Molecular genetics and applications of a Streptomyces gene amplification system

链霉菌是一组普遍存在的土壤细菌，在生态学中发挥重要作用，并产生数百种重要的生物活性化合物。它们能够在陆地生态系统中合成或降解各种化合物。它们是抗生素（超过所有抗生素的2/3）以及其他代谢物的最丰富的已知来源，其具有除草剂、抗癌药物、免疫调节剂和抗寄生虫化合物的应用。基因组测序最近表明，每个分离物都能够产生20-40种不同的潜在生物活性化合物。 不幸的是，这些细菌在实验室中产生的这些化合物不到10%。 随着多重耐药细菌的迅速出现，人们对挖掘这些途径以获得新的抗生素来治疗由细菌病原体如葡萄球菌、链球菌、分枝杆菌和沙门氏菌引起的疾病重新产生了兴趣。 我们最近已经确定了链霉菌中的分子催化剂，放大编码抗生素生物合成途径的基因簇的拷贝数。这些新工具包括两个重组位点，可以插入到靶基因簇和催化重组的伴侣酶的侧翼。 作为原理的证明，我们已经证明，我们可以使用我们的遗传工具来产生抗生素生物合成基因簇的10个串联重复序列，从而将抗生素的产量提高20倍。 值得注意的是，我们发现这个系统在E.杆菌事实上，这个系统可以催化两个不相关的细菌没有额外的辅因子的事件表明，它可能被用来扩增基因簇在广泛的细菌，也许真核宿主。据我们所知，这是第一个在任何生物体中诱导扩增基因簇的系统。这种新的基因扩增系统有望成为一种有价值的工具，用于增加各种细菌中商业上重要的生物合成或降解途径的活性，并且还可能在真核细胞中发现诱导型基因扩增的重要应用。

Streptomyces are a ubiquitous group of soil bacteria that play important roles in ecology and produce hundreds of important bioactive compounds. They are able to synthesize or degrade diverse compounds in terrestrial ecosystems. They are the most prolific known source of antibiotics (more than 2/3 of all antibiotics) as well as other metabolites having applications as herbicides, anticancer drugs, immunoregulators, and antiparasitic compounds. Genome sequencing has recently shown that each isolate is able to produce its own repertoire of 20-40 different, potentially bioactive compounds. Unfortunately, these bacteria produces less than 10% of these compounds in the laboratory. With multidrug resistant bacteria rapidly emerging, there is renewed interest in mining these pathways for new antibiotics to treat diseases caused by bacterial pathogens such as Staphylococcus, Streptococcus, Mycobacterium, and Salmonella. We have recently identified molecular catalysts in Streptomyces that amplify the copy number of gene clusters encoding antibiotic biosynthetic pathways. These new tools include two recombination sites that can be inserted to flank a targeted gene cluster and a partner enzyme catalyzing recombination. As proof of principle, we have demonstrated that we can use our genetic tools to produce 10 tandem repeats of an antibiotic biosynthetic gene cluster, thereby increasing the production of the antibiotic by 20 fold. Remarkably, we found that this system was also functional in E. coli. The fact that this system could catalyze events in two unrelated bacteria without additional cofactors suggests that it might be used to amplify gene clusters in a wide range of bacteria and perhaps eukaryotic hosts. To our knowledge, this represents the first system for inducible amplification of gene clusters in any organism. This new system of gene amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic or degradative pathways in a wide variety of bacteria and may also find important applications for inducible gene amplification in eukaryotic cells.