Site-Specific Recombination Strategies for Engineering Actinomycete Genomes
Site-Specific Recombination Strategies for Engineering Actinomycete Genomes
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DOI：
10.1128/aem.06054-11
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发表时间：
2012-03-01
影响因子：
4.4
通讯作者：
Luzhetskyy, Andriy
中科院分区：
文献类型：
--
作者：
Herrmann, Simone;Siegl, Theresa;Luzhetskyy, Andriy
在放线菌中使用基于定点重组的技术的可行性早在几年前就被证明了。尽管这些技术具有巨大的潜力，但它们大多被用于从染色体上移除简单的标记。在这篇文章中，我们提出了不同的定点重组策略，在几个放线菌属于链霉菌属，小单孢菌，和糖链霉属。基于CRE/LOXP和DRE/ROX的两种不同系统已被用于许多应用。Cre重组酶在异种loxLE和loxRE位点上的活性与其在野生型loxP位点上的活性相似。此外，与其他细菌相比，天蓝色链霉菌M145基因组中loxLERE位点两侧的安普霉素抗性标记以惊人的高频率(80%)被消除。构建了编码Dre重组酶的人工基因，并在放线菌中成功表达。我们开发了一种基于噬菌体整合系统和定点重组酶相结合的无标记表达方法。Cre重组酶已被用于链霉菌基因组大片段的缺失，包括苯丙内酯、莫能菌素和脂霉素生物合成基因簇。菌株Tu6071、肉桂链霉菌A519和金黄色链霉菌Tu117。最后，我们还证明了在Cre重组酶的催化下，质粒和粘粒DNA可以定点整合到放线菌的染色体中。我们期望本文提出的策略将被广泛用于研究放线菌的遗传学。
The feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the genera Streptomyces, Micromonospora, and Saccharothrix. Two different systems based on Cre/loxP and Dre/rox have been utilized for numerous applications. The activity of the Cre recombinase on the heterospecific loxLE and loxRE sites was similar to its activity on wild-type loxP sites. Moreover, an apramycin resistance marker flanked by the loxLERE sites was eliminated from the Streptomyces coelicolor M145 genome at a surprisingly high frequency (80%) compared to other bacteria. A synthetic gene encoding the Dre recombinase was constructed and successfully expressed in actinomycetes. We developed a marker-free expression method based on the combination of phage integration systems and site-specific recombinases. The Cre recombinase has been used in the deletion of huge genomic regions, including the phenalinolactone, monensin, and lipomycin biosynthetic gene clusters from Streptomyces sp. strain Tu6071, Streptomyces cinnamonensis A519, and Streptomyces aureofaciens Tu117, respectively. Finally, we also demonstrated the site-specific integration of plasmid and cosmid DNA into the chromosome of actinomycetes catalyzed by the Cre recombinase. We anticipate that the strategies presented here will be used extensively to study the genetics of actinomycetes.