A Strategy for co-expression of multiple proteins in Komagataella phaffii without introducing selectable markers via the Cre/loxP system

Abstract

Objective The objective was to develop a convenient strategy for co-expressing multiple proteins in Komagataella phaffii via the Cre/loxP system without introducing any markers.Results A 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. phaffii 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 ZeoR 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.Conclusions With easy manipulation, the method was effective in co-expressing multiple proteins with rescue of markers in this yeast.