Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9
Garrigos, Javier (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Weninger, Astrid (Graz University of Technology)
Montesinos Seguí, José Luis (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Schmid, Christian (Graz University of Technology)
Valero Barranco, Francisco (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Rinnofner, Claudia (Austrian Centre of Industrial Biotechnology (ACIB))
Glieder, Anton (Graz University of Technology)
Garcia-Ortega, Xavier (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)

Data:	2021
Resum:	Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy. A scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3'hydroxyibuprofen. For the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration. The online version contains supplementary material available at 10. 1186/s12934-021-01577-4.
Ajuts:	Agència de Gestió d'Ajuts Universitaris i de Recerca 2017/SGR-1462 European Commission 635734
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:	Bidirectional promoters ; Human CYP2C9 ; Ibuprofen ; Pichia pastoris ; Bioprocess optimisation ; Whole cell biocatalyst
Publicat a:	Microbial cell factories, Vol. 20 (April 2021) , art. 90, ISSN 1475-2859

DOI: 10.1186/s12934-021-01577-4
PMID: 33902608

13 p, 1.3 MB

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