Binz Tina M, Wenzel Silke C, Schnell Hans-Jörg, Bechthold Andreas, Müller Rolf
Universität des Saarlandes, Institut für Pharmazeutische Biotechnologie, 66123 Saarbrücken, Germany.
Chembiochem. 2008 Feb 15;9(3):447-54. doi: 10.1002/cbic.200700549.
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. Tü6071. 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.
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领域越来越受到关注。这种方法能够在更易处理的宿主生物体中生产复杂的代谢产物，并为通过基因工程产生新型类似物提供了基础。在此，我们描述了一种用于高度复杂的苯那内酯生物合成途径异源表达的直接策略，该途径最近从链霉菌属菌株Tü6071中克隆得到。生物合成基因簇包含至少11个转录单元，这些转录单元含有35个基因，它们共同催化具有抗菌活性的结构独特的三环萜烯糖苷的组装。通过使用Red/ET重组工程技术，从两个黏粒中重建了苯那内酯途径，并在几种链霉菌菌株中进行了异源表达。现在，通过在大肠杆菌中对该途径进行基因工程改造，所建立的表达系统为生物合成基因的功能研究和新型类似物的产生提供了一个便利的平台。从表达构建体中缺失一个修饰基因产生了一种新型的、未糖基化的苯那内酯衍生物；这证明了该方法的前景。
