A method for constructing giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase

Abstract

The present invention discloses a method for constructing giant organelles in Escherichia coli to enhance the oxygen resistance of [Fe-Fe] hydrogenase, and belongs to the field of enzyme catalysis technology. The method comprises the following steps: constructing two plasmids capable of expressing [Fe-Fe] hydrogenase and guide protein and [Fe-Fe] hydrogenase maturation-promoting protein in Escherichia coli; transforming the two plasmids into Escherichia coli BL21 (DE3) competent cells and performing pre-culture; transferring the pre-culture to a new culture medium, performing aerobic culture, and after culturing for a period of time, determining its OD600 value, transferring the culture to an anaerobic glove box, anaerobically culturing, centrifuging, and obtaining Escherichia coli containing giant organelles. The present invention provides a method for constructing giant organelles in Escherichia coli to enhance the oxygen resistance of [Fe-Fe] hydrogenase, which effectively enhances the oxygen resistance and stability of [Fe-Fe] hydrogenase by constructing giant organelles in Escherichia coli, and provides a new sustainable method for the production of hydrogen.

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 source, hydrogen plays a vital role in addressing climate change, reducing greenhouse gas emissions, and promoting the development of a low-carbon economy. Currently, hydrogen production relies primarily on fossil fuels, particularly through methods such as methane-to-hydrogen and coal-to-hydrogen, which have a high carbon footprint. To achieve sustainable hydrogen production, the development of novel, clean hydrogen production methods is crucial, and light-driven water splitting systems have attracted considerable attention due to their potential.

[0003] Although precious metal photo / electrocatalysts (such as Pt and Pd) can effectively reduce the reaction overpotential and accelerate hydrogen production, the high cost and limited resources of these materials limit the feasibility of their large-scale application. In contrast, naturally occurring hydrogenases have attracted attention due to their efficient and highly 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 research of light-driven hydrogen production. However, hydrogenases are highly sensitive to oxygen, and exposure to oxygen will lead to a significant decrease in activity or even complete inactivation. In addition, the extraction and purification process is complex 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 giant organelles in Escherichia coli to enhance the oxygen tolerance of [Fe-Fe] hydrogenase. By constructing giant organelles in Escherichia coli, the oxygen tolerance 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 solutions adopted by the present invention are 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, a guide protein, and a [Fe-Fe] hydrogenase maturation-promoting protein in Escherichia coli;

[0009] S2. The two plasmids obtained in S1 were transformed into Escherichia coli BL21 (DE3) competent cells, which were pre-cultured 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 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 cultured with shaking 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 [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 condition in S2 is 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. and 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: Using genetic engineering techniques to modify Escherichia coli, a model enzyme and a guide protein, [Fe-Fe] hydrogenase (HydA1), were selected. Following induction, giant organelles with hydrogen-producing activity formed in the E. coli cytoplasm. Strong hydrogen bonds and hydrophobic interactions between the guide proteins caused the [Fe-Fe] hydrogenase, expressed fused to them, to self-assemble into the giant organelles.

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

[0021] The present invention discloses a method for constructing a giant organelle in Escherichia coli to enhance the oxygen resistance of [Fe-Fe] hydrogenase. By genetic engineering means, [Fe-Fe] hydrogenase and a guide protein (CipA) are fused and expressed to construct a [Fe-Fe] hydrogenase giant organelle, which significantly enhances the oxygen resistance and stability of [Fe-Fe] hydrogenase in an oxygen atmosphere. Dense spherical protein nanoparticles are formed by hydrophobic and hydrogen bond interactions between the guide proteins, forming a physical barrier to oxygen penetration, and the CipA protein has a unique oxygen binding site, which enables [Fe-Fe] hydrogenase to produce hydrogen under aerobic conditions. In addition, the giant organelle can be combined with a photosensitizer as a light-driven hydrogen production platform, demonstrating that it maintains electron transfer and a certain photocatalytic activity 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 hydrogen production, which helps 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 the construction of 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, and the scale bar is 5 μm. Figure 3 b in the figure is the optical microscopic result of giant organelles, the scale bar is 5 μm, Figure 3 The c in the figure is the transmission electron microscopy result of giant organelles, and the scale bar is 1 μm. Figure 3 The d in the figure is the statistical diagram of the average diameter of giant organelles;

[0026] Figure 4 The results of the evaluation of the catalytic activity of 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 the figure is the statistical graph of hydrogen production 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, where Figure 6 a in the figure is the binding site between oxygen molecules and the 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 c in the figure represents 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 binding to giant organelles, the scale bar is 5μm, Figure 7 Figure c is the fluorescence microscopy result of EY binding to giant organelles, the scale bar is 5μm, Figure 7 Where d is the hydrogen production of the semi-artificial photosynthetic system under light over time under different oxygen atmospheres. DETAILED DESCRIPTION

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

[0031] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0032] Plasmids pETDuet-1 and pCDFDuet-1 were synthesized and purchased from Sangon Biotechnology. Other 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 this field and can be purchased through commercial channels.

[0034] Example 1

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

[0036] S2. Transform the two plasmids obtained in S1 into competent Escherichia coli BL21(DE3) cells, designating them FdH-CipA. Preculture the cells in 3 mL of Luria-Bertani medium supplemented with 100 μg / mL ampicillin and 40 μg / mL streptomycin at 37°C and 200 rpm for 6 h to obtain a preculture.

[0037] S3. The preculture obtained in S2 was transferred to new Luria Bertani medium at a ratio of 1:50 and cultured aerobically at 37°C and 200 rpm. 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 under anaerobic conditions for 20 h. 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 were added as inducers. After centrifugation at 5000 rpm / min for 5 min, Escherichia coli containing giant organelles was obtained.

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

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

[0042] Depend on Figure 3 As can be seen from the a in Example 1, 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, giant organelles with round nanoparticle characteristics were observed under transmission electron microscopy, 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 5mM MV 2+ and 20mM 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 electrons required 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, the enzyme activity of [Fe-Fe] hydrogenase encapsulated in the giant organelle of Example 1 reached 2024.21±63.31 nmol H2mg at the optimal temperature of 35°C. -1 min -1 The enzyme activity remained stable within the temperature range of 35°C to 60°C. In contrast, the free [Fe-Fe] hydrogenase in Comparative Example 1 lost almost all its activity (a decrease of 97.65%) after heating at 60°C for 60 minutes. However, the [Fe-Fe] hydrogenase encapsulated by FdH-CipA in Example 1 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 As shown in the figure c, the optimal pH for [Fe-Fe] hydrogenase in terms of pH stability 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 that of the unencapsulated [Fe-Fe] hydrogenase in Comparative Example 1. However, in an alkaline environment, particularly under the strongly alkaline condition 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 the d in Example 1, the maximum hydrogen generation rate of the [Fe-Fe] hydrogenase giant organelle 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 increased linearly over time, indicating that this process has catalytic properties. Compared with the free [Fe-Fe] hydrogenase in Comparative Example 1, Example 1 significantly improved the efficiency of hydrogen production, indicating that it has a clear advantage 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 to [Fe-Fe] hydrogenase in Example 1, the experimental scheme is as follows: a certain amount of giant organelle solution was placed in an air atmosphere and allowed to stand for 20, 40, 80, 160, and 320 minutes. It was then moved into an anaerobic glove box to remove oxygen and add 5mM MV2+ and 20mM sodium dithionite for 90min. Figure 5 .

[0049] Depend on Figure 5 As can be seen, after 320 minutes of exposure to ambient air, 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 demonstrates that the giant organelle can effectively protect [Fe-Fe] hydrogenase from oxidative damage by oxygen, significantly improving its stability and durability in practical applications. Previous studies have shown that oxidative damage in enzyme-catalyzed reactions often limits the lifespan of enzymes, while enzymes encapsulated in organelles can effectively avoid oxygen damage, 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, much 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, polarization force fields are not 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 6CipA effectively binds oxygen, slowing its entry into the protein and thus slowing the inactivation of [Fe-Fe] hydrogenase. To further elucidate the mechanism of oxygen binding, analysis revealed that oxygen resides for a prolonged period within the CipA binding pocket, and two primary binding modes were identified. The first binding mode is the "lysine-cysteine" pocket, where lysine at position 82 and cysteine at position 99 form an oxygen-binding pocket through spatial arrangement. The side chains of lysine and cysteine contain polar groups, and the amino group of lysine and the thiol group of cysteine can form hydrogen bonds or polar interactions with oxygen, helping to stabilize the oxygen within the pocket. Furthermore, the spatial arrangement of lysine and cysteine is well-suited to the size of the oxygen molecule, which stabilizes the oxygen within the pocket. The second binding mode occurs in the valine-asparagine pocket, where valine at position 39 and asparagine at position 58 form a spatially arranged pocket that interacts with oxygen. Valine is a hydrophobic amino acid, while asparagine has 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 giant organelles of E. coli provided in Example 1 in photocatalytic hydrogen production under different oxygen environments was studied. 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 performance of the system in photocatalytic hydrogen production 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 As can be seen from the a in the figure, EY can form a hybrid with the giant organelle through electrostatic interaction, giving it fluorescent properties. At the same time, the giant organelle gives the semi-artificial photosynthetic system oxygen tolerance.

[0054] Depend on Figure 7 As shown in Figure b, EY was successfully embedded or attached to the surface or interior of the giant organelle (EY@FdH-CipA), showing bright green fluorescence. Photocatalytic results showed that EY@FdH-CipA can 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 was shown within the first 2 hours. This result indicates 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 solutions of the present invention rather than to limit the same. 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 solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a giant organelle 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, a guide protein, and a [Fe-Fe] hydrogenase maturation-promoting protein in Escherichia coli; S2. The two plasmids obtained in S1 were transformed into Escherichia coli BL21 (DE3) competent cells, which were pre-cultured in 3 ml of Luria Bertani medium for 6 h to obtain a pre-culture; S3. The preculture obtained in S2 was transferred to new Luria Bertani medium at a ratio of 1:50 and cultured aerobically. After a period of culture, the OD600 value was measured. The culture was transferred to an anaerobic glove box, an inducer was added, and the culture was shaken at 25°C under anaerobic conditions for 20 hours. The culture was centrifuged to obtain Escherichia coli containing giant organelles. The [Fe-Fe] hydrogenase described in S1 is HydA1, and the initiator protein is CipA; the maturation-promoting proteins of [Fe-Fe] hydrogenase include: HydE, HydF, and HydG; 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; S1. Two plasmids were constructed from the empty plasmids pETDuet-1 and pCDFDuet-1, respectively, which can express [Fe-Fe] hydrogenase HydA1, the indicative protein CipA, and the maturation-promoting proteins HydE, HydF, and HydG of [Fe-Fe] hydrogenase in Escherichia coli: pETDuet-1 / HydE+HydA-CipA and pCDFDuet-1 / HydF+HydG.

2. The method for constructing a giant organelle 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.

3. The method for constructing a giant organelle 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.

4. The method for constructing a giant organelle 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.

5. The method for constructing a giant organelle 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.

6. The method for constructing a giant organelle 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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