Heterologous expression and genetic engineering of the phenalinolactone biosynthetic gene cluster by using Red/ET recombineering
Heterologous expression and genetic engineering of the phenalinolactone biosynthetic gene cluster by using Red/ET recombineering
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DOI：
10.1002/cbic.200700549
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发表时间：
2008-01-15
期刊：
影响因子：
3.2
通讯作者：
Mueller, Rolf
中科院分区：
文献类型：
--
作者：
Binz, Tina M.;Wenzel, Silke C.;Mueller, Rolf
天然产物生物合成途径的异源表达在生物技术和药物发现中越来越受到关注，这种方法使得能够在更顺从的宿主生物体中产生复杂的代谢物，并为通过基因工程产生新的类似物提供基础。在这里，我们描述了一个简单的策略，为异源表达的高度复杂的phenalinolactone生物合成途径，这是最近从链霉菌属Tu 6071克隆。该生物合成基因簇包含至少11个转录单元，其含有35个基因，这些基因一起催化具有抗菌活性的结构独特的三环萜苷的组装。通过Red/ET重组工程，从两个cosmetics和异源表达在几个链霉菌菌株中的phenalinolactone途径重建。建立的表达系统现在提供了一个方便的平台，生物合成基因的功能研究和新的类似物的产生，通过在大肠杆菌中的途径的基因工程。从表达构建体中删除修饰基因导致了一种新的、非糖基化的非那林内酯衍生物;这证明了这种方法的前景。
The heterologous expression of natural product biosynthetic pathways is of increasing interest in biotechnology and drug discovery, This approach enables the production of complex metabolites in more amenable host organisms and provides the basis for the generation of novel analogues through genetic engineering. Here we describe a straightforward strategy for the heterologous expression of the highly complex phenalinolactone biosynthetic pathway, which was recently cloned from Streptomyces sp. Tu6071. The biosynthetic gene cluster comprises at least 11 transcriptional units that harbor 35 genes, which together catalyze the assembly of structurally unique tricyclic terpene glycosides with antibacterial activity. By using Red/ET recombineering, the phenalinolactone pathway was reconstituted from two cosmids and heterologously expressed in several Streptomyces strains. The established expression system now provides a convenient platform for functional investigations of the biosynthetic genes and the generation of novel analogues, by genetic engineering of the pathway in Escherichia coli. Deletion of a modifying gene from the expression construct resulted in a novel, unglycosylated phenalinolactone derivative; this demonstrates the promise of this methodology.