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首页> 美国卫生研究院文献>Nucleic Acids Research >Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition
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Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition

机译：通过转座有效地将两个大的次级代谢产物途径基因簇转移到异源宿主中

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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 polyketideonribosomal 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.
机译：通过转座进行水平基因转移已广泛用于原核生物的转基因。然而，尽管宿主范围受到更大的限制，但对于大转基因的转移，结合是优选的。我们研究了转座子可用于将大型转基因传递给异源宿主的可能性。这种可能性与在各种异源宿主中大的次级代谢产物基因簇的表达特别相关。最近，我们表明，针对异源表达的大型基因簇（例如I型聚酮/非核糖体肽途径）的工程化不再是瓶颈。在这里，我们应用重组工程化埃博霉素（epohilone）（epohilone（epo））或粘氧铬（myxochromide S（mchS））基因簇，用于在异源宿主中进行转座递送和表达。 58 kb epo基因簇通过缝合从两个克隆中完全重建。然后，将epo启动子交换为在异源宿主中有活性的启动子，然后工程改造成MycoMar转座子。将类似的过程应用于mchS基因簇。工程化的基因簇被转移并在异源宿主粘球霉（Myxococcus xanthus）和恶臭假单胞菌（Pseudomonas putida）中表达。我们实现了迄今为止尚未报道的任何系统中最大的转座，并建议转座子的传递将成为传递大转基因的选择方法，特别是不仅用于代谢工程，而且还用于原核生物和真核生物的一般转基因。

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