A novel bi-directional promotor system for tunable recombinant protein expression in Pichia pastoris

Abstract

Pichia pastoris has become an important recombinant production host in research and industry. Recombinant protein production in P. pastoris is commonly regulated by the methanol inducible PAOX1 and constitutive PGAP promoter systems. However, both are attributed to various disadvantages rendering limited degree of freedom for bioprocess engineers to tune productivity. A promising and innovative approach to tune and control productivity is the combination of two different promoter systems with varying characteristics forming a bi-directional promoter system. In this study, we successfully characterized a recombinant P. pastoris strain carrying both a de-repression promoter (PDC) and an inducible promoter (PAOX1). We cloned the model enzyme cellobiohydrolase 2 (CBH2) downstream each promoter variant and analyzed how different feeding regimes in the bioreactor activated these promoters and thus productivity. In fact, we were able to tune recombinant protein production using different feeding strategies. We determined optimal operating windows for both promoters to work individually but also concomitantly. Summarizing, novel bi-directional promoter systems allow tunable recombinant protein production in P. pastoris and thus allow a high degree of freedom for bioprocess development and optimization.