Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation.
Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiologica...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0068677 |
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| author | Xia Wang Jian-Guo Yang Li Chen Ji-Long Wang Qi Cheng Ray Dixon Yi-Ping Wang |
| author_facet | Xia Wang Jian-Guo Yang Li Chen Ji-Long Wang Qi Cheng Ray Dixon Yi-Ping Wang |
| author_sort | Xia Wang |
| collection | DOAJ |
| description | Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ~100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase-LacI expression system was used to replace the σ(54)-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7-dependent promoters, ~42% of the nitrogenase activity of the σ(54)-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology. |
| format | Article |
| id | doaj-art-0a7b0a2d070148959d2be7b3fec57d06 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-0a7b0a2d070148959d2be7b3fec57d062025-08-20T02:22:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0187e6867710.1371/journal.pone.0068677Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation.Xia WangJian-Guo YangLi ChenJi-Long WangQi ChengRay DixonYi-Ping WangBiological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ~100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase-LacI expression system was used to replace the σ(54)-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7-dependent promoters, ~42% of the nitrogenase activity of the σ(54)-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology.https://doi.org/10.1371/journal.pone.0068677 |
| spellingShingle | Xia Wang Jian-Guo Yang Li Chen Ji-Long Wang Qi Cheng Ray Dixon Yi-Ping Wang Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation. PLoS ONE |
| title | Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation. |
| title_full | Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation. |
| title_fullStr | Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation. |
| title_full_unstemmed | Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation. |
| title_short | Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation. |
| title_sort | using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation |
| url | https://doi.org/10.1371/journal.pone.0068677 |
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