title: Hybrid metabolic flux analysis and recombinant protein prediction in Pichia pastoris X-33 cultures expressing a single-chain antibody fragment.
abstract: Despite the growing importance of the Pichia pastoris expression system as industrial workhorse, the literature is almost absent in systematic studies on how culture medium composition affects central carbon fluxes and heterologous protein expression. In this study we investigate how 26 variations of the BSM+PTM1 medium impact central carbon fluxes and protein expression in a P. pastoris X-33 strain expressing a single-chain antibody fragment. To achieve this goal, we adopted a hybrid metabolic flux analysis (MFA) methodology, which is a modification of standard MFA to predict the rate of synthesis of recombinant proteins. Hybrid MFA combines the traditional parametric estimation of central carbon fluxes with non-parametric statistical modeling of product-related quantitative or qualitative measurements as a function of central carbon fluxes. It was observed that protein yield variability was 53.6 % (relative standard deviation) among the different experiments. Protein yield is much more sensitive to medium composition than biomass growth, which is mainly determined by the carbon source availability and main salts. Hybrid MFA was able to describe accurately the protein yield with normalized RMSE of 6.3 % over 5 independent experiments. The metabolic state that promotes high protein yields is characterized by high overall metabolic rates through main central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy generating pathways.
authors.affiliation: LAQV-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal.|Instituto de Biologia Experimental e Tecnológica (IBET), Av. da República, EAN, 2780-157, Oeiras, Portugal.
authors.forename: Inês A
authors.initials: IA
authors.lastname: Isidro
authors.affiliation: LAQV-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: Rui M
authors.initials: RM
authors.lastname: Portela
authors.affiliation: Instituto de Biologia Experimental e Tecnológica (IBET), Av. da República, EAN, 2780-157, Oeiras, Portugal.
authors.forename: João J
authors.initials: JJ
authors.lastname: Clemente
authors.affiliation: Instituto de Biologia Experimental e Tecnológica (IBET), Av. da República, EAN, 2780-157, Oeiras, Portugal.
authors.forename: António E
authors.initials: AE
authors.lastname: Cunha
authors.affiliation: LAQV-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal. rmo@fct.unl.pt.|Instituto de Biologia Experimental e Tecnológica (IBET), Av. da República, EAN, 2780-157, Oeiras, Portugal. rmo@fct.unl.pt.|Functional enviromics technologies SA, Campus Caparica, 2829-516, Caparica, Portugal. rmo@fct.unl.pt.
authors.forename: Rui
authors.initials: R
authors.lastname: Oliveira
mesh_terms: D003470:Culture Media; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics*
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies
keywords: Central carbon fluxes; Culture media; Hybrid MFA; Pichia pastoris X-33; Protein expression prediction