Cargando…
Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria
Introducing nitrogen fixation (nif ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each co...
Autores principales: | , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
National Academy of Sciences
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450427/ https://www.ncbi.nlm.nih.gov/pubmed/37585462 http://dx.doi.org/10.1073/pnas.2305142120 |
_version_ | 1785095196035252224 |
---|---|
author | Yang, Jianguo Xiang, Nan Liu, Yiheng Guo, Chenyue Li, Chenyu Li, Hui Cai, Shuyi Dixon, Ray Wang, Yi-Ping |
author_facet | Yang, Jianguo Xiang, Nan Liu, Yiheng Guo, Chenyue Li, Chenyu Li, Hui Cai, Shuyi Dixon, Ray Wang, Yi-Ping |
author_sort | Yang, Jianguo |
collection | PubMed |
description | Introducing nitrogen fixation (nif ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in Escherichia coli, we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in E. coli, comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization. |
format | Online Article Text |
id | pubmed-10450427 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | National Academy of Sciences |
record_format | MEDLINE/PubMed |
spelling | pubmed-104504272023-08-26 Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria Yang, Jianguo Xiang, Nan Liu, Yiheng Guo, Chenyue Li, Chenyu Li, Hui Cai, Shuyi Dixon, Ray Wang, Yi-Ping Proc Natl Acad Sci U S A Biological Sciences Introducing nitrogen fixation (nif ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in Escherichia coli, we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in E. coli, comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization. National Academy of Sciences 2023-08-16 2023-08-22 /pmc/articles/PMC10450427/ /pubmed/37585462 http://dx.doi.org/10.1073/pnas.2305142120 Text en Copyright © 2023 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by/4.0/This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Biological Sciences Yang, Jianguo Xiang, Nan Liu, Yiheng Guo, Chenyue Li, Chenyu Li, Hui Cai, Shuyi Dixon, Ray Wang, Yi-Ping Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria |
title | Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria |
title_full | Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria |
title_fullStr | Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria |
title_full_unstemmed | Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria |
title_short | Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria |
title_sort | organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria |
topic | Biological Sciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450427/ https://www.ncbi.nlm.nih.gov/pubmed/37585462 http://dx.doi.org/10.1073/pnas.2305142120 |
work_keys_str_mv | AT yangjianguo organelledependentpolyproteindesignsenablestoichiometricexpressionofnitrogenfixationcomponentstargetedtomitochondria AT xiangnan organelledependentpolyproteindesignsenablestoichiometricexpressionofnitrogenfixationcomponentstargetedtomitochondria AT liuyiheng organelledependentpolyproteindesignsenablestoichiometricexpressionofnitrogenfixationcomponentstargetedtomitochondria AT guochenyue organelledependentpolyproteindesignsenablestoichiometricexpressionofnitrogenfixationcomponentstargetedtomitochondria AT lichenyu organelledependentpolyproteindesignsenablestoichiometricexpressionofnitrogenfixationcomponentstargetedtomitochondria AT lihui organelledependentpolyproteindesignsenablestoichiometricexpressionofnitrogenfixationcomponentstargetedtomitochondria AT caishuyi organelledependentpolyproteindesignsenablestoichiometricexpressionofnitrogenfixationcomponentstargetedtomitochondria AT dixonray organelledependentpolyproteindesignsenablestoichiometricexpressionofnitrogenfixationcomponentstargetedtomitochondria AT wangyiping organelledependentpolyproteindesignsenablestoichiometricexpressionofnitrogenfixationcomponentstargetedtomitochondria |