Abstract
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 (PAOX1), and the constitutive GAP promoter (PGAP). 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 (PPDF, PUPP) 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 PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Results: Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). 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 PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. Conclusions: As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-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.
| Original language | English |
|---|---|
| Article number | 74 |
| Number of pages | 12 |
| Journal | Microbial Cell Factories |
| Volume | 20 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Dec 2021 |
| MoE publication type | A1 Journal article-refereed |
Funding
The Spanish group is member of 2017-SGR-1462 and the Reference Network in Biotechnology (XRB, Generalitat de Catalunya). JGM acknowledges a PIF scholarship from the Universitat Autònoma de Barcelona. MAN acknowledges award by the National Council of Science Technology and Technological Innovation (CONCYTEC) through its executing unit and the National Fund for Scientific, Technological and Technological Innovation Development (FONDECYT). This work was performed within the EU project IBISBA1.0 including the two IBISBA partners UAB and VTT in partnership with the bisy GmbH. EU-IBISBA obtained an ESFRI status in 2018, with the aim to accelerate biotechnology and synthetic biology activities in Europe through service infrastructure and know-how. EU-IBISBA is gratefully acknowledged for facilitating part of the work in this study through its trans-national access that received funding from the European Union’s Horizon 2020 research and innovation program (IBISBA 1.0 project) under grant N° 730,976.
Keywords
- Bioprocess development
- Expression system characterisation
- Komagataella phaffii (Pichia pastoris)
- Methanol-free bioprocesses
- Promoter regulation
- Recombinant protein production