Analytical and Testing Center, Guizhou Institute of Technology, Guiyang, 550000, China.
College of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang, 550000, China.
Biotechnol Lett. 2024 Jun;46(3):399-407. doi: 10.1007/s10529-024-03466-3. Epub 2024 Feb 28.
A convenient strategy was developed to recycle selectable markers using Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.
A plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. phaffii KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recycling of Zeo and His markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures.
With easy manipulation, the method was effective in recycling of the selectable markers, and consequently two protein genes were sequential integrated chromosomally and successfully co-expressed in the yeast.
开发了一种使用 Cre/loxP 系统回收可选择标记物的便捷策略，用于构建共表达多种蛋白质的毕赤酵母菌株。
该策略中的质粒由 pPICZαA 生成，整合了 lox71-Sh ble-lox66。首先，将质粒插入一个靶蛋白基因，然后转化到 K. phaffii KM71 中。其次，将含有 CRE 重组酶基因的辅助质粒 pPICZαA/cre/his4 进一步插入 Sh ble 基因中的染色体。最后，进行甲醇诱导以产生 CRE，用于 Cre/loxP 介导的重组，从而删除 lox71 和 lox66 之间的序列，实现 Zeo 和 His 标记物的回收。然后，将表达一个靶蛋白的所得菌株用作宿主，可以通过相同的程序插入另一个靶蛋白基因。
该方法操作简单，可有效回收可选择标记物，从而将两个蛋白质基因顺序整合到染色体上，并在酵母中成功共表达。
