A high-density fermentation process for producing active nickel-iron hydrogenase

Abstract

The application discloses a two-stage high-density fermentation method for producing active nickel-iron hydrogenase. The method comprises the following steps: performing aerobic high-density fed-batch fermentation on recombinant Escherichia coli co-expressing nickel-iron hydrogenase structural genes and mature auxiliary protein genes, so that the cell density reaches OD 600 ≥40; then switching to anaerobic conditions, adding an inducer and metal ion cofactor, and inducing expression and active center assembly of the enzyme; and finally, breaking the cells in an anaerobic environment to obtain active enzymes. Through the time-decoupling strategy, the volume yield is improved to more than 900 mg / L under the premise of ensuring high specific activity (≥20 U / mg) of the enzyme, and the technical bottleneck of low cell density and limited yield in traditional anaerobic fermentation is solved, which is suitable for industrial production of nickel-iron hydrogenase and other oxygen-sensitive metal enzymes.

Description

Technical Field

[0001] This invention belongs to the fields of industrial biotechnology and enzyme engineering, and specifically relates to a method for producing recombinant nickel iron hydrogenase using genetically engineered Escherichia coli as the host cell through a time-controlled two-stage high-density fermentation technology. Background Technology

[0002] [NiFe]-hydrogenase (EC 1.12.7.2) is a highly efficient redox metalloenzyme whose active center contains a nickel-iron heterobimetallic cluster, enabling it to reversibly catalyze the oxidation of molecular hydrogen (H2O). 2H + + 2e - With its excellent catalytic efficiency and substrate specificity, nickel-iron hydrogenase has important application value in fields such as bioenergy conversion, biofuel cells, industrial waste gas treatment, and coenzyme regeneration.

[0003] However, heterologous expression of nickel-iron hydrogenase faces a technical paradox of "biomass accumulation-active assembly": First, the anaerobic dependence of active assembly: During the maturation of nickel-iron hydrogenase, the synthesis of the [NiFe] active center, the insertion of metal ions, and the correct folding of the enzyme protein all require strictly anaerobic conditions. Oxygen will cause the metal cluster to oxidize and inactivate, producing enzyme proteins with no catalytic activity. Second, the aerobic preference of the host growth: As a facultative anaerobic microorganism, *E. coli* has an energy conversion efficiency of only 5%-10% of that of aerobic respiration. Under strictly anaerobic conditions, the specific growth rate of *E. coli* is significantly reduced, and the cell density usually cannot exceed the OD. 600 =5, which limits the enzyme production per unit volume, resulting in high industrial production costs. Third, incompatibility with existing technologies: Standard high-density fed-batch fermentation technology relies on a high dissolved oxygen environment to maintain a high specific growth rate (μ=0.2-0.5 h). -1 This fundamentally contradicts the anaerobic requirements for the assembly of the active site of nickel-iron hydrogenase. While some studies have attempted to produce nickel-iron hydrogenase through high-density cultivation in *E. coli*, a systematic process scheme is lacking, particularly regarding key control parameters for a smooth transition from the aerobic high-density growth stage to the anaerobic induction expression stage, the timing of metal ion supplementation, and the specific sequence for establishing the anaerobic environment. Therefore, there is an urgent need to develop a novel fermentation process that integrates the advantages of high-density cultivation with the requirements of anaerobic assembly. Summary of the Invention

[0004] The present invention aims to solve the technical contradiction in the production of nickel-iron hydrogenase in the prior art, which is that cell density and enzyme activity cannot be simultaneously achieved. It provides a two-stage fermentation method that can achieve a synergistic improvement in high biomass accumulation and high specific activity products.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for constructing recombinant Bacillus subtilis involves using Bacillus subtilis as a chassis strain and integrating an induction control cassette and a functional expression cassette into its genome through genetic engineering techniques to obtain a recombinant strain capable of ethanol degradation.

[0007] The chassis strain is preferably Bacillus subtilis 168 or its derivatives.

[0008] The induction control cassette contains at least one ethanol-responsive promoter or an anaerobic-responsive promoter to drive the transcription of the T7 phage RNA polymerase gene. Specifically, the ethanol-responsive promoter is selected from the PyjbD promoter or the σB-dependent PrsbV promoter, which are specifically activated by ethanol stress; the anaerobic-responsive promoter is selected from the Pldh promoter, the PnarG promoter, or the Pfnr promoter, which are activated under hypoxic or anaerobic conditions. These promoters can be used alone or in combination to form a logic or gating switch, meaning that activation of any one promoter can drive the expression of T7 phage RNA polymerase, thereby achieving a dual response to alcohol consumption and the anaerobic environment of the gut.

[0009] The functional expression cassette is driven by the T7 promoter and contains tandemly expressed alcohol dehydrogenase and aldehyde dehydrogenase genes. The specific structure is: T7 phage promoter – codon-optimized alcohol dehydrogenase gene – ribosome binding site – codon-optimized aldehyde dehydrogenase gene – terminator. This design utilizes the highly efficient transcriptional activity of T7 phage RNA polymerase to achieve high-level expression of the target gene.

[0010] The gene integration method utilizes the key flagellar synthesis gene flM from Bacillus subtilis as the integration site. Homologous recombination precisely inserts the induction control cassette and functional expression cassette into the flM gene coding region, achieving simultaneous chromosomal integration of the target gene and simultaneous knockout of the flM gene. This strategy not only ensures the genetic stability of the functional gene but also renders the strain motile, reducing biosafety risks.

[0011] The recombinant Bacillus subtilis obtained by the above method was named Bacillus subtilis DE-02. This strain has the following characteristics: First, it has dual ethanol and anaerobic induction properties, meaning it can respond to high concentrations of ethanol after drinking alcohol and can also be spontaneously activated by anaerobic signals after entering the deep intestine; Second, it uses a T7 cascade amplification system, which avoids the saturation limitation of endogenous RNA polymerase and significantly increases the protein production of alcohol dehydrogenase and acetaldehyde dehydrogenase; Third, due to the knockout of the flM gene, the strain loses its motility, significantly reducing the potential risk of translocation across the intestinal mucosal barrier; Fourth, the functional genes are integrated into the chromosome, allowing for stable inheritance without the need for antibiotics, avoiding performance degradation caused by plasmid loss.

[0012] Compared with the prior art, the present invention has the following significant advantages:

[0013] 1. Volumetric yield increased by 1-2 orders of magnitude

[0014] This invention employs a time-series decoupling strategy to first achieve high-density culture (OD) under aerobic conditions. 600 The concentration can reach 40-150, which is typical for traditional anaerobic fermentation (OD). 600 Typically, the production is 20-50 times that of <5). The huge biomass base allows for an order-of-magnitude increase in the total enzyme production during the subsequent anaerobic stage, with a volumetric yield of over 900 mg / L, significantly reducing unit production costs.

[0015] 2. The product maintains a high level of specific activity.

[0016] By strictly controlling the anaerobic switching time (≤60 min), simultaneously supplementing metal ion cofactors, and co-expressing mature accessory proteins, this invention ensures the efficient assembly of the active center, resulting in a nickel-iron hydrogenase specific activity ≥20 U / mg.

[0017] 3. Process controllability and scale-up potential

[0018] This invention establishes a time-series control parameter system based on dissolved oxygen, temperature, and inducer addition. Key control points are clearly defined, and batch-to-batch repeatability is excellent (coefficient of variation <15%). This process is compatible with existing industrial fermentation equipment, requires no special reactor design, and has promising prospects for industrial scale-up.

[0019] 4. Value of a General-Purpose Technology Platform

[0020] The two-stage fermentation strategy established in this invention provides a transferable technical framework for the large-scale production of other oxygen-sensitive metalloenzymes (such as [FeFe]-hydrogenase, nitrogenase, carbon monoxide dehydrogenase, etc.). Attached Figure Description

[0021] Figure 1This is a schematic diagram of the aerobic-anaerobic two-stage fermentation process described in this invention.

[0022] Figure 2 This is an electrophoretic pattern obtained by SDS-PAGE analysis of total and soluble proteins of the bacteria after fermentation in Example 1 of this invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0024] Example 1: Two-stage high-density fermentation production of nickel-iron hydrogenase

[0025] Recombinant Escherichia coli BL21(DE3)-SHI-hyp was selected as seed culture. Its characteristic is that it simultaneously overexpresses the encoding genes of soluble hydrogenase I (SHI) PF0891, PF0892, PF0893 and PF0894 derived from Pyrococcus furiosus, as well as its complete mature gene cluster hypABFCDE.

[0026] Step 1: Seed Liquid Preparation

[0027] The above-mentioned engineered strains were inoculated into 50 mL of LB medium and cultured at 37°C and 220 rpm for 12–16 h to obtain the seed culture.

[0028] Step 2: High-density fermentation culture

[0029] Equipment and culture medium: Use a 5 L standard fermenter (working volume 2.5 L).

[0030] Basic culture medium (g / L): glucose 25.0, (NH4)2SO4 4.0, K2HPO4 14.6, NaH2PO4·2H2O 3.6, (NH4)2-H-citrate 1.0, MgSO4·7H2O 1.5, peptone 15, yeast extract 7.5, resazurite 0.1 (redox indicator), antifoaming agent 0.1. After sterilization at 121℃ for 20 min, add 1 mL / L of 1000× trace element stock solution.

[0031] Trace element stock solution (1000×, g / L): FeCl3·6H2O 2.7, ZnCl2 0.2, CoCl2·6H2O 0.2, Na2MoO4·2H2O 0.2, CaCl2·2H2O 0.1, CuCl2 0.02, H3BO3 0.05.

[0032] Feeding medium A solution: glucose 600 g / L, MgSO4·7H2O 5 g / L, trace element stock solution 1 mL / L; B solution: 28% (w / w) ammonia water (for pH control).

[0033] Aerobic growth stage:

[0034] Inoculate the seed culture into the fermenter at a 2% (v / v) inoculation rate. Initial parameters: temperature 37℃, pH 7.0 (automatically controlled by solution B), stirring 300 rpm, aeration rate 1.0 vvm.

[0035] Batch culture lasted approximately 8 hours. After glucose depletion and a surge in dissolved oxygen, an exponential fed-batch program was initiated. The feed rate was calculated using the equation F(t) = (μ_set / Y_X / S)·X0V0·e^(μ_set·t) / S_f, with the specific growth rate μ_set set to 0.15 h. -1 Dissolved oxygen is maintained at 30% ± 5% saturation by cascading control of stirring speed (up to 1000 rpm) and aeration rate (up to 2.0 vvm).

[0036] After approximately 24 hours of continuous aerobic feeding culture, the OD of the fermentation broth was... 600 It reached 125.

[0037] Anaerobic induction phase:

[0038] When OD 600 When the value reaches 125, the execution environment is switched:

[0039] Add IPTG to a final concentration of 0.1 mM;

[0040] Add sterile filtered NiSO4·6H2O, cysteine ​​and (NH4)2Fe(SO4)2·6H2O solution to final concentrations of 25 μM, 2 mM and 0.1 mM, respectively.

[0041] Immediately stop the air supply and switch to introducing sterile nitrogen (1.0 vvm).

[0042] Reduce the stirring speed to 200 rpm;

[0043] Lower the temperature setpoint to 25°C.

[0044] Under these conditions, anaerobic culture continued for 20 hours. Within 15 minutes of switching, dissolved oxygen dropped to 0%; 30 minutes after switching, the redox indicator resazan changed from pink to colorless, confirming the establishment of the anaerobic environment.

[0045] Step 3: Sample Collection and Processing

[0046] After fermentation, the fermentation broth was cooled to 10°C, and the cells were collected using a continuous flow centrifuge (10,000×g), yielding approximately 1.5 kg of wet cells. The cells were transferred to an anaerobic glove box (95% N2, 5% H2 atmosphere) and resuspended in pre-cooled, nitrogen-saturated 50 mM Tris-HCl (pH 7.5), 2 mM DTT buffer. Cell disruption was performed using a high-pressure homogenizer at 4°C under nitrogen protection (1200 bar, 3 times). The disrupted broth was centrifuged anaerobicly at 15,000×g, 4°C for 40 min, and the supernatant was collected as the crude enzyme solution containing active nickel-iron hydrogenase.

[0047] Example 3: Product Expression and Activity Analysis

[0048] Step 1: SDS-PAGE detection

[0049] Fermentation broth samples were subjected to SDS-PAGE. The results showed that after induction, highly distinct specific protein bands appeared at approximately 44.6 kDa (PF0891), 33.1 kDa (PF0892), 29.2 kDa (PF0893), and 48.3 kDa (PF0894), dominating the soluble fraction and indicating efficient soluble expression of the target protein. Gel image density scanning software analysis showed that the protein expression level accounted for approximately 30% of the total soluble protein in the bacterial cells. The total protein content was determined using a Bradford assay kit, and the volumetric yield of nickel-iron hydrogenase was calculated to be approximately 950 mg / L.

[0050] Step 2: Hydrogenase Activity Detection

[0051] Activity was determined using sealed quartz cuvettes in an anaerobic glove box. The reaction system consisted of 1 mL of 50 mM Tris-HCl (pH 8.0) buffer containing 2 mM benzyl viologen. Pure hydrogen gas was bubbled into the system until saturation, and then an appropriate amount of diluted crude enzyme solution was added to initiate the reaction. The decrease in absorbance due to benzyl viologen reduction was monitored at 578 nm, and the activity was determined based on its molar extinction coefficient (ε = 8.65 mM). -1 ·cm -1 Calculate enzyme activity. One unit of activity (U) is defined as the amount of enzyme required to reduce 2 μmol of benzyl viologen (corresponding to 1 μmol of H2) per minute under standard conditions.

[0052] The results showed that the crude enzyme solution obtained in Example 1 had a hydrogenase specific activity of 25.2 U / mg total protein and a fermentation broth volume activity of 23940 U / L.

[0053] Comparative Example 1: Traditional single-stage anaerobic fermentation

[0054] To demonstrate the superiority of the method of the present invention, a comparative example was established. The same engineered strain as in Example 1 was used.

[0055] The same engineered strain as in Example 1 was used. The seed culture was inoculated into 2.5 L of pre-denitred nitrogen-purged basal medium. OD after inoculation... 600 ≈1. The entire fermentation process (48 h) was carried out under strictly anaerobic conditions at 37 °C, pH 7.0, with gentle stirring (150 rpm) and continuous nitrogen purging. 0.1 mM IPTG, 25 μM NiSO4, 0.1 mM (NH4)2Fe(SO4)2 and 2 mM cysteine ​​were added at the start of the culture.

[0056] After 48 h of cultivation, the OD of the fermentation broth was... 600 The specific activity was only 3.2. The bacterial cells were collected and crude enzyme solution was prepared using the same method. The specific activity of the enzyme was measured to be 27.3 U / mg total protein, but due to the extremely low bacterial cell density, the final volumetric activity was only 662.6 U / L.

[0057] The results of Example 1 are compared with those of Comparative Example 1:

[0058] Cell density: the method of this invention (OD) 600 = 125) is the control group (OD 600 = 3.2) is about 39 times.

[0059] Volumetric activity: The method of this invention (23940 U / L) is 36.1 times that of the control group (662.6 U / L).

[0060] The above results demonstrate that the present invention, through a two-stage strategy of first aerobic high-density growth followed by anaerobic induced expression, increases the volumetric yield of nickel-iron hydrogenase by nearly two orders of magnitude while ensuring high specific activity of the enzyme, thus significantly improving the economics of industrial production.

[0061] 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. That is, all simple and equivalent changes and modifications made based on the claims and description of this invention fall within the protection scope of the claims. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A method for producing active nickel-iron hydrogenase, characterized in that, Includes the following steps: Aerobic high-density culture stage: the recombinant E. coli is subjected to high-density fed-batch fermentation under the condition that the dissolved oxygen is not less than 10% saturation, the carbon source feeding rate is controlled by using an exponential feeding strategy, and the cell density reaches OD 600 ≥ 40; Anaerobic induction phase: When the cell density reaches the target threshold, an inducer and metal ion cofactor are added to the fermentation system, and the system is switched to anaerobic conditions to reduce dissolved oxygen to 0% within 60 minutes, thereby inducing the expression of nickel iron hydrogenase and the assembly of the active site. Post-processing stage: After fermentation, the cells are broken under anaerobic conditions to obtain bioactive nickel-iron hydrogenase. The recombinant Escherichia coli co-expresses the nickel iron hydrogenase structural gene and the auxiliary protein gene cluster required for its maturation.

2. The method of claim 1, wherein, The high density fed-batch fermentation controls the specific growth rate μ to be 0.1-0.2 h -1 .

3. The method according to claim 1 or 2, characterized in that, The bacterial cell density target threshold is OD 600 = 40-150.

4. The method of claim 1, wherein, The switching to anaerobic conditions includes: Stop introducing air and switch to introducing sterile inert gas; reduce the stirring speed to 150-250 rpm; reduce dissolved oxygen to 0% within 5-60 minutes; adjust the incubation temperature to 20-25℃.

5. The method of claim 1, wherein, The inducing agent is isopropyl-β-D-thiogalactoside (IPTG), with a final concentration of 0.1-1.0 mM; the metal ion cofactors include nickel salts and iron salts.

6. The method according to claim 5, characterized in that, The nickel salt is a water-soluble nickel salt, selected from nickel sulfate, nickel chloride, or a combination thereof, with a final addition concentration of 10-100 μM; the iron salt is a water-soluble ferrous salt or ferric salt, selected from ferrous sulfate, ferric ammonium citrate, ferric chloride, or a combination thereof, with a final addition concentration of 0.1-0.5 mM.

7. The method according to claim 1, characterized in that, The anaerobic induction phase has a culture time of 6-24 h.

8. Active nickel-iron hydrogenase produced by the method according to any one of claims 1-7.

9. The active nickel-iron hydrogenase according to claim 8, characterized in that, The nickel-iron hydrogenase has biological activity in catalyzing the oxidation of hydrogen, with a specific activity ≥20 U / mg and a volumetric yield ≥900 mg / L.

10. The use of the method of any one of claims 1-7 in the production of an oxygen-sensitive metalloenzyme, wherein the oxygen-sensitive metalloenzyme comprises [FeFe]-hydrogenase, nitrogenase, or carbon monoxide dehydrogenase.

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