Department of Biotechnology, Delft University of Technology, Delft, The Netherlands; Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.
FEMS Yeast Res. 2013 Dec;13(8):769-81. doi: 10.1111/1567-1364.12087. Epub 2013 Oct 7.
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.
在酿酒酵母中通过同源重组进行重叠片段的体内组装是构建大型 DNA 构建体的有力方法。虽然迄今为止报道的大多数体内组装方法都导致形成环状载体，但对于代谢工程来说，通常更需要稳定的整合构建体，因为它们是大规模工业应用所必需的。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酿酒酵母中的靶向染色体整合相结合的潜力。在单个转化过程中，成功地将一个包含十个片段的 22kb 构建体组合并靶向整合到单个染色体位点，但效率较低（分析的转化体中有 5%包含正确组装的构建体）。因此，使用 meganuclease I-SceI 在靶向染色体位点处引入双链断裂，从而促进组装构建体的整合。I-SceI 辅助整合极大地提高了相同构建体的组装和整合效率，达到 95%。这项研究为酿酒酵母染色体中大型 DNA 构建体的快速、高效和稳定整合铺平了道路。
