Hydrogenase expression vector combination, recombinant bacteria, enzyme composition and application thereof

Abstract

The application provides an expression vector combination of hydrogenase, a recombinant bacterium, an enzyme composition and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. Through the combined use of formic acid dehydrogenase, formaldehyde dehydrogenase, formaldehyde enzyme and glycerol dehydrogenase, one-carbon compounds are reduced to the value-added chemical glycerol, and the rapid regeneration and efficient supply of energy cofactor NADH are realized by the biological enzyme (hydrogenase SH) method, so that the reduction equivalent is provided for the cascade catalytic reaction. In the enzyme cascade catalytic process, formic acid and methanol have strong inhibitory effect on the enzyme, the condensation of formaldehyde catalyzed by the formaldehyde enzyme can obtain neutral dihydroxy acetone, and the reaction is irreversible, and the fixation of CO2 is effectively pulled. The method guides the construction of an efficient carbon dioxide multi-enzyme immobilization system, and provides a new method and new idea for realizing carbon dioxide fixation and biological energy synthesis.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and enzyme engineering technology, specifically relating to the combination of expression vectors for hydrogenase, recombinant bacteria, enzyme compositions and their applications. Background Technology

[0002] The use of fossil fuels not only leads to resource and energy shortages but also results in the massive emission of greenhouse gases such as CO2 into the atmosphere, causing numerous environmental problems. Utilizing renewable resources such as solar energy, hydrogen energy, and biomass to produce energy and chemicals is an important direction for sustainable development. If CO2 can be rapidly and selectively converted into energy or chemicals through biotechnology, it will be of great significance for reducing greenhouse gas emissions, solving global environmental problems, and achieving a sustainable society.

[0003] Natural enzyme catalysts can promote the conversion of CO2 into various fuels and chemicals. In vitro multi-enzyme catalysis can effectively avoid side reactions, resulting in high process efficiency, strong specificity, and significant advantages. For example, formate dehydrogenase, formaldehyde dehydrogenase, and alcohol dehydrogenase from Clostridium yunnanensis, Burkholderia polyphaga, and Saccharomyces cerevisiae, respectively, can be used as catalysts for a cascade reaction. NADH is used as the electron donor, and phosphite dehydrogenase (PTDH) is used to regenerate cofactors, converting CO2 into methanol. Compared with traditional methods, enzymatic methods have advantages such as high selectivity, high yield, rapid reaction at room temperature and low pressure, and low energy consumption.

[0004] It is generally believed that the insertion of the bimetallic core and the binding of a carbonyl group and two cyanide ligands require the synergistic action of hypABCDEF. HypC associates with the N-terminal region of HoxH, and this complex is maintained until the insertion at the [NiFe] site is complete (see [(Reference: Johannes Schiffels; Thorsten Selmer. A flexible toolbox to study protein-assisted metalloenzyme assembly in vitro.[J]. Biotechnology and bioengineering. 2015, Vol. 112(No. 11): 2360-2372.)]). To coordinate the iron group, the scaffold protein HypD forms a complex with a second HypC. The transfer of the two cyanide ligands to Fe-HypCD requires the latter to interact with the cyanide-modified HypE. The modification of HypE involves interaction with HypF, which in an ATP-dependent step transfers the carbamate group from the carbamate phosphate and transfers it to HypE. Nickel transfer to HoxH is mediated by a complex between HypA and HypB, requiring GTP, followed by the dissociation of HypC from the complex. HoxW cleaves 24 residues from HoxH by recognizing the incorporated nickel ions and specific binding sites, then internalizes the [NiFe] site next to the HoxY contact site to provide activity.

[0005] Enzymatic CO2 conversion typically relies on an external electron supply from redox equivalents, most commonly nicotinamide adenine dinucleotide (NADH). This natural redox equivalent mediates the transfer of two electrons and one proton, enabling biocatalytic redox conversion. However, providing a stoichiometric amount of NADH for synthetic purposes is not cost-effective. Therefore, cofactor regeneration has become highly significant, and several cofactor regeneration methods have been developed to date, including chemical, electrochemical, and photoenzymatic methods. Currently, enzymatic regeneration of energy cofactors such as NADH uses enzymes, which is relatively slow and difficult to achieve rapid synthesis, thus becoming a significant factor limiting multi-enzyme synthesis. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a combination of expression vectors for hydrogenase, recombinant bacteria, enzyme composition and their applications. The hydrogenase expressed by the combination of expression vectors for hydrogenase of the present invention can be used for rapid regeneration and efficient supply of NADH.

[0007] This invention provides a combination of expression vectors for hydrogenase, including a first expression vector and a second expression vector;

[0008] The first expression vector uses pM1 as a backbone plasmid and inserts hoxW gene expression cassette, hoxN1 gene expression cassette and hypA2B2C1D1E1F2X gene expression cassette.

[0009] The second expression vector uses pSH6 as a backbone plasmid and inserts the hoxFUYHI2 gene expression cassette.

[0010] The present invention also provides a recombinant bacterium containing the expression vector combination described above.

[0011] This invention also provides a method for preparing hydrogenase, comprising the following steps:

[0012] The recombinant bacteria described in the above scheme were inoculated into M9 optimized medium and induced to express, resulting in an induced bacterial suspension; the bacterial suspension was then lysed to obtain a lysate containing hydrogenase;

[0013] The M9 optimized medium is based on M9 medium and also includes the following components at the following concentrations: 50 mg / L kanamycin, 50 mg / L spectinomycin, 0.5%–2% glucose, 0.5%–6% glycerol, 0.5%–6% lactose, 1–200 μM NiCl2, 1–200 μM ferric ammonium citrate, 1–200 μM riboflavin, and 1%–10% LB medium.

[0014] The present invention also provides the application of the expression vector combination described above, the recombinant bacteria, or the hydrogenase prepared by the preparation method in the synthesis of NADH.

[0015] The present invention also provides an enzyme composition comprising hydrogenase, formaldehyde dehydrogenase, formaldehyde enzyme, and glycerol dehydrogenase prepared by the preparation method described above.

[0016] Preferably, it also includes formate dehydrogenase.

[0017] The present invention also provides the application of the enzyme composition described above in the augmentation of one-carbon compounds, wherein the augmentation of one-carbon compounds preferably includes the synthesis of glycerol using one-carbon compounds.

[0018] The present invention also provides a method for synthesizing glycerol using a one-carbon compound, comprising the following steps:

[0019] A one-carbon compound, MgSO4, TPP, NAD+, and the enzyme composition described in the above scheme are mixed and subjected to an enzyme cascade catalytic reaction to obtain glycerol.

[0020] Preferably, the reaction system of the enzyme cascade catalytic reaction uses a 100mM sodium phosphate buffer as the reaction system buffer, and the reaction system contains the following components at the following concentrations: 1-5mM MgSO4, 0.1-1mM TPP, 0.6-1.2mM NAD+, 0.25-1U / ml FateDH, 0.1-0.5U / ml FaldDH, 0.05-0.5mg / ml GLDH, 0.05-0.5mg / ml FLS-M3, and 0.2-0.8mg / ml SH hydrogenase; the pH of the sodium phosphate buffer is 7.5-8.0.

[0021] Preferably, the reaction temperature of the enzyme cascade catalytic reaction is 15-26°C, H2 is introduced during the reaction at a flow rate of 20-200 ml / min, and the reaction time is 15 min-12 h.

[0022] This invention provides a combination of expression vectors for hydrogenase. In this invention, hydrogenase maturation is based on a dual-plasmid co-expression system, wherein the dual plasmids include the pM1-hoxN1 plasmid and the pSH6 plasmid. The maturation process of [NiFe]-hydrogenase is highly complex, and its catalytic center is assembled through the synergistic action of a specific set of accessory proteins. This invention achieves efficient cofactor regeneration and efficient supply by modifying the natural [NiFe]-hydrogenase SH to regenerate NADH, guiding the construction of an efficient carbon dioxide multi-enzyme immobilization system with high atom economy, providing a new method and approach for carbon dioxide fixation and bioenergy synthesis. Attached Figure Description

[0023] Figure 1 A schematic diagram of the in vitro multi-enzyme cascade catalytic synthesis of glycerol from carbon dioxide coupled with NADH regeneration;

[0024] Figure 2 The image shows the SDS-PAGE gel electrophoresis results of GLDH, FLS-M3, and SH.

[0025] Figure 3 The graph shows the detection results of the enzymatic assay for glycerol standards.

[0026] Figure 4 The graph shows the results of the enzymatic determination of glycerol concentration in the sample solution.

[0027] Figure 5 For the glycerol standard curve;

[0028] Figure 6 This is a schematic diagram of the structure of the first expression vector and the second expression vector. Detailed Implementation

[0029] This invention provides a combination of expression vectors for hydrogenase, including a first expression vector and a second expression vector;

[0030] The first expression vector uses pM1 as a backbone plasmid and inserts hoxW gene expression cassette, hoxN1 gene expression cassette and hypA2B2C1D1E1F2X gene expression cassette.

[0031] The second expression vector uses pSH6 as a backbone plasmid and inserts the hoxFUYHI2 gene expression cassette.

[0032] The present invention does not impose any special restrictions on the insertion order of the gene expression cassette of the first expression vector or the second expression vector.

[0033] [NiFe]-hydrogenase SH consists of six subunits, divided into two distinct modules: the hydrogenase portion HoxYH, accompanied by a NADH and an oxidoreductase (cardiac flavin) module, called HoxFU. The hexamer structure is completed by the homodimer of HoxI, a small subunit that binds to the cardiac flavin module and serves as the NADPH activation site for the oxidase.

[0034] The active sites of [NiFe]-hydrogenases all possess bimetallic [NiFe]-cofactors, which have a carbon monoxide moiety and two cyanide moieties linked to iron ions. These diatomic ligands regulate the redox state of nickel ions to promote H2 activation.

[0035] The biosynthesis of the NiFe(CN)₂CO catalytic cluster and its insertion into the large subunit of [NiFe]-hydrogenase require six hyp genes, hypA–hypF. HypA and GTPase HypB, along with the peptidyl prolyl cis / trans isomerase SlyD, transfer nickel ions; HypC, D, E, and F proteins are involved in Fe(CN)₂CO biosynthesis and are essential for the maturation of [NiFe]-hydrogenase. HypC is a small iron- and CO₂-binding protein, while the FeS cluster protein HypD serves as a scaffold for the partial assembly of Fe(CN)₂CO. The cyanide ligands of the active site clusters are derived from carbamoyl phosphate and are associated with HypE and HypF. HypF utilizes carbamoyl phosphate as a substrate, converting it to carbamate, then to carbamoyl adenosine, and finally transferring the carbamoyl functional group to the C-terminal cysteine ​​residue of HypE. HypX is involved in the maturation of hydrogenases in aerobic organisms and has been shown to be crucial for the oxygen tolerance of these enzymes. HoxW cleaves a small peptide from the C-terminal domain of an apolipoprotein subunit by recognizing incorporated nickel ions and specific binding sites, thus allowing the formation of oligomerases, which are essential and specific for hydrogenases. HoxN1, a high-affinity nickel permease derived from Cn, enables nickel uptake under aerobic conditions. Co-expression of hoxN1 also significantly improves maturation efficiency.

[0036] In this invention, each target gene is placed under the control of a separate T7 promoter and terminator, allowing for co-expression across a set of compatible plasmids. This ensures adequate expression levels for each target gene and addresses the issue of SH inactivity caused by IPTG-induced gene expression. Lactose triggers the expression of the T7 RNA polymerase gene after glucose consumption. This strategy, introduced by Studier, is known as "auto-induction." Growth and expression parameters can be controlled by modifying the culture medium formulation and physical conditions. *Cupriavidus necator* possesses three oxygen-tolerant [NiFe]-hydrogenases: membrane-bound hydrogenase (MBH), cytoplasmic bidirectional hydrogenase (SH), and a third hydrogenase (RH) mediating the transcriptional regulation of SH and MBH genes. The gene sequence includes a favorable combination of structural genes for MBH, SH, and RH, as well as auxiliary genes for MBH, RH, and SH maturation. This is because the SH-related operon contains partially repetitive hyp genes, and the hyp portion has overlapping functions in MBH and SH-specific maturation. Because the Cupriavidus necator gene stops translation after the translation of the first gene when applying this combination strategy, this "individual promoter strategy" is adopted.

[0037] In this invention, the structural schematic diagrams of the first expression vector and the second expression vector are as follows: Figure 6 As shown.

[0038] The present invention also provides a recombinant bacterium containing the expression vector combination described above. In the present invention, the original bacterium of the recombinant bacterium preferably includes Escherichia coli; the Escherichia coli is preferably E. coli BL21(DE3).

[0039] This invention also provides a method for preparing hydrogenase, comprising the following steps:

[0040] The recombinant bacteria described above were inoculated into M9 optimized medium and induced to express, resulting in an induced bacterial suspension. The bacterial suspension was then lysed to obtain a lysate containing hydrogenase.

[0041] In this invention, the M9 optimized medium is based on M9 medium, and preferably also includes the following components at the following concentrations: 50 mg / L kanamycin, 50 mg / L spectinomycin, 0.5%–2% glucose, 0.5%–6% glycerol, 0.5%–6% lactose, 1–200 μM NiCl2, 1–200 μM ferric ammonium citrate, 1–200 μM riboflavin, and 1%–10% LB medium.

[0042] In this invention, the M9 optimized medium is an M9 medium supplemented with necessary substances to enhance the maturation of functional hydrogenases. Compared to conventional media, the M9 optimized medium induces expression through lactose, addressing the problem that isopropyl-β-D-thiogalactopyranoside (IPTG)-induced gene expression fails to produce active functional hydrogenase SH. Ferric ammonium citrate and nickel chloride, as the addition of iron and nickel, are also crucial for the maturation of [NiFe]-hydrogenase SH. Riboflavin supplementation, as a precursor to FMN, can increase SH activity. SH maturation requires aerobic conditions, and HoxW plays a role in protecting the [NiFe] site by transferring electrons to remove oxygen or reducing FMN-a cofactors during its incorporation in the final stage of maturation. 5% (v / v) LB medium, as a substitute for the amino acid mixture, increases SH activity.

[0043] Before inducing expression in the recombinant bacteria, the present invention preferably further includes: inoculating the recombinant bacteria into the above-mentioned lactose-free M9 optimized medium and culturing it to obtain a culture; the culturing temperature is preferably 25-28℃; the culturing speed is preferably 200-220 rpm; the culturing time is preferably 12-16 h; the OD of the culture is... 578 <1.

[0044] After obtaining the culture, the present invention centrifuges the culture and collects the precipitated cells; the centrifugal force is preferably 2000g; the centrifugation time is preferably 1min.

[0045] After obtaining the precipitated cells, the present invention resuspends the precipitated cells in the M9 optimized culture medium for induced expression; the initial OD of the precipitated cells 578 The preferred concentration is 0.1–0.2; the preferred induction temperature is 22–28°C; the preferred induction rotation speed is 220 rpm; the preferred induction time is 12–16 h; after induction, the expression is further induced in the dark for 24 h by adding 200 μg / L anhydrous tetracycline (AHT). This invention induces the expression of the hoxW gene by adding anhydrous tetracycline.

[0046] The present invention also provides the application of the expression vector combination described above, the recombinant bacteria, or the hydrogenase prepared by the preparation method in the synthesis of NADH.

[0047] The present invention also provides an enzyme composition comprising hydrogenase, formaldehyde dehydrogenase, formaldehyde enzyme, and glycerol dehydrogenase prepared by the preparation method described above.

[0048] In this invention, the enzyme composition preferably further includes formate dehydrogenase.

[0049] The present invention does not have any special restrictions on the source of formaldehyde dehydrogenase, formaldehyde enzyme, glycerol dehydrogenase and formate dehydrogenase, and conventional methods in the art can be used.

[0050] In this invention, the amino acid sequence of the formaldehyde dehydrogenase is as shown in SEQ ID NO.1, specifically:

[0051] MSGNRGVVYLGSGKVEVQKIDYPKMQDPRGKKIEHGVILKVVSTNICGSDQHMVRGRTTAQVGLVLGHEITGEVIEKGRDVENLQIGDLVSVPFNVACGR CRSCKEMHTGVCLTVNPARAGGAYGYVDMGDWTGGQAEYLLVPYADFNLLKLPDRDKAMEKIRDLTCLSDILPTGYHGAVTAGVGPGSTVYVAGAGPVGL AAAASARLLGAAVVIVGDLNPARLAHAKAQGFEIADSLDTPLHEQIAALLGEPEVDCAVDAVGFEARGHGHEGAKHEAPATVLNSLMQVTRVAGKIGIPGLYVTEDPGAVDAAAKIGSLSIRFGLGWAKSHSFHTGQTPVMKYNRALMQAIMWDRINIAEVVGVQVISLDDAPRGYGEFDAGVPKKFVIDPHKTFSAA.

[0052] In this invention, the amino acid sequence of the formate dehydrogenase is as shown in SEQ ID NO.2, specifically:

[0053] MKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETSELDKHIPDADIIITTPFHPAYITKERLDKAKNLKLVVVAGVGSDHIDLDYINQTGKKISVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIGAGRIGYRVLER LLPFNPKELLYYDYQALPKEAEEKVGARRVENIEELVAQADIVTVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFTGKFDYRPQDIILLNGEYVTKAYGKHDKK.

[0054] In this invention, the formaldehyde enzyme FLS-M3 is preferably an artificial enzyme designed starting from benzaldehyde lyase (BAL), and its amino acid sequence is shown in SEQ ID NO.3, specifically:

[0055] MAMITGGELVVRTLIKAGVEHLFGLHGLHIDTIFQACLDHDVPIIDTRHEAAAGHAAEGYARAGAKLGVALVTAGGGFTNAVTPIANARLDRTPVLFLTGSGALRDDETNTLQAGIDQVAMAAPITKWAHRVMATEHIPRLVMQAIRAALSAPRGPVLLDLPWDILMNQIDEDSVIIPDLVLSAHGAHPDPADLDQALALLRKAERPVIVLGSEASRTARKTALSAFVAATGVPVFADYEGLSMLSGLPDAMRGGLVQNLYSFAKADAAPDLVLMLGARFGLHTGHGSGQLIPHSAQVIQVDPDACELGRLQGIALGIVADVGGTIEALAQATAQDAAWPDRGDWCAKVTDLAQERYASIAAKSSSEHALHPFHASQVIAKHVDAGVTVVADGGLTYLWLSEVMSRVKPGGFLCHGYLNSMGVGFGTALGAQVADLEAGRRTILVTGDGSVGYSIGEFDTLVRKQLPLIVIIMNNQSWGWTLHFQQLAVGPNRVTGTRLENGSYHGVAAAFGADGYHVDSVESFSAALAQALAHNRPACINVAVALDPIPPEELILIGMDPFAGSTENLYFQSGALEHHHHH。

[0056] In the present invention, the amino acid sequence of the glycerol dehydrogenase is as shown in SEQ ID NO.4, specifically:

[0057] LKVIQSPAKYLQGPDAAVLFGQYAKNLAESFFVIADDFVMKLAGEKVVNGLQSHDIRCHAERFNGECSHAEINRLMAILQKQGCRGVVGIGGGKTLDTAKAIGYYQKLPVVVIPTIASTDAPTSALSVIYTEAGEFEEYLIYPKNPDMVVMDTAIIAKAPVRLLVSGMGDALSTWFEAKA CYDARATSMAGGQSTEAALSLARLCYDTLLAEGEKARLAAQAGVVTEALERIIEANTYLSGIGFESSGLAAAHAIHNGFTILEECHHLYHGEKVAFGTLAQLVLQNSPMDEIETVLGFCQRAGLPVTLAQMGVKEGIDEKIAAVAKATCAEGETIHNMPFAVTPESVHAAILTADLLGQQ.

[0058] This invention achieves efficient cofactor regeneration by using a combination of formate dehydrogenase (FateDH), formaldehyde dehydrogenase (FaldDH), formaldehyde enzyme (FLS), and glycerol dehydrogenase (GLDH) to reduce one-carbon compounds, including CO2, to glycerol, and by regenerating NADH through a naturally modified hydrogenase (SH).

[0059] The present invention also provides the application of the enzyme composition described above in the augmentation of one-carbon compounds, wherein the augmentation of one-carbon compounds preferably includes the synthesis of glycerol using one-carbon compounds.

[0060] The present invention also provides a method for synthesizing glycerol using a one-carbon compound, comprising the following steps:

[0061] A one-carbon compound, MgSO4, TPP, NAD+, and the enzyme composition described in the above scheme are mixed and subjected to an enzyme cascade catalytic reaction to obtain glycerol.

[0062] In this invention, when the one-carbon compound is CO2, CO2 bubbles are bubbled into the reaction system solution of the enzyme cascade catalytic reaction until the solution is saturated.

[0063] In this invention, when the one-carbon compound is NaHCO3 and / or KHCO3, NaHCO3 and / or KHCO3 is used as the reaction substrate; the concentration of NaHCO3 and / or KHCO3 in the reaction system of the enzyme cascade catalytic reaction is preferably 10 to 100 mM.

[0064] In this invention, the reaction system for the enzyme cascade catalytic reaction preferably uses a 100mM sodium phosphate buffer as the reaction system buffer. The reaction system contains the following components at the following concentrations: 1–5mM MgSO4, 0.1–1mM TPP, 0.6–1.2mM NAD+, 0.25–1U / ml FateDH, 0.1–0.5U / ml FaldDH, 0.05–0.5mg / ml GLDH, 0.05–0.5mg / ml FLS-M3, and 0.2–0.8mg / ml SH hydrogenase; the pH of the sodium phosphate buffer is preferably 7.5–8.0. The sodium phosphate buffer of this invention is obtained through a reasonable formulation, wherein TPP (thiamine pyrophosphate) assists formaldehyde enzyme FLS in catalyzing the carbon linkage reaction, exhibiting good stereoselectivity and enantioselectivity, and NAD+... + The reduction equivalent of NADH is obtained by regeneration through hydrogenase cascade reaction.

[0065] In this invention, the reaction temperature of the enzyme cascade catalytic reaction is preferably 15–26°C, H2 is introduced during the reaction at a flow rate of 20–200 ml / min, and the reaction time is preferably 15 min–12 h, more preferably 1–10 h, and even more preferably 5 h. This invention utilizes a multi-enzyme stepwise cascade catalytic method, combining formate dehydrogenase (FateDH) and formaldehyde dehydrogenase (FaldDH), formaldehyde enzyme (FLS), and glycerol dehydrogenase (GLDH), and using hydrogenase SH to achieve rapid regeneration and efficient supply of the energy cofactor NADH, guiding the construction of a highly efficient carbon dioxide multi-enzyme immobilization system, and providing new methods and ideas for carbon dioxide fixation and bioenergy synthesis.

[0066] Biological CO2 fixation and utilization can be carried out under mild conditions, offering advantages such as simplicity and high efficiency, and is of great significance in terms of environment, energy, and resources. As an important component of biological methods, multi-enzyme catalysis can effectively avoid side reactions, exhibiting high process efficiency and specificity, with significant advantages.

[0067] C1 compounds (CO2, HCOOH, HCHO, CH3OH, and CH4) can interconvert. In methanogenic bacteria, CH4 can be continuously oxidized to CO2, which is then assimilated via the CBB cycle. In methanogenic bacteria, CO2 can be continuously reduced to CH4. CO2 can also be continuously reduced to CH3OH or HCHO in vitro. Therefore, theoretically, any new assimilation pathway constructed based on any C1 compound can be used to assimilate other C1 compounds.

[0068] Formate dehydrogenase spontaneously catalyzes HCOO _ Oxidized to Enzymatic reactions utilize various natural cofactors as electron acceptors, such as nicotinamide adenine dinucleotide (NAD). + ) and nicotinamide adenine dinucleotide phosphate (NADP) + FateDH can also readily catalyze the reverse reaction to reduce CO2 to HCOO- under mild reaction conditions, i.e. NADH (or NADPH) is used as the electron donor. Formic acid can be converted into formaldehyde by formaldehyde dehydrogenase. After converting carbon dioxide into formaldehyde, formaldehyde enzyme condenses three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone. Formaldehyde enzyme is a ThDP-dependent enzyme designed by Siege and colleagues, starting with benzaldehyde lyase. This enzyme produces "active formaldehyde," which is then fused with formaldehyde, ultimately achieving the condensation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone.

[0069] Finally, glycerol dehydrogenase converts dihydroxyacetone to glycerol. This achieves highly selective production of high-value-added chemicals from C1 molecules. Throughout the enzyme cascade reaction system, hydrogenase participates in the continuous regeneration of the NADH cofactor, providing the reducing equivalent for the cascade catalytic reaction. The pathway of this enzyme cascade catalyzing the conversion of CO2 to glycerol is as follows: Figure 1 As shown.

[0070] In the presence of ATP, glycerol is phosphorylated to glycerol 3-phosphate by glycerol kinase, and then oxidized by glycerol phosphate oxidase to produce hydrogen peroxide. Under the action of peroxidase, the chromogenic substrate is converted to benzoquinone imine, and its optical density is proportional to the glycerol concentration. By using the glycerol phosphate oxidase method and the classic GPO-Trinder enzymatic reaction method, the glycerol content in liquid samples can be determined colorimetrically, allowing the detection of the glycerol product of the enzyme cascade reaction. Furthermore, the absorbance value can be used to preliminarily determine the yield of the glycerol product in this system.

[0071] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Specifically, the formate dehydrogenase in the enzyme cascade catalytic system was purchased from Macklin Biotechnology, and the formaldehyde dehydrogenase was purchased from Sigma-Aldrich.

[0072] Example 1: Construction and transformation of FLSM3-tac_pMAL-c4X recombinant plasmid

[0073] The gene and protein information of FLS-M3 was obtained from the PDB database (www.pdb.org) and handed over to GenScript Biotech Co., Ltd. in Nanjing, Jiangsu Province for plasmid synthesis. The plasmid was cloned into the Nde I and EcoR I of the pMAL-c4X expression vector with ampicillin (Amp) as the resistance, and shipped as a lyophilized plasmid.

[0074] The obtained 4 μg lyophilized plasmid was centrifuged to precipitate the plasmid. 40 μl of ddH2O was added to dissolve the plasmid, and after mixing, it was centrifuged again. The final plasmid concentration was 100 ng / μl.

[0075] The recombinant plasmid was transformed into BL21(DE3) competent cells and plated on LB agar plates containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 1.5 g / ml agar, and 50 mg / L ampicillin. The plates were incubated overnight at 37°C, and positive clones were screened.

[0076] Selected single colonies were cultured overnight at 37°C and 220 rpm in LB liquid medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 50 mg / L ampicillin.

[0077] Example 2: Induction, Expression, and Purification Method of FLS-M3 Enzyme

[0078] The shaken bacterial culture was inoculated into 200 ml of LB liquid medium at a 2% inoculation rate and cultured at 37°C. When the OD value reached 0.08, IPTG was added to a final concentration of 0.2 mM and induced at 16°C and 220 rpm for 16–18 h.

[0079] Collect bacterial cells by centrifugation at 6000 rpm and 4°C for 20 min, discarding the supernatant. Resuspend the cells in PBS buffer (pH 7.5) containing 1 mM β-mercaptoethanol (βME), 1 mM MgSO4, 0.2 mM TPP, and 10 mM imidazole. Turn on the cooling water circulator beforehand to lower the temperature to 4°C. Then, add the resuspended bacterial solution to a high-pressure cell disruptor at 1000 bar for 5–8 cycles until the solution becomes clear.

[0080] Centrifuge at 12000 rpm, 4℃ for 30 min to remove cell debris from the lysate. Remove particles from the supernatant and sterilize using a 0.45 μm Sartorius miniart filter. Purification of the FLS-M3 protease was performed using a Ni-NTA affinity chromatography column, followed by two washes with 20 mL of PBS buffer (pH 7.5) containing 1 mM βME, 1 mM MgSO4, 0.2 mM TPP, and 10 mM imidazole.

[0081] Prepare gradient elution buffers: Lysis buffer contains 10 mM imidazole, 1 mM βME, 1 mM MgSO4, and 0.2 mM TPP in a pH 7.5 PBS buffer; Wash buffer contains 30 mM imidazole, 1 mM βME, 1 mM MgSO4, and 0.2 mM TPP in a pH 7.5 PBS buffer; Elution buffer contains 300 mM imidazole, 1 mM βME, 1 mM MgSO4, and 0.2 mM TPP in a pH 7.5 PBS buffer. Pour the protein supernatant from the filter membrane into a nickel column and perform gradient elution with the above buffers until Coomassie Brilliant Blue no longer turns blue. Collect the last 300 mM imidazole eluent, and then use a concentration tube with a molecular weight 20% lower than the protein to be concentrated for ultrafiltration to concentrate the protein.

[0082] A 30 kDa ultrafiltration tube was used, centrifuged at 3500 g, and centrifuged at 4 °C. Imidazole was removed by ultrafiltration with PBS buffer (pH 7.5) containing 1 mM βME, 1 mM MgSO4, and 0.2 mM TPP. When the remaining protein volume reached 1 ml after centrifugation, the protein was transferred to 1.5 mL centrifuge tubes and stored at -80 °C for later use. The precipitate, supernatant, and purified protein were collected and analyzed by SDS-PAGE gel electrophoresis. The results are as follows: Figure 2 As shown in the diagram. Lane 3 is for the maker; lane 4 is for the FLS-M3 supernatant; and lane 5 is for the FLS-M3 purification.

[0083] Example 3: Transformation, induced expression and purification of glycerol dehydrogenase (GLDH)

[0084] The gene and protein information of glycerol dehydrogenase (GLDH) was obtained from the PDB database (www.pdb.org) and handed over to GenScript Biotech Co., Ltd. in Nanjing, Jiangsu Province for plasmid synthesis. The plasmid was cloned into the Nde I and HindIII of the Pet-28a+ expression vector, with ampicillin (Amp) as the resistance, and shipped as a lyophilized plasmid.

[0085] The obtained 4 μg lyophilized plasmid was then centrifuged to precipitate the plasmid. 40 μl of ddH2O was added to dissolve the plasmid, and after mixing, it was centrifuged again. The final plasmid concentration was 100 ng / μl.

[0086] The recombinant plasmid was transformed into BL21(DE3) competent cells and plated on LB agar plates containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 1.5 g / ml agar, and 50 mg / L kanamycin. The plates were incubated overnight at 37°C, and positive clones were screened.

[0087] Selected single colonies were cultured overnight at 37°C and 220 rpm in LB liquid medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 50 mg / L kanamycin.

[0088] The shaken bacterial culture was inoculated into 200 ml of LB liquid medium at a 2% inoculation rate and cultured at 37°C. When the OD value reached 0.6-0.8, IPTG was added to a final concentration of 0.2 mM and induced at 22°C and 220 rpm for 16 h.

[0089] Collect bacterial cells by centrifugation at 6000 rpm and 4°C for 15 min, discarding the supernatant. Resuspend the cells in Tris-HCl buffer (pH 7.8) containing 300 mM NaCl and 10 mM imidazole. Turn on the cooling water circulator beforehand to lower the temperature to 4°C. Then, add the resuspended bacterial solution to a high-pressure cell disruptor at 1000 bar for 5–8 cycles until the solution becomes clear.

[0090] Centrifuge at 12000 rpm, 4℃ for 30 min to remove cell debris from the lysate. Remove particles from the supernatant and sterilize using a 0.45 μm Sartorius miniart filter. GLDH protease purification was performed using a Ni-NTA affinity chromatography column, followed by two washes with 20 mL of 50 mM Tris-HCl buffer (pH 7.8–8.0) containing 10 mM imidazole and 300 mM NaCl. Prepare gradient elution buffers: 1) Lysis buffer: 10mM imidazole, 300mM NaCl, 50mM Tris, pH=7.8; 2) Washbuffer: 30mM imidazole, 300mM NaCl, 50mM Tris, pH=7.8; 3) Elutionbuffer: 300mM imidazole, 300mM NaCl, 50mM Tris, pH=7.8; Pour the protein supernatant after filtration into a nickel column and perform gradient elution with the above buffers until Coomassie Brilliant Blue no longer turns blue. Collect the last 300mM imidazole concentration of elution buffer, and then use a concentration tube with a molecular weight 20% lower than that of the protein to be concentrated for ultrafiltration to concentrate the protein.

[0091] A 30 kDa ultrafiltration tube was used, centrifuged at 3500 g, and centrifuged at 4°C. Imidazole was removed by ultrafiltration with imidazole-free PBS buffer (pH 7.8). When the remaining protein volume after centrifugation reached 0.5–1 ml, the protein was transferred to 1.5 mL centrifuge tubes and stored at -80°C for later use. The precipitate, supernatant, and purified protein were collected and analyzed by SDS-PAGE gel electrophoresis. The results are as follows: Figure 2 As shown in the diagram. Lane 1 is for GLDH supernatant; lane 2 is for GLDH purification; and lane 3 is for the maker.

[0092] Example 4: Transformation and co-expression of hydrogenase (SH)

[0093] The hydrogenase SH expression vector places each target gene under the control of a separate promoter and terminator, allowing for co-expression across a set of compatible plasmids. SH generation requires five structural genes (hoxFUYHI) and eight auxiliary genes (hypC1D1E1A2B2F2X and hoxW), as well as hoxN1, a high-affinity nickel transporter from Cn. The mature SH gene was divided into two modules, resulting in pM1 and pSH6 expression plasmids. The designed expression vectors were synthesized by GenScript Biotech Co., Ltd. in Nanjing, Jiangsu Province, and shipped as lyophilized plasmids.

[0094] To co-express pM1 and pSH6, the pM1 plasmid was first transformed into *E. coli* BL21(DE3) and plated on LB agar plates containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 1.5 g / ml agar, and 50 mg / L kanamycin. The plates were incubated overnight at 37°C, and positive clones were screened. Single colonies obtained from the plates were picked and transferred to 4 ml LB agar plates containing 50 mg / L kanamycin and incubated for 12 h. Then, they were inoculated into 250 ml flasks (50 ml LB agar, 50 mg / L kanamycin) and incubated for 2 h until the OD value reached 0.5. The second plasmid, pSH6, was then transformed using electroporation. After culturing in large flasks until the OD value reaches 0.5, incubate on ice for 30 min, centrifuge at 4°C and 9000 rpm / min for 20 min, discard the supernatant, resuspend the precipitate in 100 ml of ice-cold 10% glycerol solution, incubate on ice for 15 min, centrifuge at 4°C and 9000 rpm / min for 20 min, discard the supernatant, resuspend the precipitate in 50 ml of ice-cold 10% glycerol solution, incubate on ice for 15 min, centrifuge at 4°C and 9000 rpm / min for 20 min, discard the supernatant, resuspend the precipitate in 10 ml of ice-cold 10% glycerol solution, incubate on ice for 15 min, centrifuge at 4°C and 9000 rpm / min for 20 min, discard the supernatant, and finally dilute with ice-cold 10% glycerol solution to obtain pM1 competent cells. Take 100 μl of pM1 competent cells and place them in pre-cooled electroporation cuvettes. Add 2 μl of pSH6 plasmid to each of the two electroporation cuvettes. After electroporation, quickly add 1 ml of LB medium and incubate at 37°C for 40 min. Then, spread them on plates containing 50 mg / L of double antibiotics (kanamycin and spectinomycin) and incubate overnight at 37°C.

[0095] Selected single colonies were cultured overnight at 37°C and 220 rpm in LB liquid medium containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and 50 mg / L kanamycin.

[0096] Prior to induction, the culture medium was prepared in the following lactose-free M9 optimized medium at 28°C and 220 rpm for 12 h. The expression was then analyzed at OD... 578 Transfer was performed before ≥1. Cells were pelleted by centrifugation at 2000g for 1 min and then resuspended in the following M9 optimized medium containing lactose (initial OD). 578 =0.1~0.2), induce for 12 h at 220 rpm, then add 200 μg / L anhydrous tetracycline (AHT) and induce in the dark for 24 h. Collect bacterial cells by centrifugation at 6000 rpm and 4℃ for 30 min, discarding the supernatant. Resuspend in PBS buffer at pH 7.0. Turn on the cooling water circulator beforehand to lower the temperature to 4℃, then add the resuspended bacterial solution to a high-pressure cell disruptor for disruption at 1000 bar, 5~8 cycles, until the bacterial solution becomes clear.

[0097] Centrifuge at 12000 rpm, 4℃ for 30 min to remove cell debris from the lysis buffer. Remove particles from the supernatant and sterilize by passing the solution through a 0.45 μm Sartorius miniart filter. Aliquot the enzyme solution into 1.5 mL centrifuge tubes and store at -80℃ for later use. Collect the supernatant after cell lysis and perform SDS-PAGE gel electrophoresis for analysis. Results are as follows: Figure 2 As shown. Lane 7 is maker; lane 8 is SH (M9 medium containing 5% LB); lane 9 is SH (M9 medium containing 50% LB);

[0098] M9 optimized culture medium is based on 6 g·L⁻¹ -1 Na2HPO4, 3g·L -1 KH2PO4, 1g·L -1 NH4Cl, 0.5 g·L - 1 M9 medium containing NaCl, 1 mM MgSO4, 0.1 mM CaCl2 and 1 mM thiamine, supplemented with 50 mg / L kanamycin, 50 mg / L spectinomycin, 1% (wt / vol) glucose, 2% (vol / vol) glycerol, 0.8% (wt / vol) lactose, 1 μM NiCl2, 100 μM ferric ammonium citrate, 1 μM riboflavin and 5% (vol / vol) LB medium.

[0099] Example 5: In vitro enzyme cascade reaction to generate glycerol

[0100] The enzyme cascade reaction system consisted of 6 ml of solution. Before the reaction, CO2 was bubbled into the solution for 20 minutes until saturation. The reaction system contained formate dehydrogenase (FateDH) at concentrations of 0.25 U / ml, 0.5 U / ml, and 1.0 U / ml, and formaldehyde dehydrogenase (FaldDH) at concentrations of 0.1 U / ml, 0.25 U / ml, and 0.5 U / ml. The concentrations of GLDH, FLS-M3, and other enzymes obtained from Examples 2, 3, and 4 were 10 μg / ml, 50 μg / ml, 100 μg / ml, and 500 μg / ml, respectively, as well as crude SH enzyme solution at concentrations of 0.2 mg / ml, 0.4 mg / ml, and 0.8 mg / ml. The reaction buffer was a 100 mM sodium phosphate buffer at pH 8.0. The reaction was carried out at 25°C with a pH of 8.0. Pure H2 generated by a hydrogen generator was introduced during the reaction at flow rates of 20 ml / min, 50 ml / min, 100 ml / min, and 200 ml / min. The reaction was terminated after 1–12 h in a 70°C metal bath for 20 min. The enzyme in the reaction system was removed by centrifugation at 13,000 rpm for 2 min, and the supernatant was used for product determination.

[0101] Example 6: In vitro enzyme cascade reaction to generate glycerol (using NaHCO3 as substrate)

[0102] The enzyme cascade reaction system consisted of 6 ml of substrate NaHCO3 at final concentrations of 10 mM, 20 mM, 50 mM, and 100 mM; formate dehydrogenase (FateDH) at concentrations of 0.25 U / ml, 0.5 U / ml, and 1.0 U / ml; and formaldehyde dehydrogenase (FaldDH) at concentrations of 0.1 U / ml, 0.25 U / ml, and 0.5 U / ml. The concentrations of GLDH, FLS-M3, and other enzymes obtained from Examples 2, 3, and 4 were 10 μg / ml, 50 μg / ml, 100 μg / ml, and 500 μg / ml, respectively, as well as crude SH enzyme solution at concentrations of 0.2 mg / ml, 0.4 mg / ml, and 0.8 mg / ml. The reaction buffer was a 100 mM sodium phosphate buffer at pH 8.0. The reaction was carried out at 25°C with a pH of 8.0. Pure H2 generated by a hydrogen generator was introduced during the reaction at flow rates of 20 ml / min, 50 ml / min, 100 ml / min, and 200 ml / min. The reaction was terminated after 12 hours in a 70°C metal bath for 20 minutes.

[0103] Example 7: In vitro enzyme cascade reaction to generate glycerol (using KHCO3 as substrate)

[0104] The enzyme cascade reaction system consisted of 6 ml of substrate KHCO3 at final concentrations of 10 mM, 20 mM, 50 mM, and 100 mM; formate dehydrogenase (FateDH) at concentrations of 0.25 U / ml, 0.5 U / ml, and 1.0 U / ml; and formaldehyde dehydrogenase (FaldDH) at concentrations of 0.1 U / ml, 0.25 U / ml, and 0.5 U / ml. The concentrations of GLDH, FLS-M3, and other enzymes obtained from Examples 2, 3, and 4 were 10 μg / ml, 50 μg / ml, 100 μg / ml, and 500 μg / ml, respectively, as well as crude SH enzyme solution at concentrations of 0.2 mg / ml, 0.4 mg / ml, and 0.8 mg / ml. The reaction buffer was a 100 mM sodium phosphate buffer at pH 8.0. The reaction was carried out at 25°C with a pH of 8.0. Pure H2 generated by a hydrogen generator was introduced during the reaction at flow rates of 20 ml / min, 50 ml / min, 100 ml / min, and 200 ml / min. The reaction was terminated after 12 hours in a 70°C metal bath for 20 minutes.

[0105] Example 8: Determination of the product glycerol

[0106] The product glycerol was determined using the liquid sample glycerol enzymatic assay kit from Beijing Pulilai Gene Technology Co., Ltd. The kit employs a combination of the glycerol phosphate oxidase method and the classic GPO-Trinder enzymatic reaction method, measuring the glycerol content in liquid samples colorimetrically. The detection linear range is 10–1200 μM. The principle is as follows: in the presence of ATP, glycerol is phosphorylated to glycerol 3-phosphate by glycerol kinase, and then oxidized by glycerol phosphate oxidase to produce hydrogen peroxide; under the action of peroxidase, the chromogenic substrate is converted to benzoquinone imine, and its optical density is directly proportional to the glycerol concentration.

[0107] According to the liquid sample glycerol enzymatic assay kit, the working solution was prepared by mixing R1:R2 in a 4:1 ratio and used immediately or stored at 4°C for less than 1 day; discard if discoloration occurs. 4 mM glycerol standard was serially diluted to 1000 μM, 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, 15.625 μM, and 7.8125 μM with 100 mM sodium phosphate buffer (pH 8.0, consistent with the sample buffer), as well as a 0 μM control reaction tube. The reaction was carried out in a 96-well plate with a system of 50 μL standard / sample solution + 150 μL working solution, reacted at 37°C for 15 min, and the absorbance was measured at 550 nm. Figure 3 These are the test results for glycerol standards. The concentrations of the glycerol standards, from left to right, are 1000 μM, 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, 15.625 μM, 7.8125 μM, and 0 μM. The absorbance values ​​at 550 nm are shown in Table 1.

[0108] Table 1. Glycerin Standards (Abs) 550

[0109]

[0110] Taking Example 5 as an example, the supernatant obtained after the termination reaction was diluted 10 times, and then, following the method of the liquid sample glycerol enzymatic assay kit, 50 μL of sample solution + 150 μL of working solution was added, and the reaction was carried out at 37°C for 15 min. The absorbance was then measured at 550 nm. Figure 4 This is the result of the glycerol concentration detection in the sample solution. The absorbance values ​​at 550 nm after a 10-fold dilution and the corresponding concentrations of glycerol produced are shown in Table 2.

[0111] Table 2 Sample Solution Abs 550

[0112]

[0113] A standard curve was prepared based on glycerol standards. Figure 5 It can be seen that in the 2ml system of Example 5, 10.977mM and 13.824mM of glycerol were produced after reacting for 1 and 2 hours, respectively.

[0114] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for synthesizing glycerol using a one-carbon compound, comprising the following steps: A one-carbon compound, MgSO4, TPP, NAD+, FateDH, FaldDH, GLDH, FLS-M3, and SH hydrogenase are mixed and subjected to an enzyme cascade catalytic reaction to obtain glycerol. The reaction system for the enzyme cascade catalytic reaction uses a 100mM sodium phosphate buffer as the reaction buffer. The reaction system contains the following components at the following concentrations: 1-5mM MgSO4, 0.1-1mM TPP, 0.6-1.2mM NAD+, 0.25-1U / ml FateDH, 0.1-0.5U / ml FaldDH, 0.05-0.5mg / ml GLDH, 0.05-0.5mg / ml FLS-M3, and 0.2-0.8mg / ml SH hydrogenase; the pH of the sodium phosphate buffer is 7.5-8.

0. The reaction temperature of the enzyme cascade catalytic reaction is 25℃, H2 is introduced during the reaction, the flow rate of H2 is 20~200ml / min, and the reaction time is 1~12h; The one-carbon compound is CO2, NaHCO3, or KHCO3; The preparation method of the SH hydrogenase includes the following steps: The recombinant bacteria were inoculated into M9 optimized medium and induced to express, resulting in induced bacterial suspensions. The bacterial culture was lysed to obtain a lysate containing hydrogenase; The M9 optimized medium is based on M9 medium and also includes the following components at the following concentrations: 50 mg / L kanamycin, 50 mg / L spectinomycin, 0.5%–2% glucose, 0.5%–6% glycerol, 0.5%–6% lactose, 1–200 μM NiCl2, 1–200 μM ferric ammonium citrate, 1–200 μM riboflavin, and 1%–10% LB medium. The recombinant bacteria contain a combination of expression vectors for hydrogenase; the combination of expression vectors for hydrogenase includes a first expression vector and a second expression vector; the first expression vector uses pM1 as a backbone plasmid and inserts a hoxW gene expression cassette, a hoxN1 gene expression cassette, and a hypoA2B2C1D1E1F2X gene expression cassette; the second expression vector uses pSH6 as a backbone plasmid and inserts a hoxFUYHI2 gene expression cassette.

2. The method according to claim 1, characterized in that, When the one-carbon compound is CO2, CO2 bubbles are bubbled into the reaction system solution of the enzyme cascade catalytic reaction until the solution is saturated; when the one-carbon compound is NaHCO3 or KHCO3, NaHCO3 or KHCO3 is used as the reaction substrate; the concentration of NaHCO3 or KHCO3 in the reaction system of the enzyme cascade catalytic reaction is 10~100mM.

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