Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
| Autor: | Ilya I. Tolstorukov, Miguel Angel Nieto-Taype, Francisco Valero, Merja Penttilä, Xavier Garcia-Ortega, Christian Schmid, José Luis Montesinos-Seguí, Knut Madden, Juha Tähtiharju, Kiira Vuoristo, Anton Glieder, Jaana Uusitalo, Pauliina Tukiainen, Javier Garrigós-Martínez |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Expression system characterisation
lcsh:QR1-502 Gene Expression Bioengineering Chemostat Applied Microbiology and Biotechnology Bioprocess development lcsh:Microbiology Pichia pastoris Komagataella phaffii (Pichia pastoris) Transcription (biology) Gene expression Bioprocess Promoter Regions Genetic Gene biology Chemistry Research Methanol Methanol-free bioprocesses Promoter biology.organism_classification Recombinant protein production Recombinant Proteins Kinetics Biochemistry Promoter regulation Batch Cell Culture Techniques Saccharomycetales Unfolded protein response Biotechnology |
| Zdroj: | Garrigós-Martínez, J, Vuoristo, K, Nieto-Taype, M A, Tähtiharju, J, Uusitalo, J, Tukiainen, P, Schmid, C, Tolstorukov, I, Madden, K, Penttilä, M, Montesinos-Seguí, J L, Valero, F, Glieder, A & Garcia-Ortega, X 2021, ' Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris ', Microbial Cell Factories, vol. 20, no. 1, 74 . https://doi.org/10.1186/s12934-021-01564-9 Universitat Autònoma de Barcelona Microbial Cell Factories, Vol 20, Iss 1, Pp 1-12 (2021) Dipòsit Digital de Documents de la UAB Microbial Cell Factories |
| DOI: | 10.1186/s12934-021-01564-9 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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