Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris

Background: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P_AOX1), and the constitutive GAP promoter (P_GAP). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (P_PDF, P_UPP) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter P_GAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Results: Both the P_PDF and P_UPP-based expression systems outperformed similar P_GAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (q_p). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both P_PDF and P_UPP expression systems were also successfully implemented in 15 L fed-batch cultivations where q_p and product to biomass yield (Y_P/X*) values were similar than those obtained in chemostat cultivations. Conclusions: As an outcome of the macrokinetic characterization presented, the novel P_PDF and P_UPP were observed to offer much higher efficiency for CalB production than the widely used P_GAP-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest.