Sequential deletion of Pichia pastoris genes by a self-excisable cassette.

A rapid and convenient method is presented for unmarked gene deletions in Pichia pastoris. Cre/mutated lox system, Zeocin(®) (Invitrogen) resistance marker and homologous arms were spliced together by fusion PCR to generate the gene disruption cassettes (homologous region-lox71-Cre-ZeoR-lox66-homologous region), which could be integrated into the P. pastoris genome via homologous recombination. After transferring double-cross-over recombinants to methanol induction medium, transient expression of Cre recombinase caused the recombination of lox71-Cre-ZeoR-lox66 fragment into a double-mutant lox72 site, thus excising the Cre-ZeoR cassette from the P. pastoris genome. As the double-mutant lox72 site displays strongly reduced binding affinity for Cre recombinase, this method could be used sequentially to disrupt P. pastoris genes without introducing selectable markers. The effectiveness of this strategy was verified by introducing both single and double gene deletions into the P. pastoris genome.