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

• Main Authors: Garrigos, Javier, Vuoristo, Kiira, Nieto-Taype, Miguel Angel, Tähtiharju, Juha, Uusitalo, Jaana, Tukiainen, Pauliina, Schmid, Christian, Tolstorukov, Ilya, Madden, Knut, Penttilä, Merja, Montesinos Seguí, José Luis, Valero Barranco, Francisco, Glieder, Anton, Garcia-Ortega, Xavier
• Format: Article in Journal/Newspaper
• Language: English
• Published: 2021
• Subjects: Komagataella phaffii (Pichia pastoris); Recombinant protein production; Methanol-free bioprocesses; Expression system characterisation; Promoter regulation; Bioprocess development; Antarc*; Antarctica
• Online Access: https://ddd.uab.cat/record/255373

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), and the constitutive GAP promoter (P). 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, P) 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, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Both the P and P-based expression systems outperformed similar P-based expression in chemostat cultivations, reaching ninefold higher specific production rates (q ). 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 and P expression systems were also successfully implemented in 15 L fed-batch cultivations where q and product to biomass yield (Y *) values were similar than those obtained in chemostat cultivations. As an outcome of the macrokinetic characterization presented, the novel P and P were observed to offer much higher efficiency for CalB production than the widely used P-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 ...