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 -tolerant [NiFe]-hydrogenases from (formerly ) are of particular interest as they maintain catalysis even in the presence of molecular oxygen. H...

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Bibliographic Details
Published in:Frontiers in microbiology 2022-04, Vol.13, p.894375-894375
Main Authors: Fan, Qin, Caserta, Giorgio, Lorent, Christian, Zebger, Ingo, Neubauer, Peter, Lenz, Oliver, Gimpel, Matthias
Format: Article
Language:English
Subjects:
[NiFe]-hydrogenase
Cupriavidus necator
difficult-to-express protein
Escherichia coli
Microbiology
regulatory hydrogenase
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Summary:Hydrogenases are biotechnologically relevant metalloenzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. The O -tolerant [NiFe]-hydrogenases from (formerly ) 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 as a model for the development of such a heterologous hydrogenase production process in . 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 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 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 of catalytically active RH and an up to 160-fold space-time yield in compared to that in the native host [
ISSN:1664-302X
1664-302X
DOI:10.3389/fmicb.2022.894375