mazF as a counter-selectable marker for unmarked genetic modification of Pichia pastoris

Authors

  • Junjie Yang,

    1. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
    2. Graduate University of the Chinese Academy of Sciences, Beijing, China; and
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  • Weihong Jiang,

    1. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
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  • Sheng Yang

    1. Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
    2. Huzhou Research Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, China
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  • Editor: Monique Bolotin-Fukuhara

Correspondence: Sheng Yang, Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. Tel.: +86 21 54 924 173; fax: +86 21 54 924 015; e-mail: syang@sibs.ac.cn

Abstract

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.

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