Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli

dc.contributor.authorFan, Qin
dc.contributor.authorCaserta, Giorgio
dc.contributor.authorLorent, Christian
dc.contributor.authorLenz, Oliver
dc.contributor.authorNeubauer, Peter
dc.contributor.authorGimpel, Matthias
dc.date.accessioned2021-06-30T13:55:28Z
dc.date.available2021-06-30T13:55:28Z
dc.date.issued2021-05-31
dc.date.updated2021-06-11T17:14:42Z
dc.description.abstractHydrogenases are abundant metalloenzymes that catalyze the reversible conversion of molecular H2 into protons and electrons. Important achievements have been made over the past two decades in the understanding of these highly complex enzymes. However, most hydrogenases have low production yields requiring many efforts and high costs for cultivation limiting their investigation. Heterologous production of these hydrogenases in a robust and genetically tractable expression host is an attractive strategy to make these enzymes more accessible. In the present study, we chose the oxygen-tolerant H2-sensing regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha H16 owing to its relatively simple architecture compared to other [NiFe]-hydrogenases as a model to develop a heterologous hydrogenase production system in Escherichia coli. Using screening experiments in 24 deep-well plates with 3 mL working volume, we investigated relevant cultivation parameters, including inducer concentration, expression temperature, and expression time. The RH yield could be increased from 14 mg/L up to >250 mg/L by switching from a batch to an EnPresso B-based fed-batch like cultivation in shake flasks. This yield exceeds the amount of RH purified from the homologous host R. eutropha by several 100-fold. Additionally, we report the successful overproduction of the RH single subunits HoxB and HoxC, suitable for biochemical and spectroscopic investigations. Even though both RH and HoxC proteins were isolated in an inactive, cofactor free apo-form, the proposed strategy may powerfully accelerate bioprocess development and structural studies for both basic research and applied studies. These results are discussed in the context of the regulation mechanisms governing the assembly of large and small hydrogenase subunits.en
dc.description.sponsorshipDFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"en
dc.description.sponsorshipEC/H2020/810856/EU/Twin to Illuminate Metals in Biology and Biocatalysis through Biospectroscopy/TIMB3en
dc.description.sponsorshipDFG, 414044773, Open Access Publizieren 2021 - 2022 / Technische Universität Berlinde
dc.identifier.eissn2076-2607
dc.identifier.urihttps://depositonce.tu-berlin.de/handle/11303/13332
dc.identifier.urihttp://dx.doi.org/10.14279/depositonce-12124
dc.language.isoenen
dc.relation.ispartof10.14279/depositonce-16438
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/en
dc.subject.ddc570 Biowissenschaften; Biologiede
dc.subject.other[NiFe]-hydrogenaseen
dc.subject.otherRalstonia eutrophaen
dc.subject.otherheterologous protein productionen
dc.subject.othercofactor assemblyen
dc.subject.otherdifficult-to-express proteinen
dc.subject.otherEscherichia colien
dc.titleOptimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia colien
dc.typeArticleen
dc.type.versionpublishedVersionen
dcterms.bibliographicCitation.articlenumber1195en
dcterms.bibliographicCitation.doi10.3390/microorganisms9061195en
dcterms.bibliographicCitation.issue6en
dcterms.bibliographicCitation.journaltitleMicroorganismsen
dcterms.bibliographicCitation.originalpublishernameMDPIen
dcterms.bibliographicCitation.originalpublisherplaceBaselen
dcterms.bibliographicCitation.volume9en
tub.accessrights.dnbfreeen
tub.affiliationFak. 3 Prozesswissenschaften>Inst. Biotechnologie>FG Bioverfahrenstechnikde
tub.affiliation.facultyFak. 3 Prozesswissenschaftende
tub.affiliation.groupFG Bioverfahrenstechnikde
tub.affiliation.instituteInst. Biotechnologiede
tub.publisher.universityorinstitutionTechnische Universität Berlinen
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