One-step assembly and targeted integration of multigene constructs assisted by the I-SceI meganuclease in Saccharomyces cerevisiae.
One-step assembly and targeted integration of multigene constructs assisted by the I-SceI meganuclease in Saccharomyces cerevisiae.
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DOI：
10.1111/1567-1364.12087
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发表时间：
2013-12
影响因子：
3.2
通讯作者：
Daran-Lapujade P
中科院分区：
文献类型：
--
作者：
Kuijpers NG;Chroumpi S;Vos T;Solis-Escalante D;Bosman L;Pronk JT;Daran JM;Daran-Lapujade P
在酿酒酵母中通过同源重组在体内组装重叠片段是工程化大DNA构建体的有力方法。尽管迄今为止报道的大多数体内组装方法产生环状载体，但稳定的整合构建体通常优选用于代谢工程，因为它们是大规模工业应用所需的。本研究探讨了将大型多基因表达构建体的体内组装与其在S.啤酒。在单个转化过程中成功地实现了10个片段22-kb构建体与单个染色体基因座的组合组装和靶向整合，但效率较低（5%的分析转化体含有正确组装的构建体）。因此，大范围核酸酶I-SceI用于在靶向的染色体基因座处引入双链断裂，从而促进组装的构建体的整合。I-SceI辅助的整合将相同构建体的组装和整合效率显著提高至95%。本研究为大DNA构建体在S.酿酒酵母染色体
In vivo assembly of overlapping fragments by homologous recombination in Saccharomyces cerevisiae is a powerful method to engineer large DNA constructs. Whereas most in vivo assembly methods reported to date result in circular vectors, stable integrated constructs are often preferred for metabolic engineering as they are required for large-scale industrial application. The present study explores the potential of combining in vivo assembly of large, multigene expression constructs with their targeted chromosomal integration in S. cerevisiae. Combined assembly and targeted integration of a ten-fragment 22-kb construct to a single chromosomal locus was successfully achieved in a single transformation process, but with low efficiency (5% of the analyzed transformants contained the correctly assembled construct). The meganuclease I-SceI was therefore used to introduce a double-strand break at the targeted chromosomal locus, thus to facilitate integration of the assembled construct. I-SceI-assisted integration dramatically increased the efficiency of assembly and integration of the same construct to 95%. This study paves the way for the fast, efficient, and stable integration of large DNA constructs in S. cerevisiae chromosomes.
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影响因子：
6.4
作者：
Kuijpers NG;Solis-Escalante D;Bosman L;van den Broek M;Pronk JT;Daran JM;Daran-Lapujade P
通讯作者：
Daran-Lapujade P
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
2.5
作者：
Ellis, Tom;Adie, Tom;Baldwin, Geoff S.
通讯作者：
Baldwin, Geoff S.
串联重复序列与核酸内切酶切割 (TREC) 相结合：酵母基因组工程的无缝修饰工具。
影响因子：
14.9
作者：
Noskov, Vladimir N.;Segall-Shapiro, Thomas H.;Chuang, Ray-Yuan
通讯作者：
Chuang, Ray-Yuan
影响因子：
64.8
作者：
Ro, DK;Paradise, EM;Keasling, JD
通讯作者：
Keasling, JD