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Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway

Pichia pastoris is a commonly used microbial host for recombinant protein production. It is mostly cultivated in fed‐batch mode, in which the environment of the cell is continuously changing. Hence, it is vital to understand the influence of feeding strategy parameters on the intracellular reaction...

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Autores principales: Boojari, Mohammad Amin, Rajabi Ghaledari, Fatemeh, Motamedian, Ehsan, Soleimani, Mehdi, Shojaosadati, Seyed Abbas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10221529/
https://www.ncbi.nlm.nih.gov/pubmed/37093126
http://dx.doi.org/10.1111/1751-7915.14264
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author Boojari, Mohammad Amin
Rajabi Ghaledari, Fatemeh
Motamedian, Ehsan
Soleimani, Mehdi
Shojaosadati, Seyed Abbas
author_facet Boojari, Mohammad Amin
Rajabi Ghaledari, Fatemeh
Motamedian, Ehsan
Soleimani, Mehdi
Shojaosadati, Seyed Abbas
author_sort Boojari, Mohammad Amin
collection PubMed
description Pichia pastoris is a commonly used microbial host for recombinant protein production. It is mostly cultivated in fed‐batch mode, in which the environment of the cell is continuously changing. Hence, it is vital to understand the influence of feeding strategy parameters on the intracellular reaction network to fine‐tune bioreactor performance. This study used dynamic flux balance analysis (DFBA) integrated with transcriptomics data to simulate the recombinant P. pastoris (Mut(s)) growth during the induction phase for three fed‐batch strategies, conducted at constant specific growth rates (μ‐stat). The induction phase was split into equal time intervals, and the correlated reactions with protein yield were identified in the three fed‐batch strategies using the Pearson correlation coefficient. Subsequently, principal component analysis (PCA) was applied to cluster induction phase time intervals and identify the role of correlated reactions on metabolic differentiation of time intervals. It was found that increasing fluxes through the methanol dissimilation pathway increased protein yield. By adding a methanol assimilation pathway inhibitor (HgCl(2)) to the shake flask medium growing on glycerol: methanol mixture (10%: 90%, v/v), the protein titre increased by 60%. As per DFBA, the higher the methanol to biomass flux ratio (R(meoh/Δx )), the higher the protein yield. Finally, a novel feeding strategy was developed to increase the amount of R(meoh/Δx ) compared to the three feeding strategies. The concentration of recombinant human growth hormone (rhGH), used as the model protein, increased by 16% compared to the optimal culture result obtained previously (800 mg L(−1) to 928 mg L(−1)), while production yield improved by 85% (24.8 mg g(DCW) (−1) to 46 mg g(DCW) (−1)).
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spelling pubmed-102215292023-05-28 Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway Boojari, Mohammad Amin Rajabi Ghaledari, Fatemeh Motamedian, Ehsan Soleimani, Mehdi Shojaosadati, Seyed Abbas Microb Biotechnol Research Articles Pichia pastoris is a commonly used microbial host for recombinant protein production. It is mostly cultivated in fed‐batch mode, in which the environment of the cell is continuously changing. Hence, it is vital to understand the influence of feeding strategy parameters on the intracellular reaction network to fine‐tune bioreactor performance. This study used dynamic flux balance analysis (DFBA) integrated with transcriptomics data to simulate the recombinant P. pastoris (Mut(s)) growth during the induction phase for three fed‐batch strategies, conducted at constant specific growth rates (μ‐stat). The induction phase was split into equal time intervals, and the correlated reactions with protein yield were identified in the three fed‐batch strategies using the Pearson correlation coefficient. Subsequently, principal component analysis (PCA) was applied to cluster induction phase time intervals and identify the role of correlated reactions on metabolic differentiation of time intervals. It was found that increasing fluxes through the methanol dissimilation pathway increased protein yield. By adding a methanol assimilation pathway inhibitor (HgCl(2)) to the shake flask medium growing on glycerol: methanol mixture (10%: 90%, v/v), the protein titre increased by 60%. As per DFBA, the higher the methanol to biomass flux ratio (R(meoh/Δx )), the higher the protein yield. Finally, a novel feeding strategy was developed to increase the amount of R(meoh/Δx ) compared to the three feeding strategies. The concentration of recombinant human growth hormone (rhGH), used as the model protein, increased by 16% compared to the optimal culture result obtained previously (800 mg L(−1) to 928 mg L(−1)), while production yield improved by 85% (24.8 mg g(DCW) (−1) to 46 mg g(DCW) (−1)). John Wiley and Sons Inc. 2023-04-24 /pmc/articles/PMC10221529/ /pubmed/37093126 http://dx.doi.org/10.1111/1751-7915.14264 Text en © 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd. https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
spellingShingle Research Articles
Boojari, Mohammad Amin
Rajabi Ghaledari, Fatemeh
Motamedian, Ehsan
Soleimani, Mehdi
Shojaosadati, Seyed Abbas
Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway
title Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway
title_full Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway
title_fullStr Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway
title_full_unstemmed Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway
title_short Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway
title_sort developing a metabolic model‐based fed‐batch feeding strategy for pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10221529/
https://www.ncbi.nlm.nih.gov/pubmed/37093126
http://dx.doi.org/10.1111/1751-7915.14264
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