Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
Garrigos, Javier (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Vuoristo, Kiira (VTT Technical Research Centre of Finland Ltd. Solutions for Natural Resources and Environment)
Nieto-Taype, Miguel Angel (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Tähtiharju, Juha (VTT Technical Research Centre of Finland Ltd. Solutions for Natural Resources and Environment)
Uusitalo, Jaana (VTT Technical Research Centre of Finland Ltd. Solutions for Natural Resources and Environment)
Tukiainen, Pauliina (VTT Technical Research Centre of Finland Ltd. Solutions for Natural Resources and Environment)
Schmid, Christian (Bisy GmbH, Wuenschendorf 292, 8200 Hofstaetten/Raab, Austria)
Tolstorukov, Ilya (Keck Graduate Institute)
Madden, Knut (Biogrammatics Inc, 2794 Loker Ave, West, Suite 103, Carlsbad, CA 92010 USA)
Penttilä, Merja (VTT Technical Research Centre of Finland Ltd. Solutions for Natural Resources and Environment)
Montesinos Seguí, José Luis (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Valero Barranco, Francisco (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Glieder, Anton (Bisy GmbH, Wuenschendorf 292, 8200 Hofstaetten/Raab, Austria)
Garcia-Ortega, Xavier (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)

Data:	2021
Resum:	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 factory for different products of interest. The online version contains supplementary material available at 10. 1186/s12934-021-01564-9.
Ajuts:	European Commission No 730976
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Komagataella phaffii (Pichia pastoris) ; Recombinant protein production ; Methanol-free bioprocesses ; Expression system characterisation ; Promoter regulation ; Bioprocess development
Publicat a:	Microbial cell factories, Vol. 20 (march 2021) , ISSN 1475-2859

DOI: 10.1186/s12934-021-01564-9
PMID: 33757505

12 p, 1.5 MB

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