Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition.
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.
Fu
Jun
J
Gene Bridges GmbH, BioInnovationsZentrum Dresden, Department of Genomics, Dresden, Germany.
Wenzel
Silke C
SC
Perlova
Olena
O
Wang
Junping
J
Gross
Frank
F
Tang
Zhiru
Z
Yin
Yulong
Y
Stewart
A Francis
AF
Müller
Rolf
R
Zhang
Youming
Y
D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D047630:Depsipeptides / Q000096:biosynthesis; D034261:Epothilones / Q000096:biosynthesis; D022761:Gene Transfer, Horizontal; D005818:Genetic Engineering; D016941:Myxococcus xanthus / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics; D020606:Stigmatella aurantiaca / Q000235:genetics; D014169:Transformation, Bacterial; D019076:Transgenes
D004251:DNA Transposable Elements; D047630:Depsipeptides; D034261:Epothilones