Method for enhancing oxygen resistance of [Fe-Fe] hydrogenase by constructing giant organelle in escherichia coli

Abstract

The invention discloses a method for enhancing oxygen resistance of [Fe-Fe] hydrogenase by constructing giant organelles in escherichia coli, and belongs to the technical field of enzyme catalysis. The method comprises the following steps: constructing two plasmids capable of expressing [Fe-Fe] hydrogenase and guide protein as well as maturation promoting protein of the [Fe-Fe] hydrogenase in escherichia coli; transforming the two plasmids into an escherichia coli BL21 (DE3) competent cell, and carrying out pre-culture; and transferring the pre-culture into a new culture medium, carrying out aerobic culture, measuring the OD600 value after culturing for a period of time, transferring the culture into an anaerobic glove box, and carrying out anaerobic culture and centrifugation to obtain the escherichia coli containing the giant organelle. According to the method for enhancing the oxygen resistance of the [Fe-Fe] hydrogenase by constructing the giant organelle in the escherichia coli, the oxygen resistance and the stability of the [Fe-Fe] hydrogenase are effectively enhanced by constructing the giant organelle in the escherichia coli, and a novel sustainable method is provided for hydrogen production.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme catalysis, and particularly relates to a method for constructing a giant organelle in Escherichia coli to enhance the oxygen resistance of [Fe-Fe] hydrogenase. Background Art

[0002] As a clean energy, hydrogen plays an important role in addressing climate change, reducing greenhouse gas emissions, and promoting the development of a low-carbon economy. Currently, hydrogen production mainly relies on fossil fuels, especially through methods such as methane hydrogen production and coal hydrogen production, which have a high carbon footprint. In order to achieve sustainable production of hydrogen, it is particularly important to develop new and clean hydrogen production methods, among which light-driven water splitting systems have attracted much attention due to their potential.

[0003] Although noble metal photo / electrocatalysts (such as Pt and Pd) can effectively reduce the reaction overpotential and accelerate hydrogen generation, these materials are expensive and resource-limited, limiting their feasibility for large-scale application. In contrast, naturally occurring hydrogenases have attracted attention due to their efficient and selective catalytic properties in biological systems. Hydrogenases can catalyze the production of hydrogen at low overpotentials and exhibit good stability under natural conditions, thus showing great potential in the study of light-driven hydrogen production. However, hydrogenases are highly sensitive to oxygen, and exposure to oxygen can lead to a significant decrease in activity or even complete inactivation. In addition, the extraction and purification process is complicated and costly, which increases the difficulty of their application.

[0004] In order to protect the activity of hydrogenase under aerobic conditions, researchers have tried to design microcompartments such as protein cages, carboxylases and organic frameworks through biomimetic strategies to encapsulate hydrogenase. However, existing natural protein self-assemblies (such as ferritin and virus shells) are usually between 20 and 100 nanometers in diameter, and their encapsulation capacity, oxygen isolation effect and structural stability are limited, making it difficult to meet the efficient and stable requirements of hydrogenase in the presence of oxygen. In addition, although the design of artificial bacterial aggregates to form a local hypoxic microenvironment through oxygen metabolic activities can maintain hydrogenase activity, this may increase the complexity of the system. This shows that a single structural design is difficult to meet the requirements of enhancing the ability of hydrogenase to resist oxygen, so it is particularly important to optimize the biomimetic encapsulation strategy for hydrogenase. Summary of the invention

[0005] The present invention aims to provide a method for constructing a giant organelle in Escherichia coli to enhance the oxygen resistance of [Fe-Fe] hydrogenase. By constructing a giant organelle in Escherichia coli, the oxygen resistance and stability of [Fe-Fe] hydrogenase are effectively enhanced, providing a new sustainable method for hydrogen production.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for constructing a giant organelle in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase comprises the following steps:

[0008] S1. Construct two expression plasmids capable of expressing [Fe-Fe] hydrogenase, guide protein and [Fe-Fe] hydrogenase maturation-promoting protein in Escherichia coli;

[0009] S2, transforming the two plasmids obtained in S1 into competent Escherichia coli BL21 (DE3) cells, and pre-culturing them in 3 ml of Luria Bertani medium for 6 h to obtain a pre-culture;

[0010] S3. The preculture obtained in S2 was transferred to a new Luria Bertani medium at a ratio of 1:50 and cultured aerobically. After a period of culture, the OD600 value was measured, and the culture was transferred to an anaerobic glove box, an inducer was added, and the culture was shaken at 25°C for 20 hours under anaerobic conditions. The culture was centrifuged to obtain Escherichia coli containing giant organelles.

[0011] Preferably, the [Fe-Fe] hydrogenase in S1 is HydA1, and the guiding protein is CipA; the maturation-promoting proteins of the [Fe-Fe] hydrogenase include: HydE, HydF and HydG.

[0012] Preferably, the two expression plasmids are: pETDuet-1 / HydE+HydA-CipA and pCDFDuet-1 / HydF+HydG.

[0013] Preferably, the pre-culture conditions in S2 are aerobic culture at 37° C. and 200 rpm.

[0014] Preferably, S2 is Luria Bertani medium supplemented with 100 μg / mL ampicillin and 40 μg / mL streptomycin.

[0015] Preferably, the aerobic culture conditions in S3 are 37° C., 200 rpm.

[0016] Preferably, in S3, when OD600 reaches 0.4-0.6, the culture is transferred to an anaerobic glove box.

[0017] Preferably, in S3, the inducer consists of 2.5 mmol / L sodium fumarate, 2 mmol / L L-cysteine, 250 μg / mL ammonium ferric citrate and 0.1 mmol / L isopropyl-β-D-thiogalactopyranoside.

[0018] Preferably, it is characterized in that the centrifugal process parameters in S3 are specifically 5000 rpm / min and the centrifugal time is 5 min.

[0019] Principle of the technical solution: Genetic engineering technology is used to transform E. coli, and the model enzyme and guide protein are selected: [Fe-Fe] hydrogenase (HydA1) and guide protein (CipA). After induction, giant organelles with hydrogen production activity are formed in the cytoplasm of E. coli. Strong hydrogen bonds and hydrophobic interactions between the guide proteins cause the [Fe-Fe] hydrogenase expressed in fusion with them to self-assemble to form giant organelles.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The invention discloses a method for constructing a giant organelle in Escherichia coli to enhance the oxygen resistance of [Fe-Fe] hydrogenase. By means of genetic engineering, [Fe-Fe] hydrogenase and guide protein (CipA) are fused and expressed to construct a giant organelle of [Fe-Fe] hydrogenase, which significantly enhances the oxygen resistance and stability of [Fe-Fe] hydrogenase in an oxygen atmosphere. Dense spherical protein nanoparticles are formed by the hydrophobic and hydrogen bond interactions between the guide proteins to form a physical barrier to oxygen penetration, and there are unique oxygen binding sites on the CipA protein, which realizes the production of hydrogen by [Fe-Fe] hydrogenase under aerobic conditions. In addition, the giant organelle combined with a photosensitizer can be used as a light-driven hydrogen production platform, showing that electron transfer and certain photocatalytic activity are maintained under high oxygen concentrations, and has good antioxidant properties and stability. This technology not only improves the catalytic efficiency and stability of [Fe-Fe] hydrogenase, but also reduces the cost of extraction and purification, providing a new and sustainable method for the production of hydrogen, which helps to promote the development of a low-carbon economy.

[0022] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic diagram of the structure of Escherichia coli containing giant organelles provided in Example 1 of the present invention;

[0024] Figure 2 Schematic diagram of two plasmids provided in Example 1, wherein: Figure 2 a in the figure is a schematic diagram of constructing the expression plasmid pETDuet-1 / HydE+HydA-CipA from the empty plasmid pETDuet-1. Figure 2 b is a schematic diagram of constructing the expression plasmid pCDFDuet-1 / HydF+HydG from the empty plasmid pCDFDuet-1;

[0025] Figure 3 The characterization results of Escherichia coli containing giant organelles provided in Example 1, wherein: Figure 3a in the figure is the optical microscopic result of Escherichia coli containing giant organelles. The scale bar is 5 μm. Figure 3 b is the optical microscopic result of giant organelles, the scale bar is 5μm, Figure 3 The c in the figure is the transmission electron microscopy result of giant organelles, with a scale of 1 μm. Figure 3 The d in the figure is the statistical diagram of the average diameter of giant organelles;

[0026] Figure 4 The catalytic activity evaluation results of the Escherichia coli containing giant organelles on [Fe-Fe] hydrogenase provided in Example 1, wherein: Figure 4 a in the figure is a schematic diagram for evaluating the activity of [Fe-Fe] hydrogenase in giant organelles. Figure 4 b in the figure is the statistical diagram of enzyme activity of [Fe-Fe] hydrogenase under different temperature conditions. Figure 4 c in the figure is the statistical diagram of enzyme activity of [Fe-Fe] hydrogenase under different pH conditions. Figure 4 d in is the statistical graph of hydrogen generation rate of [Fe-Fe] hydrogenase;

[0027] Figure 5 The enzyme activity of [Fe-Fe] hydrogenase in giant organelles after exposure to air for different time periods;

[0028] Figure 6 is the result of molecular dynamics simulation experiment, among which, Figure 6 a in the figure is the binding site between oxygen molecule and guide protein CipA. Figure 6 b in the figure is the oxygen binding pocket formed by lysine at position 82 and cysteine ​​at position 99 of the guide protein CipA. Figure 6 The c in the figure is the oxygen binding pocket formed by the valine at position 39 and the asparagine at position 58 of the guide protein CipA;

[0029] Figure 7 Giant organelles can be used as a platform for building semi-artificial photosynthetic systems. Figure 7 a in the figure is a schematic diagram of coupling EY with giant organelles to construct a semi-artificial photosynthetic system. Figure 7 b is the optical microscopic result of EY combined with giant organelles, the scale bar is 5μm, Figure 7 c in the figure is the fluorescence microscopy result of EY binding to giant organelles, the scale bar is 5 μm, Figure 7 The d in the figure is the hydrogen production over time of the semi-artificial photosynthetic system under light in different oxygen atmospheres. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0031] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0032] Plasmids pETDuet-1 and pCDFDuet-1 were synthesized and purchased from Sangon Biotechnology Co., Ltd. The remaining reagents were purchased from Aladdin.

[0033] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0034] Example 1

[0035] S1. Two plasmids capable of expressing [Fe-Fe] hydrogenase HydA1, guide protein CipA, and [Fe-Fe] hydrogenase maturation-promoting proteins HydE, HydF, and HydG in Escherichia coli were constructed from empty plasmids pETDuet-1 and pCDFDuet-1, respectively: pETDuet-1 / HydE+HydA-CipA and pCDFDuet-1 / HydF+HydG;

[0036] S2, transform the two plasmids obtained in S1 into competent E. coli BL21 (DE3) cells, named FdH-CipA, pre-culture them in 3 ml Luria Bertani medium for 6 h, the medium is supplemented with 100 μg / mL ampicillin and 40 μg / mL streptomycin, the pre-culture conditions are 37°C, aerobic culture at 200 rpm, to obtain pre-culture;

[0037] S3. The preculture obtained in S2 was transferred to a new Luria Bertani medium at a ratio of 1:50, and aerobically cultured at 37°C and 200rpm. After a period of culture, the OD600 value was measured. When the OD600 reached 0.6, the culture was transferred to an anaerobic glove box and cultured with shaking at 25°C for 20h under anaerobic conditions. 2.5mmol / L sodium fumarate, 2mmol / L L-cysteine, 250μg / mL ammonium ferric citrate and 0.1mmol / L isopropyl-β-D-thiogalactopyranoside were added as inducers, and the culture was centrifuged at 5000rpm / min for 5min to obtain Escherichia coli containing giant organelles.

[0038] Comparative Example 1 is the same as Example 1, except that: two plasmids pETDuet-1 and pCDFDuet-1 capable of expressing [Fe-Fe] hydrogenase HydA1 and [Fe-Fe] hydrogenase maturation-promoting proteins HydE, HydF and HydG in Escherichia coli were constructed; named FdH, which does not express the guide protein CipA, and obtains a giant organelle without [Fe-Fe] hydrogenase.

[0039] The effects of Example 1 and Comparative Example 1 were verified by the following experiments:

[0040] like Figure 2 As shown in the plasmid construction diagram, [Fe-Fe] hydrogenase HydA1 and guide protein CipA and [Fe-Fe] hydrogenase maturation-promoting proteins HydE, HydF and HydG were transferred into two plasmids pETDuet-1 and pCDFDuet-1, respectively, to construct plasmids pETDuet-1 / HydE+HydA-CipA and pCDFDuet-1 / HydF+HydG. Figure 1 As shown, the two plasmids were transformed into E. coli BL21 (DE3). After pre-culture and expansion culture, anaerobic induction was performed to obtain E. coli containing giant organelles.

[0041] 1. The Escherichia coli provided in Example 1 was characterized by microscopy, and the results were as follows: Figure 3 .

[0042] Depend on Figure 3 From the a in the figure, it can be seen that Example 1 contains protein crystal inclusion bodies, whose refractive index is different from that of other components in E. coli. Under the microscope, obvious shadows at the two poles of the bacteria were observed, indicating that the giant organelles containing [Fe-Fe] hydrogenase were successfully expressed. To further confirm the existence of giant organelles, ultrasonic disruption was used to lyse E. coli and release the giant organelles inside the cells. Figure 3 From b in the figure, we can see that giant organelles are regular spherical structures. Figure 3 c and Figure 3 As can be seen from d in the figure, under transmission electron microscopy, giant organelles with round nanoparticle characteristics were observed, and they showed obvious electron opacity. The average diameter of these organelles was 725.59±9.92nm.

[0043] 2. The catalytic activity of [Fe-Fe] hydrogenase was evaluated. The experimental scheme was as follows: In an anaerobic glove box, an appropriate amount of giant organelle solution was taken to ensure that the concentration of giant organelles in each treatment group was consistent, and 5 mM MV 2+ and 20 mM dithionite (NaDT) and placed in a constant temperature incubator at different temperatures for reaction. Figure 4 .

[0044] Depend on Figure 4 As can be seen from the a in the figure, dithionite can effectively convert methyl viologen (MV 2+ )Restore to MV +, providing the required electrons for the hydrogen production reaction of [Fe-Fe] hydrogenase. The results showed that the CipA-mediated giant artificial organelle significantly enhanced the thermal stability of the encapsulated [Fe-Fe] hydrogenase.

[0045] like Figure 4 As shown in b, at the optimum temperature of 35°C, the enzyme activity of [Fe-Fe] hydrogenase encapsulated in the giant organelle of Example 1 reached 2024.21±63.31 nmol H2mg -1 min -1 . And the enzyme activity remained stable within the temperature range of 35°C to 60°C. In contrast, the activity of the free [Fe-Fe] hydrogenase in Comparative Example 1 was almost lost (decreased by 97.65%) after being heated at 60°C for 60 minutes. However, the [Fe-Fe] hydrogenase encapsulated by FdH-CipA in Example 1 still retained 28.29% of its activity under the same conditions, indicating that the giant organelle can effectively protect the [Fe-Fe] hydrogenase from the effects of high temperature.

[0046] Depend on Figure 4 From the c in FIG. 1 , it can be seen that in terms of pH stability, the optimum pH of [Fe-Fe] hydrogenase is 7.5. The experimental results show that under weakly acidic pH conditions, the activity of the encapsulated [Fe-Fe] hydrogenase in Example 1 is not significantly different from the activity of the unencapsulated [Fe-Fe] hydrogenase in Comparative Example 1. However, in an alkaline environment, especially under strongly alkaline conditions of pH 11, the hydrogen production activity of the encapsulated [Fe-Fe] hydrogenase in Example 1 remains at 29.81%.

[0047] Depend on Figure 4 As can be seen from d, the maximum hydrogen generation rate of the [Fe-Fe] hydrogenase giant organelle in Example 1 at pH 7.5 and 35°C is 4577.75±352.82 nmol mg -1 min -1 . The amount of hydrogen produced by the artificial organelle measured at an MV of 5 mM increases linearly with time, indicating that this process has catalytic properties. Compared with the free [Fe-Fe] hydrogenase in Comparative Example 1, Example 1 significantly improves the efficiency of hydrogen production, indicating that it has obvious advantages in enhancing the catalytic activity of [Fe-Fe] hydrogenase. This enhanced catalytic activity is related to the special microenvironment within the giant organelle, which helps to improve the stability and reaction rate of [Fe-Fe] hydrogenase.

[0048] 3. To verify the oxygen resistance of E. coli containing giant organelles in Example 1 to [Fe-Fe] hydrogenase, the test scheme is as follows: a certain amount of giant organelle solution is placed in an air atmosphere and left to stand for 20, 40, 80, 160, and 320 minutes. Then it is moved into an anaerobic glove box to remove oxygen and 5 mM MV is added.2+ and 20 mM sodium dithionite for 90 min. Figure 5 .

[0049] Depend on Figure 5 It can be seen that after being exposed to ambient air for 320 minutes, the catalytic activity of the giant organelle in Example 1 remained at 18.89%, while the free [Fe-Fe] hydrogenase in Comparative Example 1 almost completely lost its activity after a few minutes. This shows that the giant organelle can effectively protect the [Fe-Fe] hydrogenase from oxidative damage by oxygen, significantly improving its stability and durability in practical applications. Previous studies have pointed out that oxidative damage in enzyme-catalyzed reactions often limits the service life of the enzyme, while enzymes encapsulated in organelles can effectively avoid damage by oxygen, thereby improving catalytic efficiency and stability.

[0050] 4. Molecular dynamics simulation experiment, the experimental scheme is as follows, the molecular dynamics software GROMACS is used to simulate the binding of oxygen and CipA, and the CipA protein structure is modeled based on the experimentally obtained crystal structure (PDB ID: 7XHS). 20 oxygen molecules (0.035M, far higher than the concentration of saturated dissolved oxygen in water) are freely distributed throughout the simulation system and interact with the CipA protein without constraints. All energy minimization and molecular dynamics simulations use the AMBER99SB-ILDN protein,nucleicABBER94 force field and are performed under periodic boundary conditions. Since all molecules used do not contain polarization force field parameters, no polarization force field is used. The temperature of the simulation system is maintained at 300K by a dual Nosé-Hoover thermostat, and the pressure is maintained at 1.0atm by an Andersen-Hoover pressure controller. The results are as follows. Figure 6 .

[0051] Depend on Figure 6It can be seen that CipA can effectively bind oxygen, slowing its entry into the protein, thereby slowing down the inactivation of [Fe-Fe] hydrogenase. To further reveal the mechanism of oxygen binding, analysis showed that oxygen stayed in the binding pocket of CipA for a long time, and two main binding modes were identified. The first binding mode is the "lysine-cysteine" pocket, in which lysine at position 82 and cysteine ​​at position 99 form an oxygen binding pocket through spatial arrangement. The side chains of lysine and cysteine ​​carry polar groups, and the amino group of lysine and the thiol group of cysteine ​​can form hydrogen bonds or polar interactions with oxygen, which helps to stabilize oxygen in the pocket. In addition, the spatial arrangement of lysine and cysteine ​​is suitable for the size of oxygen molecules, making oxygen more stably located in the pocket. The second binding mode occurs in the valine-asparagine pocket, that is, valine at position 39 and asparagine at position 58 interact with oxygen through a spatial structure to form a pocket. Valine is a hydrophobic amino acid, while asparagine carries an amide group that can form hydrogen bonds. The hydrophobicity of valine helps to repel water molecules, creating a stable hydrophobic microenvironment for oxygen, while the amide group of asparagine provides weak polar interactions for oxygen, enhancing its stability.

[0052] 5. The performance of the E. coli containing giant organelles provided in Example 1 in photocatalytic hydrogen production was studied under different oxygen environments. An aqueous solution containing 100 μM eosin Y (EY) as a photosensitizer and 100 mM triethanolamine (TEOA) as an electron donor was used in combination with giant organelles containing [Fe-Fe] hydrogenase to study the light-driven hydrogen production performance of the system in the absence of redox media and in the presence of oxygen. The results are shown in Figure 5. Figure 7 .

[0053] Depend on Figure 7 From the a in the figure, we can see that EY can form a hybrid with the giant organelle through electrostatic interaction, making it fluorescent. At the same time, the giant organelle gives the semi-artificial photosynthetic system oxygen tolerance.

[0054] Depend on Figure 7 As shown in b, EY was successfully embedded or attached to the surface or interior of the giant organelle (EY@FdH-CipA), showing bright green fluorescence. The photocatalytic results showed that EY@FdH-CipA could achieve efficient photocatalytic reaction in the absence of soluble redox mediators, with a hydrogen generation rate of 0.64±0.09μmol H2 hour -1 , and by Figure 7As can be seen from the c in the figure, a nearly linear hydrogen production trend is shown within the first 2 hours. This result shows that under stable illumination conditions, the system can maintain continuous electron transfer efficiency and promote effective hydrogen generation. As the oxygen concentration increases, the photocatalytic activity decreases significantly. However, even in an environment with an oxygen concentration of 21%, the system still retains a certain photocatalytic activity, which is equivalent to 6.34±2.53% of the activity under anaerobic conditions. This result shows that EY@FdH-CipA can still maintain electron transfer and a certain photocatalytic activity under high oxygen concentrations, demonstrating its good antioxidant properties and stability.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for constructing giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase, characterized in that: The steps include: S1. Construct two expression plasmids capable of expressing [Fe-Fe] hydrogenase, guide protein and [Fe-Fe] hydrogenase maturation-promoting protein in Escherichia coli; S2, transforming the two plasmids obtained in S1 into competent Escherichia coli BL21 (DE3) cells, and pre-culturing them in 3 ml of Luria Bertani medium for 6 h to obtain a pre-culture; S3. The preculture obtained in S2 was transferred to a new Luria Bertani medium at a ratio of 1:50 and cultured aerobically. After a period of culture, the OD600 value was measured, and the culture was transferred to an anaerobic glove box, an inducer was added, and the culture was shaken at 25°C for 20 hours under anaerobic conditions. The culture was centrifuged to obtain Escherichia coli containing giant organelles.

2. The method for constructing giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase according to claim 1, characterized in that: The [Fe-Fe] hydrogenase described in S1 is HydA1, and the guiding protein is CipA; the maturation-promoting proteins of [Fe-Fe] hydrogenase include: HydE, HydF and HydG.

3. The method for constructing giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase according to claim 1, characterized in that: The pre-culture conditions in S2 were 37°C and aerobic culture at 200 rpm.

4. The method for constructing giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase according to claim 1, characterized in that: S2 is Luria Bertani medium supplemented with 100 μg / mL ampicillin and 40 μg / mL streptomycin.

5. The method for constructing giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase according to claim 1, characterized in that: Aerobic culture conditions in S3 were 37°C and 200 rpm.

6. The method for constructing giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase according to claim 1, characterized in that: In S3, when OD600 reached 0.4-0.6, the culture was transferred to an anaerobic glove box.

7. The method for constructing giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase according to claim 1, characterized in that: In S3, the inducer consists of 2.5 mmol / L sodium fumarate, 2 mmol / L L-cysteine, 250 μg / mL ammonium ferric citrate and 0.1 mmol / L isopropyl-β-D-thiogalactopyranoside.

8. The method for constructing giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase according to claim 1, characterized in that: The centrifugal process parameters in S3 are specifically 5000 rpm / min and the centrifugal time is 5 min.

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