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.