Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Dai, Wuling, Dong, Haofan, Zhang, Zhaokun, Wu, Xin, Bao, Tongtong, Gao, Le, Chen, Xiaoyi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
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
_version_ 1785120442598555648
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
work_keys_str_mv AT daiwuling enhancingtheheterologousexpressionofathermophilicendoglucanaseanditscosteffectiveproductioninpichiapastorisusingmultiplestrategies
AT donghaofan enhancingtheheterologousexpressionofathermophilicendoglucanaseanditscosteffectiveproductioninpichiapastorisusingmultiplestrategies
AT zhangzhaokun enhancingtheheterologousexpressionofathermophilicendoglucanaseanditscosteffectiveproductioninpichiapastorisusingmultiplestrategies
AT wuxin enhancingtheheterologousexpressionofathermophilicendoglucanaseanditscosteffectiveproductioninpichiapastorisusingmultiplestrategies
AT baotongtong enhancingtheheterologousexpressionofathermophilicendoglucanaseanditscosteffectiveproductioninpichiapastorisusingmultiplestrategies
AT gaole enhancingtheheterologousexpressionofathermophilicendoglucanaseanditscosteffectiveproductioninpichiapastorisusingmultiplestrategies
AT chenxiaoyi enhancingtheheterologousexpressionofathermophilicendoglucanaseanditscosteffectiveproductioninpichiapastorisusingmultiplestrategies