Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae.
Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae.
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DOI：
10.1186/s12896-016-0241-5
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发表时间：
2016-02-09
影响因子：
3.5
通讯作者：
Brown WR
中科院分区：
文献类型：
--
作者：
Xu Z;Brown WR
噬菌体编码的丝氨酸整合酶，如ϕC31整合酶，被广泛用于基因组工程，但尚未被优化用于酿酒酵母，尽管该生物在生物技术中应用广泛。利用标准化的重组酶介导盒交换反应，比较了具有或不具有核定位信号的14种丝氨酸整合酶衍生物的活性。这些整合酶在酿酒酵母和哺乳动物细胞中的相对活性表明，整合酶活性的主要决定因素是酶本身，而不是它所在的细胞。我们使用了诱导型启动子来证明六种整合酶对转化酵母的增殖能力的影响是有毒的。我们表明，一般来说，活性的噬菌体编码的丝氨酸整合酶在促进基因组整合反应方面比简单的同源重组有效一个数量级。我们的研究结果使我们能够确定噬菌体ϕBt1、TP901 ~ Nls、R4、Bxb1、MR11、A118、ϕK38、ϕC31 ~ Nls、Wβ和SPBC ~ Nls在酿酒酵母中具有整合酶活性，并表明脊椎动物细胞在整合酶活性方面比酵母受到更多的限制。本文的在线版本(doi：10.1186/s12896-0160241-5)包含补充材料，授权用户可以使用。
Phage-encoded serine integrases, such as ϕC31 integrase, are widely used for genome engineering but have not been optimized for use in Saccharomyces cerevisiae although this organism is a widely used organism in biotechnology. The activities of derivatives of fourteen serine integrases that either possess or lack a nuclear localization signal were compared using a standardized recombinase mediated cassette exchange reaction. The relative activities of these integrases in S. cerevisiae and in mammalian cells suggested that the major determinant of the activity of an integrase is the enzyme itself and not the cell in which it is working. We used an inducible promoter to show that six integrases were toxic as judged by their effects upon the proliferative ability of transformed yeast. We show that in general the active phage-encoded serine integrases were an order of magnitude more efficient in promoting genome integration reactions than a simple homologous recombination. The results of our study allow us to identify the integrases of the phage ϕBT1, TP901 ~ nls, R4, Bxb1, MR11, A118, ϕK38, ϕC31 ~ nls, Wβ and SPBC ~ nls as active in S. cerevisiae and indicate that vertebrate cells are more restricted than yeast in terms of which integrases are active. The online version of this article (doi:10.1186/s12896-016-0241-5) contains supplementary material, which is available to authorized users.
影响因子：
14.9
作者：
Malla, S;Dafhnis-Calas, F;Brookfield, JFY;Smith, MCM;Brown, WRA
通讯作者：
Brown, WRA