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Hybrid metabolic flux analysis and recombinant protein prediction in Pichia pastoris X-33 cultures expressing a single-chain antibody fragment

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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.

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Abbreviations

MFA:

Metabolic flux analysis

PLS:

Partial least squares

LV:

Latent variable

RMSE:

Root mean squared error

nRMSE:

Normalized root mean squared error

DCW:

Dry cell weight

MFD:

Metabolic flux distribution

EMP:

Embden-Meyerhof-Parnas

PPP:

Pentose phosphate pathway

TCA:

Tricarboxylic acid cycle

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Acknowledgments

Financial support for this work was provided by the Portuguese Foundation for Science and Technology through individual grant SFRH/BD/70768/2010 and project grant PTDC/BBB-BSS/2800/2012.

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Authors and Affiliations

  1. LAQV-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal

    Inês A. Isidro, Rui M. Portela & Rui Oliveira

  2. Instituto de Biologia Experimental e Tecnológica (IBET), Av. da República, EAN, 2780-157, Oeiras, Portugal

    Inês A. Isidro, João J. Clemente, António E. Cunha & Rui Oliveira

  3. Functional enviromics technologies SA, Campus Caparica, 2829-516, Caparica, Portugal

    Rui Oliveira

Authors
  1. Inês A. Isidro
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  2. Rui M. Portela
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  3. João J. Clemente
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  4. António E. Cunha
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  5. Rui Oliveira
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Correspondence to Rui Oliveira.

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Isidro, I.A., Portela, R.M., Clemente, J.J. et al. Hybrid metabolic flux analysis and recombinant protein prediction in Pichia pastoris X-33 cultures expressing a single-chain antibody fragment. Bioprocess Biosyst Eng 39, 1351–1363 (2016). https://doi.org/10.1007/s00449-016-1611-z

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  • DOI: https://doi.org/10.1007/s00449-016-1611-z

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