Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins

Abstract

ObjectiveThe objective was to develop a convenient strategy for co-expressing multiple proteins in Komagataella pha i via the Cre/loxP system without introducing any markers. ResultsA 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. pha i 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 recovery of Zeo R 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. ConclusionsWith easy manipulation, the method was effective in co-expressing multiple proteins with rescue of markers in this yeast.