introduce the gene cluster into P. putida, an additional cassette containing oriT and the tetracycline-resistant gene (oriT-tetR-tet, vii) was inserted between amp and the pUC origin (pUC) to form the final construct (viii) for P. putida expression. See remarks in the Materials and Methods section.
Mentions: The pUC-mchS was derived from a SuperCos 1 vector that contains most of the mchS pathway. The missing gene at the 3′ end was added, along with a cassette for conjugation as described (29). Two rounds of recombineering were used for engineering of the pUC-mchS plasmid. We used our recently developed technology named triple recombination for the first round. By electroporation, PCR products of IR-Tps cassette (IR, inverted repeat; Tps, MycoMar transposase gene) and the ampicillin resistance gene (0.3 μg each in 2 μl) were cotransformed into recombineering proficient competent cells in which the pUC-mchS is resident. Recombinants were selected on LB plates containing 100 μg/ml of Amp. For second round recombineering, the IR-Tn5-kan cassette (Tn5, Tn5 promoter; kan, kanamycin resistance gene) flanked with homology arms was generated by PCR. The PCR product (0.3 μg) were used for recombineering and the recombinants were selected on LB plates with 15 μg/ml of Km (Figure 1).Figure 1.

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