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Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies
Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10573353/ https://www.ncbi.nlm.nih.gov/pubmed/37834464 http://dx.doi.org/10.3390/ijms241915017 |
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author | Dai, Wuling Dong, Haofan Zhang, Zhaokun Wu, Xin Bao, Tongtong Gao, Le Chen, Xiaoyi |
author_facet | Dai, Wuling Dong, Haofan Zhang, Zhaokun Wu, Xin Bao, Tongtong Gao, Le Chen, Xiaoyi |
author_sort | Dai, Wuling |
collection | PubMed |
description | Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process. |
format | Online Article Text |
id | pubmed-10573353 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-105733532023-10-14 Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies Dai, Wuling Dong, Haofan Zhang, Zhaokun Wu, Xin Bao, Tongtong Gao, Le Chen, Xiaoyi Int J Mol Sci Article Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process. MDPI 2023-10-09 /pmc/articles/PMC10573353/ /pubmed/37834464 http://dx.doi.org/10.3390/ijms241915017 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Dai, Wuling Dong, Haofan Zhang, Zhaokun Wu, Xin Bao, Tongtong Gao, Le Chen, Xiaoyi Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies |
title | Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies |
title_full | Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies |
title_fullStr | Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies |
title_full_unstemmed | Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies |
title_short | Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies |
title_sort | enhancing the heterologous expression of a thermophilic endoglucanase and its cost-effective production in pichia pastoris using multiple strategies |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10573353/ https://www.ncbi.nlm.nih.gov/pubmed/37834464 http://dx.doi.org/10.3390/ijms241915017 |
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