mazF as a counter-selectable marker for unmarked genetic modification of Pichia pastoris
mazF as a counter-selectable marker for unmarked genetic modification of Pichia pastoris
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DOI：
10.1111/j.1567-1364.2009.00503.x
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发表时间：
2009-06-01
影响因子：
3.2
通讯作者：
Yang, Sheng
中科院分区：
文献类型：
--
作者：
Yang, Junjie;Jiang, Weihong;Yang, Sheng
在这项研究中，我们展示了一种新的方法对甲基营养酵母毕赤酵母进行无标记的遗传修饰，其中大肠杆菌毒素基因mazF被用作反选择标记。将mazF置于严格控制的AOX1启动子下，在P. pastoris中诱导mazF的表达使细胞停止生长。构建了一个模块化质粒，其中mazF和Zeocin抗性基因分别作为反选择和主动选择标记，并且在mazF - zeor盒的两侧有两个直接重复序列，用于标记循环。利用模块化质粒构建线性化的传递载体，通过同源重组，引入遗传修饰，将其整合到巴斯德酵母基因组中。在诱导AOX1启动子的甲醇培养基中进行反选择，通过直接重复序列之间的同源重组有效地回收了标记。我们使用这种方法成功地敲除了ARG1和MET2基因，敲入了一个绿色荧光蛋白表达盒，并对ARG1基因进行了定点诱变，所有这些都没有引入不必要的选择标记。这种新方法允许重复使用可选择的标记基因进行多次修饰，将成为一种有用的研究工具。
In this study, we demonstrate a novel method for unmarked genetic modification of the methylotrophic yeast Pichia pastoris, in which the Escherichia coli toxin gene mazF was used as a counter-selectable marker. mazF was placed under the tightly controlled AOX1 promoter, and the induced expression of MazF in P. pastoris halted cell growth. A modular plasmid was constructed in which mazF and a Zeocin resistance gene acted as counter-selectable and active-selectable markers, respectively, and the MazF-ZeoR cassette was flanked by two direct repeats for marker recycling. Linearized delivery vectors constructed from the modular plasmid were integrated into the P. pastoris genome via homologous recombination, introducing genetic modifications. Upon counter-selection with methanol medium, which induces the AOX1 promoter, the markers were recycled efficiently via homologous recombination between the direct repeats. We used this method successfully to knock-out the ARG1 and MET2 genes, knock-in a green fluorescent protein expression cassette, and perform site-directed mutagenesis on the ARG1 gene, all without introducing unwanted selection markers. The novel method allows repeated use of the selectable marker gene for multiple modifications and will be a useful tool for P. pastoris studies.