Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition.
Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition.
复制标题
通过转座有效地将两个大型次级代谢物途径基因簇转移到异源宿主中。
DOI：
10.1093/nar/gkn499
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发表时间：
2008-10
影响因子：
14.9
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Fu J;Wenzel SC;Perlova O;Wang J;Gross F;Tang Z;Yin Y;Stewart AF;Müller R;Zhang Y
通过转座的水平基因转移已广泛用于原核生物的转基因。然而，尽管宿主范围有更大的限制，但接合仍是大型转基因转移的首选。我们研究了转座子可用于将大转基因传递至异源宿主的可能性。这种可能性与各种异源宿主中大型次级代谢基因簇的表达特别相关。最近，我们表明，用于异源表达的大型基因簇（如 I 型聚酮化合物/非核糖体肽途径）的工程不再是瓶颈。在这里，我们应用重组工程来改造埃坡霉素 (epo) 或粘铬胺 S (mchS) 基因簇，以便在异源宿主中进行转座递送和表达。 58-kb epo 基因簇由两个克隆通过拼接完全重建。然后，将 epo 启动子替换为在异源宿主中有活性的启动子，然后工程化到 MycoMar 转座子中。 mchS 基因簇也应用了类似的过程。工程基因簇被转移并在异源宿主黄色粘球菌和恶臭假单胞菌中表达。我们实现了迄今为止报道的任何系统中最大的转座，并表明通过转座子递送将成为递送大型转基因的选择方法，特别是不仅对于代谢工程而且对于原核生物和真核生物中的一般转基因。
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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DOI：
10.1016/s0167-4781(99)00041-x
发表时间：
1999-05-14
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION
影响因子：
--
作者：
Beyer, S;Kunze, B;Müller, R
通讯作者：
Müller, R
影响因子：
14.9
作者：
Muyrers, JPP;Zhang, YM;Stewart, AF
通讯作者：
Stewart, AF
影响因子：
3.2
作者：
Kofoid, E;Rappleye, C;Roth, J
通讯作者：
Roth, J
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
--
作者：
Gross, Frank;Ring, Michael W.;Mueller, Rolf
通讯作者：
Mueller, Rolf