Harnessing the potential of chloroplast-derived expression elements for enhanced production of cellulases in Escherichia coli
Thermophilic cellulases can play a crucial part in the efficient breakdown of cellulose—a major component of lignocellulosic plant biomass, however, their commercial production needs simple and robust biomanufacturing biosystems. In this study, two cellulases (β-glucosidase and endoglucanase) were h...
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PeerJ Inc.
2025-01-01
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| author | Ayesha Siddiqui Muhammad Mudassar Iqbal Asad Ali Iqra Fatima Hazrat Ali Aamir Shehzad Sameer H. Qari Ghulam Raza Muhammad Aamer Mehmood Peter J. Nixon Niaz Ahmad |
| author_facet | Ayesha Siddiqui Muhammad Mudassar Iqbal Asad Ali Iqra Fatima Hazrat Ali Aamir Shehzad Sameer H. Qari Ghulam Raza Muhammad Aamer Mehmood Peter J. Nixon Niaz Ahmad |
| author_sort | Ayesha Siddiqui |
| collection | DOAJ |
| description | Thermophilic cellulases can play a crucial part in the efficient breakdown of cellulose—a major component of lignocellulosic plant biomass, however, their commercial production needs simple and robust biomanufacturing biosystems. In this study, two cellulases (β-glucosidase and endoglucanase) were heterologously expressed in Escherichia coli under a chloroplast-derived constitutive promoter and expression-enhancing terminator. The genes encoding the cellulases were sourced from a thermophilic bacterium Thermotoga maritima to exploit their industrially needed thermotolerance potential. The codon-optimized gene sequences were synthesized and placed under a tobacco chloroplast 16S rRNA promoter (Prrn), along with the 5′ UTR (untranslated region) from gene 10 of phage T7 (T7g10). A six-residue long histidine tag (His6-tag) was attached to the N-terminus for protein detection. A high-level of expression of β-glucosidase and endoglucanase in E. coli was recorded from the chloroplast promoter and terminator. Furthermore, the activity assays confirmed that the recombinant enzymes maintained their activity at elevated temperatures. Thermostability analysis showed that recombinant enzymes retained their thermotolerance even after being expressed in a non-native host. Where, β-glucosidase and endoglucanase showed their optimum activities at 90 °C and 100 °C, respectively. Examination of the 3D structures of T. maritima cellulases revealed differential ionic interactions contributing to this high degree of thermotolerance. The study highlights the feasibility of producing thermostable versions of recombinant enzymes in E. coli at high levels. Our finding underscores the potential of this approach to meet industrial demands for efficient enzyme production employing E. coli as a robust biomanufacturing platform. |
| format | Article |
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| institution | Kabale University |
| issn | 2167-8359 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | PeerJ Inc. |
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| spelling | doaj-art-0c1a1ba867d8416fb265a728c9bf66f42025-02-02T15:05:30ZengPeerJ Inc.PeerJ2167-83592025-01-0113e1861610.7717/peerj.18616Harnessing the potential of chloroplast-derived expression elements for enhanced production of cellulases in Escherichia coliAyesha Siddiqui0Muhammad Mudassar Iqbal1Asad Ali2Iqra Fatima3Hazrat Ali4Aamir Shehzad5Sameer H. Qari6Ghulam Raza7Muhammad Aamer Mehmood8Peter J. Nixon9Niaz Ahmad10National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, PakistanNational Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, PakistanNational Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, PakistanNational Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, PakistanNational Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, PakistanNational Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, PakistanBiology Department, Al-Jumum University College, Umm Al-Qura University, Makkah, Saudi ArabiaNational Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, PakistanDepartment of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad, PakistanDepartment of Life Sciences, Imperial College London, London, United KingdomNational Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, PakistanThermophilic cellulases can play a crucial part in the efficient breakdown of cellulose—a major component of lignocellulosic plant biomass, however, their commercial production needs simple and robust biomanufacturing biosystems. In this study, two cellulases (β-glucosidase and endoglucanase) were heterologously expressed in Escherichia coli under a chloroplast-derived constitutive promoter and expression-enhancing terminator. The genes encoding the cellulases were sourced from a thermophilic bacterium Thermotoga maritima to exploit their industrially needed thermotolerance potential. The codon-optimized gene sequences were synthesized and placed under a tobacco chloroplast 16S rRNA promoter (Prrn), along with the 5′ UTR (untranslated region) from gene 10 of phage T7 (T7g10). A six-residue long histidine tag (His6-tag) was attached to the N-terminus for protein detection. A high-level of expression of β-glucosidase and endoglucanase in E. coli was recorded from the chloroplast promoter and terminator. Furthermore, the activity assays confirmed that the recombinant enzymes maintained their activity at elevated temperatures. Thermostability analysis showed that recombinant enzymes retained their thermotolerance even after being expressed in a non-native host. Where, β-glucosidase and endoglucanase showed their optimum activities at 90 °C and 100 °C, respectively. Examination of the 3D structures of T. maritima cellulases revealed differential ionic interactions contributing to this high degree of thermotolerance. The study highlights the feasibility of producing thermostable versions of recombinant enzymes in E. coli at high levels. Our finding underscores the potential of this approach to meet industrial demands for efficient enzyme production employing E. coli as a robust biomanufacturing platform.https://peerj.com/articles/18616.pdfCellulasesHeterologous expressionE. coliChloroplast promoterThermotoga meritimaBiomanufacturing |
| spellingShingle | Ayesha Siddiqui Muhammad Mudassar Iqbal Asad Ali Iqra Fatima Hazrat Ali Aamir Shehzad Sameer H. Qari Ghulam Raza Muhammad Aamer Mehmood Peter J. Nixon Niaz Ahmad Harnessing the potential of chloroplast-derived expression elements for enhanced production of cellulases in Escherichia coli PeerJ Cellulases Heterologous expression E. coli Chloroplast promoter Thermotoga meritima Biomanufacturing |
| title | Harnessing the potential of chloroplast-derived expression elements for enhanced production of cellulases in Escherichia coli |
| title_full | Harnessing the potential of chloroplast-derived expression elements for enhanced production of cellulases in Escherichia coli |
| title_fullStr | Harnessing the potential of chloroplast-derived expression elements for enhanced production of cellulases in Escherichia coli |
| title_full_unstemmed | Harnessing the potential of chloroplast-derived expression elements for enhanced production of cellulases in Escherichia coli |
| title_short | Harnessing the potential of chloroplast-derived expression elements for enhanced production of cellulases in Escherichia coli |
| title_sort | harnessing the potential of chloroplast derived expression elements for enhanced production of cellulases in escherichia coli |
| topic | Cellulases Heterologous expression E. coli Chloroplast promoter Thermotoga meritima Biomanufacturing |
| url | https://peerj.com/articles/18616.pdf |
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