NDLI: An Innovative Cloning Platform Enables Large-Scale Production and Maturation of an Oxygen-Tolerant [NiFe]-Hydrogenase from Cupriavidus necator in Escherichia coli

Content Provider	PubMed Central
Author	Johannes, Schiffels Pinkenburg, Olaf Maximilian, Schelden Aboulnaga, El-hussiny A. A. Baumann, Marcus E. M. Selmer, Thorsten
Editor	Battista, John R.
Copyright Year	2013
Abstract	Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD+reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)−1 yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg−1 were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg−1. Owing to the combinatorial power exhibited by the presented cloning platform, the system might represent an important step towards new routes in synthetic biology.
Related Links	http://dx.doi.org/10.1371/journal.pone.0068812
Starting Page	68812
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	7
Volume Number	8
Language	English
Publisher	Public Library of Science
Publisher Date	2013-07-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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High-Yield Production of Catalytically Active Regulatory [NiFe]-Hydrogenase From Cupriavidus necator in Escherichia coli

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

Interaction between Hydrogenase Maturation Factors HypA and HypB Is Required for [NiFe]-Hydrogenase Maturation

Heterologous Expression and Maturation of an NADP-Dependent [NiFe]-Hydrogenase: A Key Enzyme in Biofuel Production

Minimal Influence of [NiFe] Hydrogenase on Hydrogen Isotope Fractionation in H2-Oxidizing Cupriavidus necator

X-ray crystallographic and computational studies of the $O_{2}-tolerant$ [NiFe]-hydrogenase 1 from Escherichia coli

Spectroscopic insights into the oxygen-tolerant membrane-associated [NiFe] hydrogenase of Ralstonia eutropha H16.

A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases.

How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases

An Innovative Cloning Platform Enables Large-Scale Production and Maturation of an Oxygen-Tolerant [NiFe]-Hydrogenase from Cupriavidus necator in Escherichia coli

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