[NiFe] hydrogenase microbial expression technology based on cosolvent peptide fusion expression

Abstract

This invention discloses a method for improving the soluble expression of the large subunit of [NiFe] hydrogenase in *Escherichia coli*, belonging to the fields of protein engineering and industrial microbiology. This method involves fusing a short-chain solubilizing peptide XXA to the C-terminus of the target [NiFe] hydrogenase large subunit to form a fusion protein. This design significantly promotes the soluble expression of the target protein while preserving the accessibility of the C-terminal hydrolysis site, enabling it to transform from an inclusion body form to a soluble form under anaerobic high-density fermentation conditions. This invention overcomes the long-standing technical bias in the field that C-terminal fusion hinders the maturation and processing of [NiFe] hydrogenase, achieving a balance between solubilizing effect and processing compatibility. It provides a new technical pathway for the recombinant production of complex metalloenzymes and has significant application value in the industrial preparation of hydrogenase, basic enzymology research, and bioenergy.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering and industrial microbiology, specifically relating to a method for achieving efficient and soluble expression of the large subunit of [NiFe] hydrogenase through a C-terminal short peptide fusion strategy. Background Technology

[0002] Hydrogenase is a core metalloenzyme in nature that catalyzes the proton reduction and hydrogen oxidation reactions. Its catalytic reaction formula is 2H₂O₂. + + 2e - H2. Based on the metal composition of the active center, hydrogenases are mainly divided into three categories: [FeFe] hydrogenase, [NiFe] hydrogenase, and Fe hydrogenase. Among them, [NiFe] hydrogenase is the most widely distributed in microorganisms due to its high catalytic stability and relatively strong oxygen tolerance, and has important application value in fields such as biohydrogen production, construction of microbial electrochemical systems, and reconstruction of energy metabolism in synthetic biology.

[0003] The catalytic large subunit of [NiFe] hydrogenase contains a nickel-iron heterobimetallic active center. The iron ion in this center is coordinated with carbonyl and cyano ligands, and its assembly process is mediated by the Hyp series of mature enzyme systems. More importantly, the vast majority of [NiFe] hydrogenase large subunits require precise cleavage of the C-terminal elongation peptide by a specific endopeptidase after translation to be finally activated. This hydrolytic event is a necessary and final step in acquiring enzyme activity; precursor proteins without C-terminal cleavage are completely inactive.

[0004] Due to the complex structure of the active site and the sophisticated maturation mechanism, the recombinant expression of [NiFe] hydrogenase in heterologous hosts has long faced technical bottlenecks. While commonly used expression hosts, such as *E. coli*, offer advantages like clear genetic background, rapid growth, and ease of operation, the large subunit of exogenous [NiFe] hydrogenase readily forms inactive inclusion body aggregates within them. Multiple factors contributing to this predicament include: incomplete or missing host maturation enzyme systems, the high sensitivity of complex folding pathways to the intracellular environment, and the aggregation of C-terminal extension peptides as folding regulatory elements under erroneous conditions.

[0005] To overcome these obstacles, researchers have tried various strategies. Co-expression of the complete or partial mature enzymes from the source host can improve cofactor assembly to some extent, but genetic manipulation is complex and has limited effect on improving solubility. Optimizing expression conditions, such as lowering the induction temperature and adjusting the concentration of metal ions, can only alleviate symptoms and cannot fundamentally solve the folding efficiency problem. Regarding fusion expression strategies, attaching macromolecular solubilizing tags such as maltose-binding protein, thioredoxin, glutathione transferase, or SUMO to the N- or C-terminus of the target protein is a common approach. However, for the large subunit of [NiFe] hydrogenase, N-terminal fusion may interfere with signal peptide recognition or initial folding, while C-terminal fusion directly conflicts with the key maturation step, C-terminal hydrolysis.

[0006] For a long time, those skilled in the art have held a clear technical bias based on their understanding of the sensitivity of C-terminal processing: any sequence fusion at the C-terminus of the large subunit of [NiFe] hydrogenase is considered a high-risk design, which is highly likely to mask or alter the protease recognition site, thereby completely blocking the hydrolysis of the C-terminal peptide and resulting in a permanently inactivated precursor protein. Therefore, C-terminal fusion strategies have been systematically avoided in the field of [NiFe] hydrogenase expression, and related research has focused on alternative solutions such as N-terminal fusion or molecular chaperone co-expression, but the results have been unsatisfactory.

[0007] The soluble [NiFe] hydrogenase PfSHI from the hyperthermophilic archaea Pyrococcus furiosus possesses immense value for both basic research and industrial applications. Its optimal reaction temperature is close to 100°C, and its thermal stability far exceeds that of mesophilic enzymes, giving it unique advantages in industrial biocatalysis. However, exogenous expression of this enzyme in Escherichia coli is particularly difficult. Even with co-expression of a mature enzyme system, the soluble yield remains extremely low, and the target protein primarily exists as inactive inclusion bodies, severely hindering related research and industrialization progress.

[0008] Therefore, developing a novel strategy that can effectively break through the aforementioned expression barriers without interfering with the key maturation process is of urgent practical need and important scientific significance for achieving efficient recombinant production of [NiFe] hydrogenase. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fusion expression strategy that can significantly improve the soluble expression level of the [NiFe] hydrogenase large subunit in Escherichia coli while maintaining its C-terminal processability.

[0010] The technical solution of the present invention to achieve the above objectives is as follows.

[0011] First, *Escherichia coli* S17-3-T7P was identified as the recombinant expression host. The originating strain, S17-3, is a patent-preserved strain (accession number CCTCC 2018200), exhibiting acid resistance and high-density growth characteristics. By integrating the T7 RNA polymerase gene into its genome, the modified strain S17-3-T7P was obtained. This strain can utilize the T7 promoter to drive efficient transcription of exogenous genes and possesses crucial anaerobic induction expression capability, meaning it can maintain active transcription of exogenous genes even under anaerobic or microaerobic conditions. This characteristic is particularly important for the expression of anaerobic enzymes such as [NiFe] hydrogenase.

[0012] Second, codon optimization was performed on the PfSHI gene cluster to construct a recombinant plasmid suitable for expression in *E. coli*. Using pETDuet as the backbone vector, seamless cloning technology was employed to insert the codon-optimized PfSHI structural gene and the mature enzyme genome into the same plasmid.

[0013] Third, a short C-terminal peptide fusion strategy was designed. The reverse sequence XXA of the antifreeze protein was fused to the C-terminus of the large subunit 0894 of PfSHI, and linked to the target protein via a flexible linker to construct the fusion expression plasmid pETDuet-PfSHI-xxa. The core innovation of this design lies in the fact that XXA, as a short-chain solubilizing peptide, interacts with the early folding intermediates of the target protein during translation through its unique amino acid composition and physicochemical properties, effectively inhibiting mis-aggregation pathways and promoting soluble expression. At the same time, the short chain characteristic ensures the spatial accessibility of the C-terminal cleavage site, allowing mature endopeptidases to normally recognize and cleave the extended peptide, thus balancing solubilizing effect and processing compatibility.

[0014] Compared with existing technologies, it has the following significant technical advantages and novelty.

[0015] Regarding the breakthrough of technical bias, this invention is the first to demonstrate that short peptide fusion at the C-terminus of the large subunit of [NiFe] hydrogenase not only does not hinder maturation processing, but can also significantly promote soluble expression, breaking the long-standing taboo of C-terminal fusion in the field and providing a brand-new design idea for the recombinant production of complex metalloenzymes.

[0016] In terms of host system adaptation, the anaerobic inducible expression ability of the S17-3-T7P strain is highly matched with the anaerobic activity characteristics of [NiFe] hydrogenase. Combined with the efficient transcription of the T7 system, high-level expression of the target protein under anaerobic conditions was achieved.

[0017] In terms of the sophistication of the fusion design, the XXA short peptide achieves the maximum solubilization effect with the smallest sequence length, avoiding the steric hindrance problem of traditional macromolecular tags. Its sequence-derived antifreeze protein properties may give the fusion protein additional stability advantages.

[0018] Regarding process scalability, the high-density fermentation process established in this invention can achieve OD (Oxygen Demand). 600 A cell density greater than 50, combined with a soluble expression strategy, significantly increased the enzyme and protein yield per unit volume of fermentation broth, laying the foundation for industrial-scale production.

[0019] In summary, this invention achieves efficient and soluble recombinant production of high-temperature [NiFe] hydrogenase PfSHI through systematic innovation in host selection, gene optimization, fusion design, and process integration. This solves a long-standing technical bottleneck in the field and has significant scientific value and industrialization prospects for promoting basic research, enzymatic characterization, and industrial catalytic applications of [NiFe] hydrogenase. Attached Figure Description

[0020] Figure 1 This is a plasmid map of the [NiFe] hydrogenase expressed in this invention;

[0021] Figure 2 Electrophoretic comparison of recombinant hydrogenase protein without and with XXA fusion peptide. Detailed Implementation

[0022] The technical solution of the present invention will be described in detail below with reference to specific embodiments. Operations not specifically described in the embodiments shall be performed according to conventional molecular biology experimental conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents and biological materials used are commercial products.

[0023] The biological materials used in the examples were sourced as follows: Escherichia coli S17-3-T7P was the modified microbial strain of the inventor, and its original strain S17-3 was a patent-deposited strain with accession number CCTCC 2018200. The relevant modification methods and strain characteristics can be found in patent CN114806987B. DNA synthesis and sequencing were commissioned to Sangon Biotech (Shanghai) Co., Ltd. All chemical reagents and biomass raw materials used in the fermentation were purchased from conventional commercial suppliers.

[0024] Example 1: Construction of a plasmid for expressing hydrogenase

[0025] Using pETDuet-1 as the backbone vector, two parallel plasmids were constructed using a modular seamless cloning strategy.

[0026] For the control plasmid pETDuet-PfSHI, the codon-optimized Pyrococcus furiosus [NiFe] hydrogenase gene cluster was assembled. Specifically, it included the coding gene for the large subunit 0894 and the coding genes for the other three subunits 0891, 0892, and 0893, along with the electron transport-related gene frxA. Appropriate ribosome binding sites were used to separate the genes to ensure co-expression. The coding genes for 0891 and 0892 were placed under the control of the first T7 promoter, and the remaining genes were placed under the control of the second T7 promoter, forming the plasmid pETduet-PfSHI, the sequence of which is shown in SEQ ID No. 1.

[0027] For the fusion expression plasmid pETDuet-PfSHI-xxa, nucleotide sequences encoding a flexible linker peptide and a short peptide XXA were introduced at the 3' end of the open reading frame of the 0894 gene. The XXA sequence is the reverse sequence of the antifreeze protein, and its coding sequence is shown in SEQ ID No. 2. The sequence of plasmid pETDuet-PfSHI-xxa is shown in SEQ ID No. 3. This design ensures that XXA is located at the C-terminus of the fusion protein, while preserving the original C-terminal cleavage site sequence of the 0894 gene, allowing it to still be recognized by mature endopeptidases after translation.

[0028] During plasmid construction, each gene module is amplified by PCR, assembled into the pETDuet backbone using a seamless cloning method, transformed into a cloning host for screening, and after verification by colony PCR and sequencing, the plasmid is extracted for subsequent transformation.

[0029] Example 2: Fermentation of high-temperature hydrogenase expression strain and SDS-PAGE detection

[0030] The correctly constructed recombinant plasmids pETDuet-PfSHI and pETDuet-PfSHI-XXA were transformed into Escherichia coli S17-3-T7P competent cells to obtain engineered strains S173-rSHI and S173-rSHI-XXA.

[0031] During the seed culture stage, the engineered bacteria preserved in glycerol were inoculated into LB liquid medium containing appropriate antibiotics and cultured at 37°C and 200 r / min for 12 to 16 h with shaking until the middle of the logarithmic growth phase.

[0032] During the fermentation stage, the seed culture was transferred to fermentation flasks at an inoculation rate of 1% to 5%. The culture medium consisted of 10 g / L glucose, 20 g / L yeast extract, 10 g / L peptone, 5 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and trace element solution per liter, with a pH of 6.8 to 7.2. The culture was carried out at 220 rpm at a controlled temperature of 37°C.

[0033] When the bacterial cell density reaches OD 600 When the concentration is ≥1.0, transfer the sample to an anaerobic bottle for anaerobic induction culture. Add isopropyl-β-D-thiogalactoside to a final concentration of 0.4 mmol / L, and simultaneously add ferrous ammonium sulfate to a final concentration of 0.1 mM, nickel sulfate to a final concentration of 20 μM, and cysteine ​​hydrochloride to a final concentration of 1 mM. Reduce the induction temperature to 25°C and continue culturing for 20 to 24 h.

[0034] After harvesting the bacterial cells, cell lysis was performed by ultrasonic disruption or high-pressure homogenization. The lysis buffer contained 50 mmol / L sodium phosphate, pH 8.0, 300 mmol / L sodium chloride, 10 mmol / L imidazole, and a protease inhibitor. The lysis buffer was centrifuged at 15,000 r / min for 30 min to separate the soluble components and inclusion body precipitate.

[0035] SDS-PAGE electrophoresis was performed on both the supernatant and precipitate samples. The expression level and soluble proportion of the target protein were quantified by grayscale scanning or Western blotting. Expected results showed that the target protein of strain S173-rSHI was mainly present in the precipitate, with a soluble proportion of less than 15%, while the target protein of strain S173-rSHI-XXA was dominant in the supernatant, with a soluble proportion of over 60%.

[0036] For C-terminal processing validation, the soluble supernatant can be purified by nickel affinity chromatography to obtain the fusion protein. The N-terminal sequence of the mature large subunit can then be detected by N-terminal sequencing or mass spectrometry to confirm that the C-terminal extended peptide has been effectively cleaved. Simultaneously, under anaerobic conditions, using methyl viologen as an electron donor and trisulfonic acid as an electron acceptor, the oxidative activity of the hydrogenase can be measured, or the hydrogen production capacity can be detected by gas chromatography to verify the catalytic function of the mature enzyme.

[0037] Through the above embodiments, the present invention successfully achieved efficient soluble expression of the large subunit of [NiFe] hydrogenase in Escherichia coli while maintaining the normal processing capability of the C-terminus, providing a feasible technical solution for the large-scale production of this type of complex metalloenzyme.

[0038] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A fusion protein for enhancing the soluble expression of the large subunit of [NiFe] hydrogenase in Escherichia coli, characterized in that... It contains the amino acid sequence of the large subunit of [NiFe] hydrogenase and the short chain solubilizing peptide XXA fused to its C-terminus.

2. An isolated nucleic acid molecule, characterized in that... Encodes the fusion protein as described in claim 1.

3. A recombinant expression vector, characterized in that... It includes the nucleic acid molecule as described in claim 2 and the expression regulatory element operatively connected thereto.

4. A host cell, characterized in that... It includes the recombinant expression vector as described in claim 3, wherein the host cell is Escherichia coli.

5. A fermentation method for producing the large subunit of [NiFe] hydrogenase, characterized in that... The method includes the following steps: culturing the host cell as described in claim 4 under suitable expression conditions; and recovering the soluble fusion protein or the large subunit of [NiFe] hydrogenase obtained by hydrolysis and maturation of the host cell or culture.

6. The application of short-chain solubilizing peptide XXA in improving the soluble expression of the large subunit of [NiFe] hydrogenase in Escherichia coli, characterized in that... The coding sequence of the solubilizing peptide is fused to the C-terminus of the coding sequence of the target [NiFe] hydrogenase large subunit.

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