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Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

BACKGROUND: Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability o...

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Autores principales: Navone, Laura, Vogl, Thomas, Luangthongkam, Pawarisa, Blinco, Jo-Anne, Luna-Flores, Carlos H., Chen, Xiaojing, von Hellens, Juhani, Mahler, Stephen, Speight, Robert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010977/
https://www.ncbi.nlm.nih.gov/pubmed/33789740
http://dx.doi.org/10.1186/s13068-021-01936-8
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author Navone, Laura
Vogl, Thomas
Luangthongkam, Pawarisa
Blinco, Jo-Anne
Luna-Flores, Carlos H.
Chen, Xiaojing
von Hellens, Juhani
Mahler, Stephen
Speight, Robert
author_facet Navone, Laura
Vogl, Thomas
Luangthongkam, Pawarisa
Blinco, Jo-Anne
Luna-Flores, Carlos H.
Chen, Xiaojing
von Hellens, Juhani
Mahler, Stephen
Speight, Robert
author_sort Navone, Laura
collection PubMed
description BACKGROUND: Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. RESULTS: In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in k(cat) and K(M) for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. CONCLUSIONS: Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13068-021-01936-8.
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spelling pubmed-80109772021-03-31 Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris Navone, Laura Vogl, Thomas Luangthongkam, Pawarisa Blinco, Jo-Anne Luna-Flores, Carlos H. Chen, Xiaojing von Hellens, Juhani Mahler, Stephen Speight, Robert Biotechnol Biofuels Research BACKGROUND: Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. RESULTS: In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in k(cat) and K(M) for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. CONCLUSIONS: Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13068-021-01936-8. BioMed Central 2021-03-31 /pmc/articles/PMC8010977/ /pubmed/33789740 http://dx.doi.org/10.1186/s13068-021-01936-8 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Navone, Laura
Vogl, Thomas
Luangthongkam, Pawarisa
Blinco, Jo-Anne
Luna-Flores, Carlos H.
Chen, Xiaojing
von Hellens, Juhani
Mahler, Stephen
Speight, Robert
Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris
title Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris
title_full Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris
title_fullStr Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris
title_full_unstemmed Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris
title_short Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris
title_sort disulfide bond engineering of appa phytase for increased thermostability requires co-expression of protein disulfide isomerase in pichia pastoris
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010977/
https://www.ncbi.nlm.nih.gov/pubmed/33789740
http://dx.doi.org/10.1186/s13068-021-01936-8
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