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Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii

The current trend in industrial biotechnology is to move from batch or fed‐batch fermentations to continuous operations. The success of this transition will require the development of genetically stable production strains, the use of strong constitutive promoters, and the development of new medium f...

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Autores principales: Cankorur‐Cetinkaya, Ayca, Narraidoo, Nathalie, Kasavi, Ceyda, Slater, Nigel K.H., Archer, David B., Oliver, Stephen G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283250/
https://www.ncbi.nlm.nih.gov/pubmed/30267565
http://dx.doi.org/10.1002/bit.26846
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author Cankorur‐Cetinkaya, Ayca
Narraidoo, Nathalie
Kasavi, Ceyda
Slater, Nigel K.H.
Archer, David B.
Oliver, Stephen G.
author_facet Cankorur‐Cetinkaya, Ayca
Narraidoo, Nathalie
Kasavi, Ceyda
Slater, Nigel K.H.
Archer, David B.
Oliver, Stephen G.
author_sort Cankorur‐Cetinkaya, Ayca
collection PubMed
description The current trend in industrial biotechnology is to move from batch or fed‐batch fermentations to continuous operations. The success of this transition will require the development of genetically stable production strains, the use of strong constitutive promoters, and the development of new medium formulations that allow an appropriate balance between cell growth and product formation. We identified genes that showed high expression in Komagataella phaffii during different steady‐state conditions and explored the utility of promoters of these genes (Chr1–4_0586 and FragB_0052) in optimizing the expression of two different r‐proteins, human lysozyme (HuLy), and the anti‐idiotypic antibody fragment, Fab‐3H6, in comparison with the widely used glyceraldehyde‐3‐phosphate dehydrogenase promoter. Our results showed that the promoter strength was highly dependent on the cultivation conditions and thus constructs should be tested under a range of conditions to determine both the best performing clone and the ideal promoter for the expression of the protein of interest. An important benefit of continuous production is that it facilitates the use of the genome‐scale metabolic models in the design of strains and cultivation media. In silico flux distributions showed that production of either protein increased the flux through aromatic amino acid biosynthesis. Tyrosine supplementation increased the productivity for both proteins, whereas tryptophan addition did not cause any significant change and, phenylalanine addition increased the expression of HuLy but decreased that of Fab‐3H6. These results showed that a genome‐scale metabolic model can be used to assess the metabolic burden imposed by the synthesis of a specific r‐protein and then this information can be used to tailor a cultivation medium to increase production.
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spelling pubmed-62832502018-12-14 Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii Cankorur‐Cetinkaya, Ayca Narraidoo, Nathalie Kasavi, Ceyda Slater, Nigel K.H. Archer, David B. Oliver, Stephen G. Biotechnol Bioeng ARTICLES The current trend in industrial biotechnology is to move from batch or fed‐batch fermentations to continuous operations. The success of this transition will require the development of genetically stable production strains, the use of strong constitutive promoters, and the development of new medium formulations that allow an appropriate balance between cell growth and product formation. We identified genes that showed high expression in Komagataella phaffii during different steady‐state conditions and explored the utility of promoters of these genes (Chr1–4_0586 and FragB_0052) in optimizing the expression of two different r‐proteins, human lysozyme (HuLy), and the anti‐idiotypic antibody fragment, Fab‐3H6, in comparison with the widely used glyceraldehyde‐3‐phosphate dehydrogenase promoter. Our results showed that the promoter strength was highly dependent on the cultivation conditions and thus constructs should be tested under a range of conditions to determine both the best performing clone and the ideal promoter for the expression of the protein of interest. An important benefit of continuous production is that it facilitates the use of the genome‐scale metabolic models in the design of strains and cultivation media. In silico flux distributions showed that production of either protein increased the flux through aromatic amino acid biosynthesis. Tyrosine supplementation increased the productivity for both proteins, whereas tryptophan addition did not cause any significant change and, phenylalanine addition increased the expression of HuLy but decreased that of Fab‐3H6. These results showed that a genome‐scale metabolic model can be used to assess the metabolic burden imposed by the synthesis of a specific r‐protein and then this information can be used to tailor a cultivation medium to increase production. John Wiley and Sons Inc. 2018-10-24 2018-12 /pmc/articles/PMC6283250/ /pubmed/30267565 http://dx.doi.org/10.1002/bit.26846 Text en © 2018 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle ARTICLES
Cankorur‐Cetinkaya, Ayca
Narraidoo, Nathalie
Kasavi, Ceyda
Slater, Nigel K.H.
Archer, David B.
Oliver, Stephen G.
Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii
title Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii
title_full Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii
title_fullStr Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii
title_full_unstemmed Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii
title_short Process development for the continuous production of heterologous proteins by the industrial yeast, Komagataella phaffii
title_sort process development for the continuous production of heterologous proteins by the industrial yeast, komagataella phaffii
topic ARTICLES
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283250/
https://www.ncbi.nlm.nih.gov/pubmed/30267565
http://dx.doi.org/10.1002/bit.26846
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