Fusion hydrogenase and application thereof

Abstract

The invention belongs to the technical field of hydrogenase biology. The invention provides a fusion hydrogenase. The fusion hydrogenase contains subunits HoxF, HoxU-HhyS and HhyL, wherein the subunits are HoxF, HoxU-HhyS and HhyL; the amino acid sequence of the subunit HoxF is as shown in SEQ ID NO. 1 in a sequence table; the amino acid sequence of the subunit HoxU-HhyS is as shown in SEQ ID NO. 3 in the sequence table; the amino acid sequence of the subunit HhyL is as shown in SEQ ID NO.5 in the sequence table. The fusion hydrogenase provided by the invention can utilize low-concentration hydrogen and even hydrogen in air to regenerate NADH (Nicotinamide Adenine Dinucleotide Horse) so as to reduce the risk of operation; the NADH regeneration method has the advantages that the NADH regeneration method is simple and convenient to operate, has tolerance to oxygen and is difficult to inactivate under the aerobic condition (still has the activity of regenerating NADH), so that the application range is wider, and the operation is simpler and more convenient; nAD + can be combined more easily, and the capacity of regenerating reducing power is higher.

Description

Technical Field

[0001] This invention belongs to the field of hydrogenase biotechnology. Background Technology

[0002] The production of many bio-based chemicals requires large amounts of the cofactor NADH (reduced nicotinamide adenine dinucleotide, also known as reduced coenzyme I), such as the microbial fermentation process for producing bulk chemicals like butanol, 1,3-propanediol, and succinic acid. This necessitates an efficient intracellular NADH regeneration system. Soluble [NiFe] hydrogenase can be used to regenerate NADH intracellularly, offering the following advantages: it eliminates the need to construct new carbon metabolic pathways for NADH regeneration, allowing valuable carbon resources to be used primarily for the synthesis of carbon-based products; the substrate hydrogen is abundant, generated through water electrolysis or obtained from industrial waste gases; hydrogen itself does not interfere with substances in existing metabolic pathways; excess hydrogen escapes from cells or culture media without interfering with intracellular reactions; and the hydrogenase can be transferred in parallel to multiple chassis strains, providing reducing power for existing metabolic pathways.

[0003] However, there are several difficulties in the application of hydrogenases: (1) Most hydrogenases require a high concentration of hydrogen to regenerate NADH. For example, hydrogenase from Ralstonia eutropha requires 85% hydrogen to regenerate NADH. However, hydrogen in the air is prone to explosion when exposed to fire when its concentration is between 4.0% and 75.6%, which limits the application of these hydrogenases. (2) Hydrogen has a solubility of only 740 μmol / L in water, making it a poorly soluble substance. Soluble [NiFe] hydrogenases that can regenerate NADH have a low affinity for hydrogen and are reversible, which results in very low activity of these hydrogenases in catalyzing the regeneration of NADH from hydrogen in atmospheric pressure systems. To solve the problem of hydrogen explosion, hydrogen concentrations below 4% should be used as much as possible. However, reducing the concentration of hydrogen will reduce the amount of hydrogen dissolved in water. In addition, the low affinity of soluble [NiFe] hydrogenases for hydrogen and the reversibility of the reaction make it even more difficult for hydrogenases to absorb and utilize hydrogen to regenerate NADH. Summary of the Invention

[0004] To address this problem, the present invention provides a hydrogenase that regenerates NADH using low concentrations of hydrogen or even hydrogen in the air. This hydrogenase is achieved by fusing high-affinity subunits from different hydrogenases with NADH regeneration subunits.

[0005] This invention provides a fusion hydrogenase containing subunits HoxF, HoxU-HhyS, and HhyL; the amino acid sequence of the HoxF subunit is shown in SEQ ID NO.1 of the sequence listing; the amino acid sequence of the HoxU-HhyS subunit is shown in SEQ ID NO.3 of the sequence listing; and the amino acid sequence of the HhyL subunit is shown in SEQ ID NO.5 of the sequence listing.

[0006] The present invention also provides another fusion hydrogenase containing subunits HoxF, HoxU-HhyS-his, and HhyL; the amino acid sequence of the subunit HoxF is shown in SEQ ID NO.1 of the sequence listing; the amino acid sequence of the subunit HoxU-HhyS-his is shown in SEQ ID NO.7 of the sequence listing; and the amino acid sequence of the subunit HhyL is shown in SEQ ID NO.5 of the sequence listing.

[0007] Furthermore, this invention provides a method for constructing the fusion hydrogenase, which is obtained by transforming *E. coli* DH5α with the HhySL fragment, HypABFCDE fragment, and pET-FU fragment ligated using Gibson ligation. The method for constructing the HhySL fragment includes: using the genome of *S. avermitlis* as a template, amplifying the HhyL gene fragment using primers HhyL-F and HhyL-R; amplifying the HhyS gene fragment using primers HhyS-F and HhyS-R; and fusing the two fragments using primers HhyS-F and HhyL-R to obtain the HhySL fragment. The base sequence of primer HhyL-F is shown in SEQ ID NO. 10 of the sequence listing; the base sequence of primer HhyL-R is shown in SEQ ID NO. 11 of the sequence listing; and the base sequence of primer HhyS-F is shown in SEQ ID NO. 11 of the sequence listing. As shown in NO.12; the base sequence of primer HhyS-R is shown in SEQ ID NO.13 of the sequence listing; the construction method of the HypABFCDE fragment includes: using the genome of S. avermitlis as a template, amplifying the HypABF gene fragment using primers HypABF-F and HypABF-R; amplifying the HypCDE gene fragment using primers HypCDE-F and HypCDE-R; ligating the two fragments using HypABF-F and HypCDE-R as primers to obtain the HypABFCDE fragment; the base sequence of primer HypABF-F is shown in SEQ ID NO.14 of the sequence listing; the base sequence of primer HypABF-R is shown in SEQ ID NO.15 of the sequence listing; the base sequence of primer HypCDE-F is shown in SEQ ID NO.16 of the sequence listing; the base sequence of primer HypCDE-R is shown in SEQ ID NO.15 of the sequence listing. As shown in ID NO. 17; the method for constructing the pET-FU fragment includes: using plasmid pET-FF2 as a template, using primers pET-FU-F1 and pET-FU-R to amplify the fragment, and then using this fragment as a template, using primers pET-FU-F2 and pET-FU-R to amplify the pET-FU fragment; the base sequence of primer pET-FU-F1 is shown in SEQ ID NO. 19 of the sequence listing; the base sequence of primer pET-FU-F2 is shown in SEQ ID NO. 20 of the sequence listing; and the base sequence of primer pET-FU-R is shown in SEQ ID NO. 21 of the sequence listing.

[0008] Furthermore, this invention provides another method for constructing the fusion hydrogenase, which is obtained by transforming *E. coli* DH5α with the HhySL-his fragment, HypABFCDE fragment, and pET-FU fragment ligated using Gibson chromatography. The method for constructing the HhySL-his fragment includes: using the genome of *S. avermitlis* (GenBank: BA000030.4) as a template, amplifying the HhyS-his gene fragment using primers HhyS-F and HhyS-his-R; using the HhyS-his and HhyL fragments as templates, fusing the two fragments using primers HhyS-F and HhyL-R to obtain the HhySL-his fragment; the base sequence of primer HhyS-his-R is shown in SEQ ID NO. 18 of the sequence listing; the base sequence of primer HhyS-R is shown in SEQ ID NO. 13 of the sequence listing; the base sequence of primer HhyS-F is shown in SEQ ID NO. 18 of the sequence listing. As shown in NO.12; the base sequence of primer HhyL-R is shown in SEQ ID NO.11 of the sequence listing; the construction method of the HypABFCDE fragment includes: using the genome of S. avermitlis as a template, amplifying the HypABF gene fragment using primers HypABF-F and HypABF-R; amplifying the HypCDE gene fragment using primers HypCDE-F and HypCDE-R; ligating the two fragments using HypABF-F and HypCDE-R as primers to obtain the HypABFCDE fragment; the base sequence of primer HypABF-F is shown in SEQ ID NO.14 of the sequence listing; the base sequence of primer HypABF-R is shown in SEQ ID NO.15 of the sequence listing; the base sequence of primer HypCDE-F is shown in SEQ ID NO.16 of the sequence listing; the base sequence of primer HypCDE-R is shown in SEQ ID NO.14 of the sequence listing. As shown in NO.17; the method for constructing the pET-FU fragment includes: using plasmid pET-FF2 as a template, using primers pET-FU-F1 and pET-FU-R to amplify the fragment, and then using this fragment as a template, using primers pET-FU-F2 and pET-FU-R to amplify the pET-FU fragment; the base sequence of primer pET-FU-F1 is shown in SEQ ID NO.19 of the sequence listing; the base sequence of primer pET-FU-F2 is shown in SEQ ID NO.20 of the sequence listing; and the base sequence of primer pET-FU-R is shown in SEQ ID NO.21 of the sequence listing.

[0009] The fusion hydrogenase provided by this invention can regenerate NADH using low concentrations of hydrogen gas or even hydrogen gas from the air, thus reducing operational risks; it is oxygen-tolerant and not easily inactivated under aerobic conditions (still retaining NADH regeneration activity), making its application range wider and its operation simpler; it also has a greater ability to bind NAD+. + It has a stronger ability to regenerate and restore. Attached Figure Description

[0010] Figure 1 Schematic diagram of soluble [NiFe] hydrogenase in Ralstonia eutropha.

[0011] Figure 2 Comparison of NADH regeneration by soluble [NiFe] hydrogenase from R. eutropha under different dissolved oxygen conditions.

[0012] Figure 3 A schematic diagram of the construction of a fusion hydrogenase with NADH regeneration function under low hydrogen conditions. Detailed Implementation

[0013] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited to the scope of the embodiments described herein.

[0014] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0015] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0016] To achieve NADH regeneration using hydrogenases under low hydrogen conditions, this example selected hydrogenases from different bacterial genera with significant evolutionary differences, namely *Ralstonia eutropha* and *Streptomyces avermitilis*. After expression using appropriate expression vectors, the regeneration capacity of NADH and the application of hydrogenases to drive the synthesis of compounds were tested.

[0017] This invention provides the following technical solutions:

[0018] First, the present invention provides the amino acid sequence of a fusion hydrogenase capable of regenerating NADH in a low-hydrogen environment and its encoding DNA molecule.

[0019] Second, the present invention also provides an expression vector for hydrogenase.

[0020] Third, the present invention further provides an engineered strain containing NADH hydrogenase for regeneration.

[0021] This invention discloses a regenerable NADH hydrogenase, as well as an expression vector, engineered strain, and production method for expressing the hydrogenase.

[0022] The hydrogenase subunits are derived from the eukaryotic alkaloid bacterium *Ralstonia eutropha* and the mycobacterium *Streptomyces avermitilis*, respectively. The hydrogenase synthesized by fusing these subunits can regenerate NADH under low hydrogen conditions to drive compound synthesis. Utilizing this enzyme and its encoding gene offers advantages such as operational safety and ease of fermentation process.

[0023] The primers and sequence information used in this invention are as follows: The underlined and bolded parts in the table are the overlapping parts of gene fragments, and the slanted and enlarged parts are the linker parts.

[0024]

[0025]

[0026] Example 1

[0027] Construction of expression vectors and engineered strains of R. eutropha-derived soluble [NiFe] hydrogenase.

[0028] The soluble [NiFe] hydrogenase R. eutropha, which can regenerate NADH at room temperature, is a hexamer with five subunits, HoxHYFUI2 (e.g. Figure 1 ).

[0029] In the genome of *R. eutropha* (Genbank AY305378.1), the gene cluster of soluble [NiFe] hydrogenase contains nine genes, HoxFUYHWI and HypA2B2F2, whose expression is tightly regulated. It consists of two catalytic units: HoxHY, which functions in the hydrogen cycle, and HoxFU, which contains NAD+. + The HoxI gene provides a binding domain for NADPH, enabling HoxHYFUI2 to catalyze NADPH regeneration. HypA2B2F2 is the mature gene for hydrogenase, conferring hydrogenase function.

[0030] Using primers hoxF-hypF2-F and hoxF-hypF2-R, the genome of R. eutropha (Genbank AY305378.1) was used as a template, and the high-fidelity PCR enzyme PrimeSTAR GXL DNAPolymerase (TaKaRa / Takara Bio, catalog number R050A) was used to amplify the gene fragment of soluble [NiFe] hydrogenase, which is 9579 bp in length.

[0031] Amplify the fragment containing the vector gene backbone. Using pET30a-F and pET30a-R, with pET30a(+) as a template, amplify the fragment containing the vector gene backbone.

[0032] The two fragments obtained above were recovered using a PCR recovery kit. The recovered fragment containing the vector gene backbone was then processed with the restriction endonuclease DpnI (Thermo Scientific). TM The original vector template was completely removed by processing with FD1703, and then recovered to obtain the processed vector gene backbone fragment.

[0033] Take 50 to 300 ng of each of the two processed fragments and add them to Gibson assembly premix (NEB, E2611S) according to the product instructions. Incubate the prepared premix at 50 degrees Celsius for 10 to 30 minutes. Then, take 10 μL of the incubated liquid and transform it into Escherichia coli DH5α (Tiangen, CB101-01) using chemical transformation. After recovery for 30 minutes, take an appropriate amount of bacterial suspension and spread it on LB agar plates containing kanamycin. Incubate overnight at 28 to 38 degrees Celsius. Collect single clones for identification. Extract plasmids from positive single clones and sequence them. The plasmid that is correctly identified is the expression plasmid pED30a-FF2.

[0034] The expression plasmid pED30a-FF2 was chemically transformed into commercially competent cells BL21(DE3) (TRANS / Full Gold, CD601-02) to obtain the engineered strain BL21(DE3)pED30a-FF2.

[0035] The control plasmid pET30a(+) was chemically transformed into commercially competent cells BL21(DE3) to obtain the control (blank) strain BL21(DE3)pED30a.

[0036] Example 2

[0037] Enzyme activity assay of soluble [NiFe] hydrogenase derived from R. eutropha.

[0038] The strain BL21(DE3)pED30a-FF2 containing soluble [NiFe] hydrogenase from R. eutropha and the control strain BL21(DE3)pED30a were inoculated into LB medium and cultured under three dissolved oxygen conditions: aerobic, anaerobic and microaerobic.

[0039] Aerobic culture involves incubating at 30°C and 220 rpm for 4 to 6 hours, followed by induction with 0.05 mM IPTG, and then overnight induction expression at 16°C and 220 rpm. Anaerobic culture involves placing the culture with the inducer into an anaerobic flask, replacing the headspace gas with nitrogen, and then inducing expression at 16°C overnight. Microaerobic culture involves adding the inducer and then allowing the culture to stand at 16°C overnight for induction expression.

[0040] The cultured cells were collected at 5000 rpm and suspended in 50 mM Tris-HCl buffer at pH 8 to adjust the cell concentration to 30 D. The cell suspension was then disrupted using an ultrasonic disruptor with a 6 mm diameter ultrasonic probe, sonication for 5 seconds, pause for 5 seconds, and a total time of 10 minutes. The temperature was maintained at 0 degrees Celsius throughout the ultrasonic disruption process. After ultrasonic disruption, the disrupted solution was centrifuged at 14000 rpm for 5 minutes to obtain the crude enzyme solution, which was used to determine enzyme activity.

[0041] The assay system for hydrogenase activity contained: 20 mM Tris-HCl at pH 8 and 800 μM NAD. + The crude enzyme solution accounted for 10% of the determination system. The reaction was carried out in an anaerobic bottle. During the determination, 80% hydrogen gas was introduced to replace the air in the bottle. Samples were taken at 0 min and 30 min, and the difference in absorbance at 340 nm was measured to calculate the amount of NADH generated.

[0042] The measurement results are shown in Table 1 and Figure 2 The amount of NADH obtained by regeneration of hydrogenase obtained under microaerobic and anaerobic conditions was greater than that obtained by hydrogenase induced under aerobic conditions, indicating that soluble [NiFe] hydrogenase from R. eutropha can tolerate oxygen under specific conditions, and microaerobic conditions can be used to culture and express hydrogenase.

[0043] Table 1. Effects of hydrogenase on NADH regeneration

[0044]

[0045] Example 3

[0046] Construction of plasmids and strains expressing low-hydrogen fusion hydrogenase.

[0047] The soluble [NiFe] hydrogenase from R. eutropha has a low affinity for hydrogen, which is due to the low affinity of the HoxH subunit, which has the function of cycling hydrogen, for hydrogen.

[0048] The hydrogenase from Streptomyces avermitilis (GenBank: BA000030.4) has a high affinity for hydrogen gas but does not have the ability to regenerate NADH.

[0049] This invention replaces the hydrogen cycling subunits HhyS and HhyL from the hydrogenase of *S. avermitilis* with the subunits HoxH and HoxY in the hydrogenase of *R. eutropha* to construct a fusion hydrogenase with enzymatic activity under low hydrogen conditions. A schematic diagram of the plasmid expressing the fusion hydrogenase is shown below. Figure 3 HypABFCDE is a hydrogenase maturation enzyme from S. avermitilis (GenBank: BA000030.4).

[0050] The primers were designed to incorporate overlapping gene fragments so that specific fragments could be joined using Gibson ligation in subsequent experiments to construct plasmids expressing fusion hydrogenase.

[0051] Using the genome of S. avermitlis (GenBank: BA000030.4) as a template, the HhyL gene fragment was amplified using primers HhyL-F and HhyL-R; the HhyS gene fragment was amplified using primers HhyS-F and HhyS-R; primers were designed to use these two fragments as templates and to fuse the two fragments using fusion PCR with primers HhyS-F and HhyL-R to obtain the HhySL fragment.

[0052] The base sequence of the primer HhyL-F is shown in SEQ ID NO.10 of the sequence listing.

[0053] The base sequence of the primer HhyL-R is shown in SEQ ID NO.11 in the sequence listing.

[0054] The base sequence of the primer HhyS-F is shown in SEQ ID NO.12 in the sequence listing.

[0055] The base sequence of the primer HhyS-R is shown in SEQ ID NO.13 in the sequence listing.

[0056] Using the genome of S. avermitlis (GenBank: BA000030.4) as a template, the HypABF gene fragment was amplified using primers HypABF-F and HypABF-R; the HypCDE gene fragment was amplified using primers HypCDE-F and HypCDE-R; using these two fragments as templates, fusion PCR was performed using primers HypABF-F and HypCDE-R to ligate the two fragments, yielding the HypABFCDE fragment.

[0057] The base sequence of the primer HypABF-F is shown in SEQ ID NO.14 of the sequence listing.

[0058] The base sequence of the primer HypABF-R is shown in SEQ ID NO.15 of the sequence listing.

[0059] The base sequence of the primer HypCDE-F is shown in SEQ ID NO.16 of the sequence listing.

[0060] The base sequence of the primer HypCDE-R is shown in SEQ ID NO.17 of the sequence listing.

[0061] Using plasmid pET-FF2 as a template, primers pET-FU-F1 and pET-FU-R were used to amplify the fragment. Then, using this fragment as a template, primers pET-FU-F2 and pET-FU-R were used to amplify the pET-FU fragment.

[0062] The base sequence of the primer pET-FU-F1 is shown in SEQ ID NO.19 of the sequence listing.

[0063] The base sequence of the primer pET-FU-F2 is shown in SEQ ID NO.20 in the sequence listing.

[0064] The base sequence of the primer pET-FU-R is shown in SEQ ID NO.21 in the sequence listing.

[0065] Construct the expression plasmid pET-FUSL-ABFCDE for the fusion hydrogenase.

[0066] Take 50 to 300 ng each of the three fragments obtained above—HhySL, HypABFCDE, and pET-FU—and add them to Gibson assembly premix (NEB, E2611S) according to the product instructions. Incubate the prepared premix at 50°C for 10 to 30 minutes. Then, take 10 μL of the incubated liquid and transform it into *E. coli* DH5α (Tiangen, CB101-01) using chemical transformation. After recovery for 30 minutes, take an appropriate amount of bacterial suspension and spread it on LB agar plates containing kanamycin. Incubate overnight at 28 to 38°C. Collect single clones for identification and sequencing. A schematic diagram of the correctly constructed plasmid pET-FUSL-ABFCDE is shown below. Figure 3 As shown, the plasmid contains gene fragments HoxF, HoxU-HhyS, and HhyL that can express fusion hydrogenase subunits.

[0067] The amino acid sequence of the HoxF subunit is shown in SEQ ID NO.1 of the sequence listing; the nucleic acid sequence of the HoxF subunit is shown in SEQ ID NO.2 of the sequence listing.

[0068] The subunit HoxU-HhyS has a linker between the two subunits HoxU and HhyS. The nucleic acid sequence of the linker is as shown in SEQ ID NO.9 in the sequence list. The amino acid sequence of the subunit HoxU-HhyS is as shown in SEQ ID NO.3 in the sequence list. The nucleic acid sequence of the subunit HoxU-HhyS is as shown in SEQ ID NO.4 in the sequence list.

[0069] The amino acid sequence of the subunit HhyL is as shown in SEQ ID NO.5 in the sequence list. The nucleic acid sequence of the subunit HhyL is as shown in SEQ ID NO.6 in the sequence list.

[0070] Using the genome of S. avermitlis (GenBank: BA000030.4) as a template, and using the primers HhyS-F and HhyS-his-R, the gene fragment of HhyS-his was amplified.

[0071] The base sequence of the primer HhyS-his-R is as shown in SEQ ID NO.18 in the sequence list.

[0072] The base sequence of the primer HhyS-R is as shown in SEQ ID NO.13 in the sequence list.

[0073] Using this HhyS-his and HhyL fragment as templates, and using fusion PCR, with HhyS-F and HhyL-R as primers, the two fragments were fused to obtain the HhySL-his fragment.

[0074] The base sequence of the primer HhyS-F is as shown in SEQ ID NO.12 in the sequence list.

[0075] The base sequence of the primer HhyL-R is as shown in SEQ ID NO.11 in the sequence list.

[0076] Construct the fusion hydrogenase expression plasmid pET-FUSL-ABFCDE(his). Take 50 to 300 ng of each of the HhySL-his fragment, HypABFCDE fragment, and pET-FU fragment, add them to the Gibson assembly premix (NEB, E2611S), prepare according to the configuration table in the product manual, and incubate the prepared premix at 50 °C for 10 to 30 min. Then take 10 μL of the incubated liquid and transform Escherichia coli DH5α (Tiangen / Bioteke, CB101-01) by chemical transformation method, recover for 30 min, take an appropriate amount of the bacterial suspension and spread it on the LB plate containing kanamycin, culture overnight at 28 °C to 38 °C, take monoclonal for identification and sequencing. The schematic diagram of the constructed plasmid pET-FUSL-ABFCDE(his) is as Figure 3As shown, the plasmid contains gene fragments HoxF, HoxU-HhyS-his, and HhyL that can express fusion hydrogenase subunits.

[0077] The amino acid sequence of the HoxF subunit is shown in SEQ ID NO.1 of the sequence listing; the nucleic acid sequence of the HoxF subunit is shown in SEQ ID NO.2 of the sequence listing.

[0078] The subunit HoxU-HhyS-his has a linker protein between the two subunits HoxU and HhyS-his. The nucleic acid sequence of the linker protein is shown in SEQ ID NO.9 of the sequence listing. The amino acid sequence of the subunit HoxU-HhyS-his is shown in SEQ ID NO.7 of the sequence listing. The nucleic acid sequence of the subunit HoxU-HhyS-his is shown in SEQ ID NO.8 of the sequence listing.

[0079] The amino acid sequence of the subunit HhyL is shown in SEQ ID NO.5 of the sequence listing, and the nucleic acid sequence of the subunit HhyL is shown in SEQ ID NO.6 of the sequence listing.

[0080] The expression plasmids pET-FUSL-ABFCDE and pET-FUSL-ABFCDE(his) were chemically transformed into commercially competent cells BL21(DE3) (TRANS / Taiwan Gold, CD601-02) to obtain engineered strains BL21(DE3)pET-FUSL-ABFCDE and BL21(DE3)pET-FUSL-ABFCDE(his).

[0081] Example 4

[0082] Enzyme activity assay of low-hydrogen fusion hydrogenase in 9% hydrogen gas

[0083] Strains BL21(DE3)pET30a, BL21(DE3)pET30a-FF2, BL21(DE3)pET-FUSL-ABFCDE, and BL21(DE3)pET-FUSL-ABFCDE(his) were cultured using the microaerobic culture method described in Example 2. Crude enzyme solutions were prepared, and enzyme activity and NAD+ levels in the solutions were determined according to the method described in Example 2. + The concentration was 25 μM, and the hydrogen concentration was 9%.

[0084] The enzyme activity results of the crude enzyme solution are shown in Table 2. pET30a is the empty plasmid strain, pET-FF2 is the soluble [NiFe] hydrogenase from *R. eutropha*, and pET-FUSL-ABFCDE is the constructed fusion hydrogenase. Under 9% hydrogen gas purging, the crude enzyme solution with fusion hydrogenase showed a greater increase in absorbance at 340 nm after 20 min, indicating that the crude enzyme solution can regenerate NADH under 9% conditions. Preliminary calculations of the enzyme activity of the crude enzyme solution showed that the highest activity of the fusion hydrogenase was 0.11 nmol NADH / min / mg. After adding the his tag, the activity of the fusion hydrogenase reached 0.21 nmol NADH / min / mg. The soluble [NiFe] hydrogenase from *R. eutropha* showed the same value as that of the empty plasmid pET30a, indicating that NADH cannot be regenerated under these conditions.

[0085] Table 2. Results of NADH regeneration using 9% hydrogen gas via hydrogenase.

[0086] Strains used in crude enzyme solution NADH / μmol / L BL21(DE3)pET30a 3.5 BL21(DE3)pET-FF2 3.3 BL21(DE3)pET-FUSL-ABFCDE 5.9 BL21(DE3)pET-FUSL-ABFCDE(his) 8.1

[0087] Example 5

[0088] Assay of the activity of a low-hydrogen fusion hydrogenase that regenerates NADH using hydrogen from the air.

[0089] Strains BL21(DE3)pET30a, BL21(DE3)pET30a-FF2, and BL21(DE3)pET-FUSL-ABFCDE(his) were cultured using the microaerobic culture method described in Example 2, and crude enzyme solutions were prepared. Enzyme activity and NAD+ levels in the solutions were determined according to the method described in Example 2. + The concentration was 25 μM. Air was introduced to test the ability to regenerate NADH, and nitrogen was introduced as a control. The difference between the two conditions was used to calculate the hydrogenase activity.

[0090] The enzyme activity assay results are shown in Table 3. pET30a is an empty plasmid strain, pET-FF2 is a soluble [NiFe] hydrogenase from R. eutropha, and pET-FUSL-ABFCDE(his) is a constructed fusion hydrogenase. Under the condition of air introduction and nitrogen as a control, the empty plasmid strain showed almost no NADH regeneration activity, while the fusion hydrogenase strain could regenerate NADH using air.

[0091] Table 3. Results of NADH regeneration using hydrogenase from air.

[0092] Strains used in crude enzyme solution NADH / μmol / L BL21(DE3)pET30a 0.1 BL21(DE3)pET30a-FF2 0 BL21(DE3)pET-FUSL-ABFCDE(his) 1.4

Claims

1. Fusion hydrogenase, characterized by, Contains subunits HoxF, HoxU-HhyS, and HhyL; The amino acid sequence of the subunit HoxF is shown in SEQ ID NO.1 of the sequence listing; The amino acid sequence of the subunit HoxU-HhyS is shown in SEQ ID NO.3 in the sequence listing; The amino acid sequence of the subunit HhyL is shown in SEQ ID NO.5 of the sequence listing.

2. Fusion hydrogenase, characterized by, Contains subunits HoxF, HoxU-HhyS-his, and HhyL; The amino acid sequence of the subunit HoxF is shown in SEQ ID NO.1 of the sequence listing; The amino acid sequence of the subunit HoxU-HhyS-his is shown in SEQ ID NO.7 of the sequence listing; The amino acid sequence of the subunit HhyL is shown in SEQ ID NO.5 of the sequence listing.

3. The method for constructing the fusion hydrogenase according to claim 1, characterized in that, The HhySL, HypABFCDE, and pET-FU fragments were obtained by ligating them using Gibson and then transforming them into E. coli DH5α. The method for constructing the HhySL fragment includes: a method for constructing the HhySL fragment, HypABFCDE fragment, and pET-FU fragment, comprising: using the genome of Streptomyces avermitilis (GenBank: BA000030.4) as a template, amplifying the HhyL gene fragment using primers HhyL-F and HhyL-R; amplifying the HhyS gene fragment using primers HhyS-F and HhyS-R; fusing the two fragments using primers HhyS-F and HhyL-R to obtain the HhySL fragment; the base sequence of primer HhyL-F is shown in SEQ ID NO.10 of the sequence listing; the base sequence of primer HhyL-R is shown in SEQ ID NO.11 of the sequence listing; the base sequence of primer HhyS-F is shown in SEQ ID NO.12 of the sequence listing; and the base sequence of primer HhyS-R is shown in SEQ ID NO.13 of the sequence listing; The method for constructing the HypABFCDE fragment includes: using the genome of *S. avermitlis* as a template, amplifying the HypABF gene fragment using primers HypABF-F and HypABF-R; amplifying the HypCDE gene fragment using primers HypCDE-F and HypCDE-R; and ligating the two fragments using primers HypABF-F and HypCDE-R to obtain the HypABFCDE fragment; the base sequence of primer HypABF-F is shown in SEQ ID NO. 14 of the sequence listing; the base sequence of primer HypABF-R is shown in SEQ ID NO. 15 of the sequence listing; the base sequence of primer HypCDE-F is shown in SEQ ID NO. 16 of the sequence listing; and the base sequence of primer HypCDE-R is shown in SEQ ID NO. 17 of the sequence listing. The method for constructing the pET-FU fragment includes: using plasmid pET-FF2 as a template, amplifying the fragment using primers pET-FU-F1 and pET-FU-R, and then using this fragment as a template, amplifying the pET-FU fragment using primers pET-FU-F2 and pET-FU-R; the base sequence of primer pET-FU-F1 is shown in SEQ ID NO.19 of the sequence listing; the base sequence of primer pET-FU-F2 is shown in SEQ ID NO.20 of the sequence listing; and the base sequence of primer pET-FU-R is shown in SEQ ID NO.21 of the sequence listing.

4. The method for constructing the fusion hydrogenase according to claim 3, characterized in that, The HhySL-his fragment, HypABFCDE fragment, and pET-FU fragment were obtained by transforming E. coli DH5α after Gibson ligation. The method for constructing the HhySL-his fragment includes: using the genome of S. avermitlis (GenBank: BA000030.4) as a template, amplifying the HhyS-his gene fragment using primers HhyS-F and HhyS-his-R; using the HhyS-his and HhyL fragments as templates, fusing the two fragments using primers HhyS-F and HhyL-R to obtain the HhySL-his fragment; the base sequence of primer HhyS-his-R is shown in SEQ ID NO.18 of the sequence listing; the base sequence of primer HhyS-R is shown in SEQ ID NO.13 of the sequence listing; the base sequence of primer HhyS-F is shown in SEQ ID NO.12 of the sequence listing; and the base sequence of primer HhyL-R is shown in SEQ ID NO.11 of the sequence listing. The method for constructing the HypABFCDE fragment includes: using the genome of *S. avermitlis* as a template, amplifying the HypABF gene fragment using primers HypABF-F and HypABF-R; amplifying the HypCDE gene fragment using primers HypCDE-F and HypCDE-R; and ligating the two fragments using primers HypABF-F and HypCDE-R to obtain the HypABFCDE fragment; the base sequence of primer HypABF-F is shown in SEQ ID NO. 14 of the sequence listing; the base sequence of primer HypABF-R is shown in SEQ ID NO. 15 of the sequence listing; the base sequence of primer HypCDE-F is shown in SEQ ID NO. 16 of the sequence listing; and the base sequence of primer HypCDE-R is shown in SEQ ID NO. 17 of the sequence listing. The method for constructing the pET-FU fragment includes: using plasmid pET-FF2 as a template, amplifying the fragment using primers pET-FU-F1 and pET-FU-R, and then using this fragment as a template, amplifying the pET-FU fragment using primers pET-FU-F2 and pET-FU-R; the base sequence of primer pET-FU-F1 is shown in SEQ ID NO.19 of the sequence listing; the base sequence of primer pET-FU-F2 is shown in SEQ ID NO.20 of the sequence listing; and the base sequence of primer pET-FU-R is shown in SEQ ID NO.21 of the sequence listing.

5. Strain BL21(DE3)pET-FUSL-ABFCDE, characterized by: The fusion hydrogenase described in claim 1 was obtained by transferring it into competent cells BL21(DE3).

6. Strain BL21(DE3)pET-FUSL-ABFCDE(his), characterized by: The fusion hydrogenase described in claim 2 was obtained by transferring it into competent cells BL21(DE3).

7. The application of the strain BL21(DE3)pET-FUSL-ABFCDE according to claim 5 in the preparation of NADH regeneration enzyme under low hydrogen conditions.

8. The application of the strain BL21(DE3)pET-FUSL-ABFCDE(his) according to claim 6 in the preparation of NADH regeneration enzyme under low hydrogen conditions.

9. The use of the strain BL21(DE3)pET-FUSL-ABFCDE(his) according to claim 6 in the preparation of air-regenerated NADH enzyme.

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