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High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli
Hydrogenases are biotechnologically relevant metalloenzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. The O(2)-tolerant [NiFe]-hydrogenases from Cupriavidus necator (formerly Ralstonia eutropha) are of particular interest as they maintain catalysis eve...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9100943/ https://www.ncbi.nlm.nih.gov/pubmed/35572669 http://dx.doi.org/10.3389/fmicb.2022.894375 |
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author | Fan, Qin Caserta, Giorgio Lorent, Christian Zebger, Ingo Neubauer, Peter Lenz, Oliver Gimpel, Matthias |
author_facet | Fan, Qin Caserta, Giorgio Lorent, Christian Zebger, Ingo Neubauer, Peter Lenz, Oliver Gimpel, Matthias |
author_sort | Fan, Qin |
collection | PubMed |
description | Hydrogenases are biotechnologically relevant metalloenzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. The O(2)-tolerant [NiFe]-hydrogenases from Cupriavidus necator (formerly Ralstonia eutropha) are of particular interest as they maintain catalysis even in the presence of molecular oxygen. However, to meet the demands of biotechnological applications and scientific research, a heterologous production strategy is required to overcome the low production yields in their native host. We have previously used the regulatory hydrogenase (RH) from C. necator as a model for the development of such a heterologous hydrogenase production process in E. coli. Although high protein yields were obtained, the purified enzyme was inactive due to the lack of the catalytic center, which contains an inorganic nickel-iron cofactor. In the present study, we significantly improved the production process to obtain catalytically active RH. We optimized important factors such as O(2) content, metal availability, production temperature and time as well as the co-expression of RH-specific maturase genes. The RH was successfully matured during aerobic cultivation of E. coli by co-production of seven hydrogenase-specific maturases and a nickel permease, which was confirmed by activity measurements and spectroscopic investigations of the purified enzyme. The improved production conditions resulted in a high yield of about 80 mg L(–1) of catalytically active RH and an up to 160-fold space-time yield in E. coli compared to that in the native host C. necator [<0.1 U (L d) (–1)]. Our strategy has important implications for the use of E. coli K-12 and B strains in the recombinant production of complex metalloenzymes, and provides a blueprint for the production of catalytically active [NiFe]-hydrogenases in biotechnologically relevant quantities. |
format | Online Article Text |
id | pubmed-9100943 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-91009432022-05-14 High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli Fan, Qin Caserta, Giorgio Lorent, Christian Zebger, Ingo Neubauer, Peter Lenz, Oliver Gimpel, Matthias Front Microbiol Microbiology Hydrogenases are biotechnologically relevant metalloenzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. The O(2)-tolerant [NiFe]-hydrogenases from Cupriavidus necator (formerly Ralstonia eutropha) are of particular interest as they maintain catalysis even in the presence of molecular oxygen. However, to meet the demands of biotechnological applications and scientific research, a heterologous production strategy is required to overcome the low production yields in their native host. We have previously used the regulatory hydrogenase (RH) from C. necator as a model for the development of such a heterologous hydrogenase production process in E. coli. Although high protein yields were obtained, the purified enzyme was inactive due to the lack of the catalytic center, which contains an inorganic nickel-iron cofactor. In the present study, we significantly improved the production process to obtain catalytically active RH. We optimized important factors such as O(2) content, metal availability, production temperature and time as well as the co-expression of RH-specific maturase genes. The RH was successfully matured during aerobic cultivation of E. coli by co-production of seven hydrogenase-specific maturases and a nickel permease, which was confirmed by activity measurements and spectroscopic investigations of the purified enzyme. The improved production conditions resulted in a high yield of about 80 mg L(–1) of catalytically active RH and an up to 160-fold space-time yield in E. coli compared to that in the native host C. necator [<0.1 U (L d) (–1)]. Our strategy has important implications for the use of E. coli K-12 and B strains in the recombinant production of complex metalloenzymes, and provides a blueprint for the production of catalytically active [NiFe]-hydrogenases in biotechnologically relevant quantities. Frontiers Media S.A. 2022-04-29 /pmc/articles/PMC9100943/ /pubmed/35572669 http://dx.doi.org/10.3389/fmicb.2022.894375 Text en Copyright © 2022 Fan, Caserta, Lorent, Zebger, Neubauer, Lenz and Gimpel. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Fan, Qin Caserta, Giorgio Lorent, Christian Zebger, Ingo Neubauer, Peter Lenz, Oliver Gimpel, Matthias High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli |
title | High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli |
title_full | High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli |
title_fullStr | High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli |
title_full_unstemmed | High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli |
title_short | High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli |
title_sort | high-yield production of catalytically active regulatory [nife]-hydrogenase from cupriavidus necator in escherichia coli |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9100943/ https://www.ncbi.nlm.nih.gov/pubmed/35572669 http://dx.doi.org/10.3389/fmicb.2022.894375 |
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