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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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Detection of myxochromide S compounds in M. xanthus. (A) The color of M. xanthus colonies transformed with the mchS gene cluster is shown. Colonies from M. xanthus::pTps-mchS (1–7) and M. xanthus::pTps-lacZ (LacZ) were picked and replated on a kanamycin plate. The photo was taken after 2 days incubation. (B) the chemical structures of myxochromides S1–3. (C) HPLC profiles from extracts of the M. xanthus::pTpS-mchS mutant strain (a) in comparison to M. xanthus wild-type (b); diode array detection at 400 nm. Numbers correspond to substances as follows: 1, Myxochromide S1; 2, Myxochromide S2; 3, Myxochromide S3. Peaks marked with an asterisk are assumed to be Myxochromide S derivatives as well.
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Figure 2: Detection of myxochromide S compounds in M. xanthus. (A) The color of M. xanthus colonies transformed with the mchS gene cluster is shown. Colonies from M. xanthus::pTps-mchS (1–7) and M. xanthus::pTps-lacZ (LacZ) were picked and replated on a kanamycin plate. The photo was taken after 2 days incubation. (B) the chemical structures of myxochromides S1–3. (C) HPLC profiles from extracts of the M. xanthus::pTpS-mchS mutant strain (a) in comparison to M. xanthus wild-type (b); diode array detection at 400 nm. Numbers correspond to substances as follows: 1, Myxochromide S1; 2, Myxochromide S2; 3, Myxochromide S3. Peaks marked with an asterisk are assumed to be Myxochromide S derivatives as well.

Mentions: Myxochromide S compounds are characterized by their yellow–orange color and are easily observed in culture. Colonies from pTps-mchS transformation were reddish (Figure 2A) and the liquid cultures are reddish as well (data not shown). A methanol extract from M. xanthus DK1622::pTps-mchS was analyzed with HPLC and HPLC/MS for the production of myxochromides S. Myxochromides S1–3, known from S. aurantica, could be identified in extracts of the M. xanthus mutant strains via HPLC [Figure 2B, peaks 1 (S1), 2 (S2), 3 (S3)], which could also be verified via HPLC/MS analysis (data not shown). Due to the high production of myxochromides S in M. xanthus (∼500 mg/l), minor myxochromide S derivatives could also be detected (peaks marked with an asterix, Figure 2B).Figure 2.


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)

Detection of myxochromide S compounds in M. xanthus. (A) The color of M. xanthus colonies transformed with the mchS gene cluster is shown. Colonies from M. xanthus::pTps-mchS (1–7) and M. xanthus::pTps-lacZ (LacZ) were picked and replated on a kanamycin plate. The photo was taken after 2 days incubation. (B) the chemical structures of myxochromides S1–3. (C) HPLC profiles from extracts of the M. xanthus::pTpS-mchS mutant strain (a) in comparison to M. xanthus wild-type (b); diode array detection at 400 nm. Numbers correspond to substances as follows: 1, Myxochromide S1; 2, Myxochromide S2; 3, Myxochromide S3. Peaks marked with an asterisk are assumed to be Myxochromide S derivatives as well.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Detection of myxochromide S compounds in M. xanthus. (A) The color of M. xanthus colonies transformed with the mchS gene cluster is shown. Colonies from M. xanthus::pTps-mchS (1–7) and M. xanthus::pTps-lacZ (LacZ) were picked and replated on a kanamycin plate. The photo was taken after 2 days incubation. (B) the chemical structures of myxochromides S1–3. (C) HPLC profiles from extracts of the M. xanthus::pTpS-mchS mutant strain (a) in comparison to M. xanthus wild-type (b); diode array detection at 400 nm. Numbers correspond to substances as follows: 1, Myxochromide S1; 2, Myxochromide S2; 3, Myxochromide S3. Peaks marked with an asterisk are assumed to be Myxochromide S derivatives as well.
Mentions: Myxochromide S compounds are characterized by their yellow–orange color and are easily observed in culture. Colonies from pTps-mchS transformation were reddish (Figure 2A) and the liquid cultures are reddish as well (data not shown). A methanol extract from M. xanthus DK1622::pTps-mchS was analyzed with HPLC and HPLC/MS for the production of myxochromides S. Myxochromides S1–3, known from S. aurantica, could be identified in extracts of the M. xanthus mutant strains via HPLC [Figure 2B, peaks 1 (S1), 2 (S2), 3 (S3)], which could also be verified via HPLC/MS analysis (data not shown). Due to the high production of myxochromides S in M. xanthus (∼500 mg/l), minor myxochromide S derivatives could also be detected (peaks marked with an asterix, Figure 2B).Figure 2.

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