An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli

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.

Figure 1. Current model of SH maturation in Cupriavidus necator.

Steps: 1. HypC binds to HoxH, preventing improper folding prior metal-center assembly , ; 2. Delivery of the iron center comprising the diatomic ligands: The cyanide ligands are derived from carbamoyl phosphate (CMP) in a transcarbamoylation/dehydration reaction catalyzed by HypF/HypE, thereby transferring the carbamoyl moiety to a C-terminal cysteine of HypE –. Modified HypE forms a complex with the preassembled HypCD complex, which is likely to “store” the iron complex until the ligand coordination is completed and subsequently delivers it to apo-HoxH. This step probably involves a redox reaction , ; 3. Nickel insertion is mediated by the concerted action of HypA and HypB in a GTP dependent reaction –; 4. HypC detaches from HoxH; 5. HoxW cleaves a 24 amino acid peptide off the C-terminus of the HoxH apoprotein. HoxW activity requires pre-incorporated nickel , ; 6. HoxH folds and thereby buries the bimetallic core inside the protein (at the hydrophobic contact surface to HoxY). The SH subunits assemble; Prior to this step, FMN cofactors and the iron-sulfur clusters are delivered by the cellular machineries. Unresolved reactions, which include the action of HypX and the origin of the carbonyl ligand are indicated (*).

Figure 2. Gene selection for heterologous SH expression in Escherichia coli.

a) Distribution of hydrogenase related genes and putative SH related genes on the pHG1 megaplasmid of Cupriavidus necator (Cn). pHG1 comprises three distinct hydrogenase clusters (locus MBH cluster: 100–22390; locus hyd4 cluster: 59962–74032; locus SH cluster: 79712–89227) . The MBH cluster contains the MBH and RH structural genes along with numerous accessory genes for MBH, RH and SH maturation. A partially duplicated set of maturation related genes is present in vicinity to the SH structural genes. The putative structural and auxiliary genes localized in the hyd4 cluster have not been characterized to date. The hyp genes present in this cluster have been tested as an alternative set for SH maturation in this study. Thicker arrows indicate structural genes. b) Main expression constructs designed in this study for recombinant SH production in E. coli. Plasmids used for maturation and SH production trials were derivatives of the three depicted constructs. The pM1 and pM2 derivatives differ in the origin of the hypABCDEF genes (see Table 1). Two independent genes hypX and hoxN1, as well as the HoxH specific endopeptidase gene hoxW, were included in both pM1 and pM2 derivatives. All genes were placed under control of individual T7-promoters and -terminators.

Figure 3. Selection of Optimization Trials for Maximized SH Production in Recombinant Strains.

Specific activities were determined in extracts of cells from three independent cultivation trials. Error bars indicated represent standard deviations. Parameters were modified as follows: Temperature (1∶18°C; 2∶25°C; 3∶30°C; 4∶37°C), Time (1∶24 h; 2∶30 h; 3∶36 h; 4∶42 h), Medium (1: LB; 2: TB; 3: HEM; 4: M9), Ferric ammonium citrate (1∶0 µM; 2∶50 µM; 3∶100 µM; 4∶500 µM), C-sources (given as % (wt/vol) glucose/% (vol/vol) glycerol/% (wt/vol) lactose; 1∶0.05/1/0.2; 2∶0.1/2/0.4; 3∶0.1/2/0.8; 4∶0.2/2/0.8), NiCl₂ (1∶0 µM; 2∶1 µM; 3∶25 µM; 4∶100 µM).

Figure 4. SDS-PAGE Analysis of Enzyme Preparations.

5 µg of protein were applied to each lane and separated on a 12% SDS-gel; Legend: SH_hex,wt = wildtype six-subunit SH purified from Cn; SH_tet,var1 = four-subunit SH purified from recombinant strain SH1F (N-terminally-StrepII tagged HoxF; HoxF*); SH_hex,var2 = six-subunit SH purified from recombinant strain SH2F (N-terminally-StrepII tagged HoxI; HoxI*).

Figure 5. UV/Vis Spectroscopy of Recombinant SH.

Main: Spectrum of purified, oxidized SH_var2 (1 mg·mL⁻¹); Inset: Difference spectrum between oxidized and dithionite (500 µM) reduced enzyme.

Figure 6. SDS-PAGE analyses of SH preparations from cells, which were harvested after different cultivation times.

Cells from strain SH2F (six-subunit SHvar2; 5′-StrepII tagged HoxI; HoxI*), cultivated under ‘optimized autoinduction conditions’, were harvested after 38 hours (lanes 1–3) and 44 hours (lanes 4–6) from the same culture. 1 gram of cells thus obtained was used for preparation of the cell-free extracts (lanes 1 & 4; 25 µg applied). Purification was carried out using a 1 mL StrepTactin Superflow® gravity flow column (lanes 2 & 5; 15 µg and 5 µg applied, respectively) and subsequently a Superdex 200 HR 10/300 gel filtration column for polishing (lanes 3 & 6; 4 µg and 5 µg applied, respectively). Notably, the HoxFUI₂ diaphorase module appears to be stable over time, while the HoxYH hydrogenase moiety dissociates from the complex after 38–44 hours of cultivation. Activities of the preparations after gel filtration: SH_{38 h}: 103 U·mg⁻¹; SH_{44 h}: 21 U·mg⁻¹. M = Protein marker.

Figure 7. Deletion experiments for in vivo maturation of the SH in Escherichia coli.

Strains and values are listed in Table S3; Control strains: K0: pSH4.wt (structural genes without maturation related genes); K1A: pSH4.wt and pM1 (structural genes and complete M1 set; 100%; 1.95±0.24 U·mg⁻¹). Deleted complexes or proteins omitted are indicated for the K1A deletion strains. K1B represents the substitution strain, in which the M1 Hyp proteins are replaced by the M2 analogs (pSH4.wt and pM2). Results are given as specific activities exhibited by extracts obtained from three independent experiments, which were normalized for control strain K1A. Error bars indicated represent standard deviations.