Rationale-based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffii (Pichia pastoris)
Nieto-Taype, Miguel Angel (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Garrigos, Javier (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Sánchez-Farrando, Marc (Purac Bioquímica S.A.)
Valero Barranco, Francisco (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Garcia-Ortega, Xavier (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Montesinos Seguí, José Luis (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)

Data:	2020
Resum:	Its features as a microbial and eukaryotic organism have turned Komagataella phaffii (Pichia pastoris) into an emerging cell factory for recombinant protein production (RPP). As a key step of the bioprocess development, this work aimed to demonstrate the importance of tailor designing the cultivation strategy according to the production kinetics of the cell factory. For this purpose, K. phaffii clones constitutively expressing (P ) Candida rugosa lipase 1 (Crl1) with different gene dosage were used as models in continuous and fed-batch cultures. Production parameters were much greater with a multicopy clone (MCC) than with the single-copy clone (SCC). Regarding production kinetics, the specific product generation rate (q ) increased linearly with increasing specific growth rate (µ) in SCC; by contrast, q exhibited saturation in MCC. A transcriptional analysis in chemostat cultures suggested the presence of eventual post-transcriptional bottlenecks in MCC. After the strain characterization, in order to fulfil overall development of the bioprocess, the performance of both clones was also evaluated in fed-batch mode. Strikingly, different optimal strategies were determined for both models due to the different production kinetic patterns observed as a trade-off for product titre, yields and productivity. The combined effect of gene dosage and adequate µ enables rational process development with a view to optimize K. phaffii RPP bioprocesses. The combined effect of strain and bioprocess engineering should be considered during a rational process development towards bioprocess optimization. The synergy between gene dosage and specific growth rate have been evaluated for the Komagataella phaffii (Pichia pastoris) recombinant protein production (RPP) of an industrial lipase in chemostat and fed-batch cultures. Different optimal strategies were designed from a trade-off for product titer, yields and productivity for two clone models with different gene dosage and production kinetics, from which further transcription analysis supported potential bottlenecks after transcription.
Ajuts:	Agència de Gestió d'Ajuts Universitaris i de Recerca 2017/SGR-146 Agencia Estatal de Investigación CTQ2016-74959-R
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
Publicat a:	Microbial biotechnology, Vol. 13, Issue 2 (Març 2020) , p. 315-327, ISSN 1751-7915

DOI: 10.1111/1751-7915.13498
PMID: 31657146

13 p, 416.2 KB

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