Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes.
Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes.
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DOI：
10.1038/srep07100
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发表时间：
2014-11-18
影响因子：
4.6
通讯作者：
Bibb MJ
中科院分区：
文献类型：
--
作者：
Fernández-Martínez LT;Bibb MJ
对新的天然产物的探索导致了新放线菌物种的分离，其中许多最终需要遗传分析。其中一些分离株可能表现出同源重组的固有频率较低，并且在实验室条件下无法形成孢子，从而加剧了在遗传上未表征的菌株中靶向基因缺失和替换的构建。为了促进这些物种的遗传操作，我们已经开发了一种有效的方法，通过同源重组在放线菌中产生基因或基因簇缺失，该方法不会对靶生物的基因组引入任何其他变化。我们已经合成了用于在放线菌中表达的密码子优化的I-SceI基因，其导致产生酵母I-SceI归巢核酸内切酶，其在独特的引入的18个碱基对识别序列处产生双链断裂。只有那些经历同源重组的基因组才能存活，为重组体提供了强大的选择，其中大约一半具有所需的突变基因型。为了证明该系统的功效和效率，我们删除了天蓝色链霉菌M1141中化合物的红色十一烷基普地吉宁复合物的基因簇的一部分。我们相信，我们开发的系统将广泛适用于广泛的放线菌。
The search for new natural products is leading to the isolation of novel actinomycete species, many of which will ultimately require genetic analysis. Some of these isolates will likely exhibit low intrinsic frequencies of homologous recombination and fail to sporulate under laboratory conditions, exacerbating the construction of targeted gene deletions and replacements in genetically uncharacterised strains. To facilitate the genetic manipulation of such species, we have developed an efficient method to generate gene or gene cluster deletions in actinomycetes by homologous recombination that does not introduce any other changes to the targeted organism's genome. We have synthesised a codon optimised I-SceI gene for expression in actinomycetes that results in the production of the yeast I-SceI homing endonuclease which produces double strand breaks at a unique introduced 18 base pair recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype. To demonstrate the efficacy and efficiency of the system, we deleted part of the gene cluster for the red-pigmented undecylprodiginine complex of compounds in Streptomyces coelicolor M1141. We believe that the system we have developed will be broadly applicable across a wide range of actinomycetes.
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影响因子：
3.6
作者：
TAKANO, E;GRAMAJO, HC;BIBB, MJ
通讯作者：
BIBB, MJ
天然和合成四环素诱导的启动子用于抗生素产生细菌链霉菌。
影响因子：
14.9
作者：
Rodriguez-Garcia, Antonio;Combes, Patricia;Perez-Redondo, Rosario;Smith, Matthew C A;Smith, Margaret C M
通讯作者：
Smith, Margaret C M
影响因子：
3.2
作者：
MURAKAMI, T;HOLT, TG;THOMPSON, CJ
通讯作者：
THOMPSON, CJ
DOI：
10.1007/bf00259605
发表时间：
1989-11-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
MUTH, G;NUSSBAUMER, B;PUHLER, A
通讯作者：
PUHLER, A
影响因子：
64.5
作者：
JACQUIER, A;DUJON, B
通讯作者：
DUJON, B