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.
Highlights
Hydrogenases are ancient, highly complex metalloenzymes which catalyze the conversion of molecular hydrogen (H2) into protons and electrons
In this study, we addressed the major requirements for a multigene expression system, aiming at the ability to either reliably produce such enzymes at high yields, or implement their production circuit into a primary metabolic route, e. g. in synthetic biology approaches
Design of the Cloning and Expression System The cloning system presented in this paper is based on a methodology previously developed in our lab, which utilizes type IIS restriction-ligation [35]
Summary
Hydrogenases are ancient, highly complex metalloenzymes which catalyze the conversion of molecular hydrogen (H2) into protons and electrons. Most hydrogenases catalyze either the activation or production of H2 in vivo, giving its host the ability to utilize H2 as a source of low potential electrons or alternatively dispense excess reducing equivalents as molecular hydrogen to control the cellular redox balance. The largest and most manifold group is represented by the [NiFe]hydrogenases, found in archaea and diverse groups of bacteria [3]. With regard to their properties and sensitivity towards oxygen, they may be classified in accordance to the environment their hosts were exposed to throughout evolution [4]. A small group of [NiFe]-hydrogenases are termed ‘oxygen tolerant’, since their catalytic cycle proceeds even under air, which makes these catalysts important targets for the biotechnological industry [5,6,7]
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