Open AccessmazF as a counter‐selectable marker for unmarked genetic modification of Pichia pastoris
Author(s) -
Yang Junjie,
Jiang Weihong,
Yang Sheng
Publication year - 2009
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/j.1567-1364.2009.00503.x
Subject(s) - selectable marker , pichia pastoris , biology , plasmid , homologous recombination , gene , expression cassette , genetics , mutagenesis , reporter gene , electroporation , gene cassette , marker gene , computational biology , recombinant dna , gene expression , mutation , vector (molecular biology) , integron
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.