Xu Zhengyao, Brown William R A
School of Life Sciences, Queens Medical Centre, Nottingham University, Nottingham, NG7 2UH, UK.
BMC Biotechnol. 2016 Feb 9;16:13. doi: 10.1186/s12896-016-0241-5.
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.
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，但尚未针对酿酒酵母进行优化，尽管该生物体是生物技术中广泛使用的生物体。
使用标准化的重组酶介导的盒式交换反应比较了十四种具有或缺乏核定位信号的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，而不是其作用的细胞。我们使用诱导型启动子表明，六种整合酶根据其对转化酵母增殖能力的影响判断是有毒的。我们表明，一般来说，活性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效率高一个数量级。
我们的研究结果使我们能够鉴定出噬菌体ϕBT1、TP901 ~ nls、R4、Bxb1、MR11、A118、ϕK38、ϕC31 ~ nls、Wβ和SPBC ~ nls的整合酶在酿酒酵母中具有活性，并表明脊椎动物细胞在活性整合酶方面比酵母受到更多限制。
