Escherichia coli expressing chloroplast chaperones as a proxy to test heterologous Rubisco production in leaves
Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Esche...
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| Veröffentlicht in: | Journal of experimental botany Jg. 74; H. 2; S. 664 |
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11.01.2023
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| Abstract | Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Escherichia coli. While this enhances opportunities for catalytic improvement, there remain limitations in the utility of the expression system. Here we generate, optimize, and test a robust Golden Gate cloning E. coli expression system incorporating the protein folding machinery of tobacco chloroplasts. By comparing the expression of different plant Rubiscos in both E. coli and plastome-transformed tobacco, we show that the E. coli expression system can accurately predict high level Rubisco production in chloroplasts but poorly forecasts the biogenesis potential of isoforms with impaired production in planta. We reveal that heterologous Rubisco production in E. coli and tobacco plastids poorly correlates with Rubisco large subunit phylogeny. Our findings highlight the need to fully understand the factors governing Rubisco biogenesis if we are to deliver an efficient, low-cost screening tool that can accurately emulate chloroplast expression. |
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| AbstractList | Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Escherichia coli. While this enhances opportunities for catalytic improvement, there remain limitations in the utility of the expression system. Here we generate, optimize, and test a robust Golden Gate cloning E. coli expression system incorporating the protein folding machinery of tobacco chloroplasts. By comparing the expression of different plant Rubiscos in both E. coli and plastome-transformed tobacco, we show that the E. coli expression system can accurately predict high level Rubisco production in chloroplasts but poorly forecasts the biogenesis potential of isoforms with impaired production in planta. We reveal that heterologous Rubisco production in E. coli and tobacco plastids poorly correlates with Rubisco large subunit phylogeny. Our findings highlight the need to fully understand the factors governing Rubisco biogenesis if we are to deliver an efficient, low-cost screening tool that can accurately emulate chloroplast expression.Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Escherichia coli. While this enhances opportunities for catalytic improvement, there remain limitations in the utility of the expression system. Here we generate, optimize, and test a robust Golden Gate cloning E. coli expression system incorporating the protein folding machinery of tobacco chloroplasts. By comparing the expression of different plant Rubiscos in both E. coli and plastome-transformed tobacco, we show that the E. coli expression system can accurately predict high level Rubisco production in chloroplasts but poorly forecasts the biogenesis potential of isoforms with impaired production in planta. We reveal that heterologous Rubisco production in E. coli and tobacco plastids poorly correlates with Rubisco large subunit phylogeny. Our findings highlight the need to fully understand the factors governing Rubisco biogenesis if we are to deliver an efficient, low-cost screening tool that can accurately emulate chloroplast expression. Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Escherichia coli. While this enhances opportunities for catalytic improvement, there remain limitations in the utility of the expression system. Here we generate, optimize, and test a robust Golden Gate cloning E. coli expression system incorporating the protein folding machinery of tobacco chloroplasts. By comparing the expression of different plant Rubiscos in both E. coli and plastome-transformed tobacco, we show that the E. coli expression system can accurately predict high level Rubisco production in chloroplasts but poorly forecasts the biogenesis potential of isoforms with impaired production in planta. We reveal that heterologous Rubisco production in E. coli and tobacco plastids poorly correlates with Rubisco large subunit phylogeny. Our findings highlight the need to fully understand the factors governing Rubisco biogenesis if we are to deliver an efficient, low-cost screening tool that can accurately emulate chloroplast expression. |
| Author | Birch, Rosemary Kapralov, Maxim V Buck, Sally Whitney, Spencer M Gionfriddo, Matteo Skinner, Tanya Rhodes, Tim Yuan, Ding |
| Author_xml | – sequence: 1 givenname: Sally orcidid: 0000-0001-9569-0160 surname: Buck fullname: Buck, Sally organization: ARC Centre of Excellence in Translational Photosynthesis, Australian National University, Canberra 2000, Australia – sequence: 2 givenname: Tim orcidid: 0000-0002-4146-7913 surname: Rhodes fullname: Rhodes, Tim organization: ARC Centre of Excellence in Translational Photosynthesis, Australian National University, Canberra 2000, Australia – sequence: 3 givenname: Matteo orcidid: 0000-0003-2364-0812 surname: Gionfriddo fullname: Gionfriddo, Matteo organization: ARC Centre of Excellence in Translational Photosynthesis, Australian National University, Canberra 2000, Australia – sequence: 4 givenname: Tanya orcidid: 0000-0002-2661-6415 surname: Skinner fullname: Skinner, Tanya organization: ARC Centre of Excellence in Translational Photosynthesis, Australian National University, Canberra 2000, Australia – sequence: 5 givenname: Ding orcidid: 0000-0001-7895-0944 surname: Yuan fullname: Yuan, Ding organization: ARC Centre of Excellence in Translational Photosynthesis, Australian National University, Canberra 2000, Australia – sequence: 6 givenname: Rosemary orcidid: 0000-0002-3050-7406 surname: Birch fullname: Birch, Rosemary organization: ARC Centre of Excellence in Translational Photosynthesis, Australian National University, Canberra 2000, Australia – sequence: 7 givenname: Maxim V orcidid: 0000-0001-7966-0295 surname: Kapralov fullname: Kapralov, Maxim V organization: School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne NE1 7RU, UK – sequence: 8 givenname: Spencer M orcidid: 0000-0003-2954-2359 surname: Whitney fullname: Whitney, Spencer M organization: ARC Centre of Excellence in Translational Photosynthesis, Australian National University, Canberra 2000, Australia |
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| Copyright | The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com. |
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| Keywords | Chloroplast transformation synthetic biology CO2 fixation photosynthesis Golden Gate cloning |
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| Snippet | Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes'... |
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| SubjectTerms | Carbon Dioxide - metabolism Chloroplasts - metabolism Escherichia coli - genetics Escherichia coli - metabolism Molecular Chaperones - metabolism Nicotiana - metabolism Photosynthesis Plant Leaves - metabolism Plants, Genetically Modified - metabolism Ribulose-Bisphosphate Carboxylase - genetics Ribulose-Bisphosphate Carboxylase - metabolism |
| Title | Escherichia coli expressing chloroplast chaperones as a proxy to test heterologous Rubisco production in leaves |
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