Ajinomoto-Genetika Research Institute, 1st Dorozhny Proezd 1-1, Moscow, Russia 117545.
Mol Biotechnol. 2011 Jul;48(3):218-27. doi: 10.1007/s12033-010-9362-6.
A method for repeated PCR-mediated promoter replacement in the yeast Saccharomyces cerevisiae is described. It was proposed to use the DNA fragment comprising the marker gene that enables both positive and negative selection (a selectable/counter-selectable marker) surrounded by direct repeats of the desired promoter as a promoter replacement cassette. This fragment is integrated upstream of the target gene because of PCR-added terminal sequences for homologous recombination with the target locus. Subsequent marker excision via homologous recombination between the copies of the two promoters leaves one copy of the desired promoter upstream of the target genes, without any heterologous scar sequence. To test this method, a set of plasmids bearing the S. cerevisiae URA3 gene surrounded by two copies of the ADH1 or PGK1 promoter was constructed. Using these cassettes, the native promoters of the GSH1 and GSH2 genes were replaced in the ura3Δ0 recipient strains. The proposed method is useful for research applications due to simple marker excision, and for construction of "self-cloning" industrial strains, because no heterologous DNA is retained in the genome of the resulting strain after marker excision.
介绍了一种在酵母酿酒酵母中重复 PCR 介导启动子替换的方法。该方法建议使用包含标记基因的 DNA 片段作为启动子替换盒，该标记基因可同时进行阳性和阴性选择（可选择/可消除标记），并被所需启动子的直接重复序列包围。由于 PCR 添加了与靶基因座同源重组的末端序列，该片段整合到靶基因的上游。随后通过两个启动子之间的拷贝之间的同源重组进行标记物切除，在不留下任何异源疤痕序列的情况下，在靶基因的上游留下一个所需启动子的拷贝。为了测试该方法，构建了一组带有 S. cerevisiae URA3 基因的质粒，该基因被两个 ADH1 或 PGK1 启动子的拷贝包围。使用这些盒，在 ura3Δ0 受体菌株中替换了 GSH1 和 GSH2 基因的天然启动子。由于简单的标记物切除，该方法对于研究应用很有用，并且对于构建“自我克隆”工业菌株也很有用，因为在标记物切除后，在产生的菌株的基因组中不保留任何异源 DNA。
