Hydrogen dependent carbon dioxide reductase mutants for carbon dioxide reduction

Abstract

The invention discloses a hydrogen-dependent carbon dioxide reductase mutant for carbon dioxide reduction, and belongs to the field of enzyme engineering and carbon dioxide reduction. According to the present invention, the engineering modification is performed on the hydrogen-dependent carbon dioxide reductase (HDCR) from the thermoanaerobacter kivui so as to obtain the HDCR mutant, and the HDCR mutant can be used to synthesize the carbon dioxide reductase, such that the carbon dioxide reductase, the carbon dioxide reductase, the carbon dioxide reductase, the carbon dioxide reductase, the carbon dioxide reductase, the carbon dioxide reductase, and the carbon dioxide reductase, the carbon dioxide reductase, the carbon dioxide reductase and the carbon dioxide reductase are subjected to the engineering modification, compared with reported formate dehydrogenase, the HDCR mutant obtained by the invention does not depend on hydrogen, efficient reduction of carbon dioxide is realized through an enzyme method and an electrotransformation mode, and the generated product is single and easy to separate and purify. The HDCR mutant provided by the invention shows huge potential value in the carbon dioxide immobilization industry.

Description

Technical Field

[0001] The present invention relates to a hydrogen-dependent carbon dioxide reductase mutant for carbon dioxide reduction, specifically a hydrogen-dependent carbon dioxide reductase mutant with improved activity and its application, belonging to the fields of enzyme engineering and carbon dioxide reduction. Background Art

[0002] Carbon dioxide is the most abundant carbon source on Earth, with an annual anthropogenic emission of up to 33 billion tons. The resource utilization of carbon dioxide has very broad prospects. Developing efficient carbon fixation technologies to convert carbon dioxide into high-value chemicals, fuels, polymeric materials, etc. is of great significance for alleviating severe environmental problems and shortages of energy and resources (Liu Z, Wang K, Chen Y, et al. Third-generation biorefineries as the means to produce fuels and chemicals from CO2. Nature catalysis, 2020, 3(3): 274-288.).

[0003] Carbon dioxide is an inert gas in a fully oxidized state. The binding energy of the C=O bond is as high as 803 kJ / mol, and it has high thermodynamic stability, so it is difficult to be reduced and fixed. Currently, the methods for fixing carbon dioxide mainly include electrochemistry, photochemistry, and thermochemistry. However, the low catalytic selectivity of these methods results in non-single products, low energy utilization efficiency, and the catalysts used all rely on noble metals such as In, Sn, Bi, Hg, Cu, Zn, Ni, Cd, etc. (Wang G, Chen J, Ding Y, et al. Electrocatalysis for CO2 conversion: from fundamentals to value-added products. Chemical Society Reviews, 2021, 50(8): 4993-5061; CN116288469A), and the technical cost is difficult to control, making it difficult to be applied on a large scale in industry. Biological catalysts such as formate dehydrogenase have high catalytic selectivity and mild reaction conditions, but the reported catalytic efficiency of formate dehydrogenase is currently low (CN116676283A). The hydrogen-dependent carbon dioxide reductase from Thermoanaerobacter kivui, which is in the form of long fibers, can catalyze the reduction of carbon dioxide to formic acid with hydrogen as an electron donor, and its activity is as high as 930 U / mg, which is 10 times higher than the catalytic efficiency of the currently reported optimal chemical catalyst (Schwarz F M, Schuchmann K, Müller V. Hydrogenation of CO2 at ambient pressure catalyzed by a highly active thermostable biocatalyst. Biotechnology for biofuels, 2018, 11(1): 1-11). The enzyme molecule is composed of four subunit units, namely formate dehydrogenase subunit (FdhF), two ferredoxin subunits (HycB3, HycB4), and hydrogenase subunit (HydA2) (Dietrich HM, Righetto R D, Kumar A, et al. Membrane-anchored HDCR nanowires drive hydrogen-powered CO2 fixation. Nature, 2022, 607(7920): 823-830.). This enzyme needs to use hydrogen as a substrate during use, and its safety is difficult to guarantee, which causes great limitations in the practical application of this enzyme in carbon dioxide.

[0004] Therefore, it is urgent to modify hydrogen-dependent carbon dioxide reductase to obtain highly efficient formate dehydrogenase and promote the application of enzyme catalysis in industrial carbon dioxide fixation. Summary of the Invention

[0005] Problems to be Solved by the Invention

[0006] At present, most of the formate dehydrogenases available for carbon dioxide reduction are achieved by microbial whole-cell transformation, using hydrogen as a substrate to reduce carbon dioxide to produce formate, and the products are not single, which is not conducive to separation; at the same time, there are also some formate dehydrogenases that can achieve carbon dioxide conversion through electrochemistry without relying on hydrogen, but they are limited by low enzyme activity and low conversion efficiency. If the currently reported hydrogen-dependent carbon dioxide reductase (HDCR) with the highest enzyme activity is directly used for electrochemical conversion, since HDCR is a complex with a very large molecular weight, it is difficult to be immobilized on the electrode.

[0007] Solutions for Solving the Problems

[0008] According to the existing problems, the inventors found through research that the hydrogenase subunit contained in HDCR will use electrical energy to reduce protons to produce hydrogen, which will divert the electrical energy of the formate dehydrogenase subunit and reduce the energy efficiency. Therefore, in the present invention, HDCR is modified in order to be able to immobilize it on the electrode and at the same time maintain its high enzyme activity, and then be able to efficiently convert carbon dioxide into formate through electrochemistry.

[0009] [1]. Hydrogen-dependent carbon dioxide reductase mutant, wherein the hydrogen-dependent carbon dioxide reductase comprises a formate dehydrogenase subunit, HycB3 subunit, HycB4 subunit and HydA2 subunit;

[0010] Compared with the hydrogen-dependent carbon dioxide reductase, the mutant lacks one or more of the following (a1) to (a3):

[0011] (a1) HycB3 subunit or a part thereof,

[0012] (a2) HycB4 subunit or a part thereof,

[0013] (a3) HydA2 subunit or a part thereof;

[0014] Optionally, the hydrogen-dependent carbon dioxide reductase is derived from Thermoanaerobacter kivui.

[0015] [2]. The hydrogen-dependent carbon dioxide reductase mutant according to [1], wherein the hydrogen-dependent carbon dioxide reductase mutant is a mutant selected from any one of the following (m1) to (m4):

[0016] (m1) lacks the HydA2 subunit compared to the hydrogen-dependent carbon dioxide reductase;

[0017] (m2) lacks the HycB4 subunit and the HydA2 subunit compared to the hydrogen-dependent carbon dioxide reductase;

[0018] (m3) lacks the HycB3 subunit, the HycB4 subunit and the HydA2 subunit compared to the hydrogen-dependent carbon dioxide reductase;

[0019] (m4) lacks the HycB4 subunit, the HydA2 subunit and amino acid residues 159 - 184 of the HycB3 subunit compared to the hydrogen-dependent carbon dioxide reductase.

[0020] [3]. The hydrogen-dependent carbon dioxide reductase mutant according to [1] or [2], wherein,

[0021] the formate dehydrogenase subunit comprises the polypeptide shown in SEQ ID NO: 1, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID NO: 1;

[0022] the HycB3 subunit comprises the polypeptide shown in SEQ ID NO: 2, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID NO: 2;

[0023] the HycB4 subunit comprises the polypeptide shown in SEQ ID NO: 3, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID NO: 3;

[0024] the HydA2 subunit comprises the polypeptide shown in SEQ ID NO: 4, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID NO: 4.

[0025] [4]. An isolated polynucleotide, wherein the polynucleotide encodes the hydrogen-dependent carbon dioxide reductase mutant according to any one of [1] - [3].

[0026] [5]. A recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide according to [4].

[0027] [6]. A recombinant host cell, wherein the recombinant host cell comprises a hydrogen-dependent carbon dioxide reductase mutant as described in any one of [1] to [3], an isolated polynucleotide as described in [3], or a recombinant expression vector as described in [4];

[0028] Optionally, the recombinant host cell is derived from a microorganism of the genus Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus, or Corynebacterium;

[0029] Preferably, the recombinant host cell is derived from Escherichia coli.

[0030] [7]. A cell culture comprising the recombinant host cell as described in [6].

[0031] [8]. A product, which comprises a hydrogen-dependent carbon dioxide reductase mutant as described in any one of [1] to [3], a polynucleotide as described in [4], a recombinant expression vector as described in [5], a recombinant host cell as described in [6], or a cell culture as described in [7].

[0032] [9]. A method for catalyzing carbon dioxide reduction, which includes the step of using a hydrogen-dependent carbon dioxide reductase mutant as described in any one of [1] to [3], a polynucleotide as described in [4], a recombinant expression vector as described in [5], a recombinant host cell as described in [6], a cell culture as described in [7], or a product as described in [8];

[0033] Optionally, carbon dioxide is added to the system containing the hydrogen-dependent carbon dioxide reductase mutant.

[0034]

[10] . According to the method as described in [9], wherein the system containing the hydrogen-dependent carbon dioxide reductase mutant further comprises an electron donor;

[0035] Optionally, the products obtained by catalyzing carbon dioxide reduction include formic acid and / or formate.

[0036]

[11] . According to the method as described in [9], wherein the method catalyzes carbon dioxide reduction through a three-electrode system;

[0037] Preferably, the working electrode in the three - electrode system is a carbon paper containing the hydrogen - dependent carbon dioxide reductase mutant described in any one of [1] to [3].

[0038] Optionally, the products obtained by catalyzing carbon dioxide reduction include formic acid and / or formate.

[0039]

[12] . Use of the hydrogen - dependent carbon dioxide reductase mutant described in any one of [1] to [3], the polynucleotide described in [4], the recombinant expression vector described in [5], the recombinant host cell described in [6], the cell culture described in [7], or the product described in [8] in catalyzing carbon dioxide reduction;

[0040] Optionally, the products obtained by catalyzing carbon dioxide reduction include formic acid and / or formate.

[0041] Effects of the Invention

[0042] 1. The HDCR mutant provided by the present invention can achieve the conversion of carbon dioxide without relying on hydrogen.

[0043] 2. The HDCR mutant provided by the present invention can generate a single product through the enzyme conversion method, and the product is easy to separate and purify.

[0044] 3. The efficient reduction of carbon dioxide is achieved through the enzyme - catalysis method, solving the technical problem of carbon dioxide to C1 products. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Shows the specific enzyme activity of HDCR and mutants in catalyzing carbon dioxide reduction.

[0046] Figure 2 Shows that the electro - driven HDCR mutant catalyzes carbon dioxide reduction to produce formic acid. DETAILED DESCRIPTION OF THE INVENTION

[0047] The following will detail various exemplary embodiments, features and aspects of the present invention. The special word "exemplary" here means "serving as an example, embodiment or illustration". Any embodiment described as "exemplary" here does not have to be construed as superior or better than other embodiments.

[0048] In addition, for better illustration of the present invention, numerous specific details are given in the following detailed description. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In other instances, methods, means, equipment and steps well - known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

[0049] Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the systematic errors inevitable in industrial production.

[0050] In this specification, the meaning expressed by "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0051] In this specification, the "some specific / preferred embodiments", "some other specific / preferred embodiments", "embodiments", etc. mentioned refer to the specific elements (e.g., features, structures, properties, and / or characteristics) related to the embodiment, which are included in at least one of the embodiments described herein, and may or may not exist in other embodiments. Additionally, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0052] In this specification, the numerical range expressed by "numerical value A to numerical value B" refers to the range including the endpoint numerical values A and B.

[0053] In this specification, the term "hydrogen-dependent carbon dioxide reductase" (HDCR) refers to a complex enzyme in the form of nanofibers, which is composed of four subunits, including formate dehydrogenase subunit (FdhF), ferredoxin subunits HycB3 and HycB4, and [Fe-Fe] hydrogenase subunit (HydA2). Among them, HycB3 and HycB4 subunits aggregate with each other non-covalently to form the core of nanofibers, and then FdhF subunit and HydA2 are respectively bound to HycB3 and HycB4 of the nanofiber core to form the final complex. The four genes encoding the four subunits are arranged in the order of fdhf-hycb3-hycb4-hyda2 and a fdhd gene is connected downstream. This gene expresses the FdhD protein that assists in the maturation of the active center of FdhF. The expression genes of the four subunits and the fdhd gene together constitute the gene cluster fdhf-hycb3-hycb4-hyda2-fdhd of HDCR. In some embodiments of the present invention, the hydrogen-dependent carbon dioxide reductase is derived from Thermoanaerobacter kivui, which is composed of four subunits, including formate dehydrogenase subunit (FdhF), ferredoxin subunits HycB3 and HycB4, and [Fe-Fe] hydrogenase subunit (HydA2). The mutants of the hydrogen-dependent carbon dioxide reductase are: (1) truncating HydA2, or (2) truncating both HydA2 and HycB4 subunits simultaneously, or (3) truncating HycB3 subunit, HycB4 subunit and HydA2 subunit simultaneously, or (4) truncating the α-helix between positions 159-184 at the C-terminus of HycB3 on the basis of truncating both HydA2 and HycB4 subunits. The mutants are mainly composed of the remaining subunits to form heterotrimers, heterodimers or monomers.

[0054] In this specification, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other operation, such as conjugation with a labeled component).

[0055] In this specification, the term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a natural source and has not been intentionally modified by humans in the laboratory is naturally occurring. As used in the present invention, "naturally occurring" and "wild-type" are synonyms.

[0056] As used herein, the term "mutant" refers to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to a "wild-type" or "comparative" polynucleotide or polypeptide, where a substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid, a deletion refers to removing a nucleotide or amino acid occupying a position, and an insertion refers to adding a nucleotide or amino acid adjacent to and immediately following a nucleotide or amino acid occupying a position. In some specific embodiments of the present invention, there may be a deletion of a subunit or a partial subunit compared to the wild-type hydrogen-dependent carbon dioxide reductase, such as deletion of the HycB3 subunit or a part thereof, the HycB4 subunit or a part thereof, or the HydA2 subunit or a part thereof in the wild-type hydrogen-dependent carbon dioxide reductase, so as to retain the carbon dioxide reduction performance of the mutant and further improve the catalytic efficiency of the mutant.

[0057] As used herein, the term "amino acid mutation" or "nucleotide mutation" includes "substituting, repeating, deleting, or adding one or more amino acids or nucleotides". In the present invention, the term "mutation" refers to a change in a nucleotide sequence or an amino acid sequence. In a specific embodiment, the term "mutation" refers to "deletion".

[0058] In some embodiments, the "mutation" of the present invention may be selected from "conservative mutations". As used herein, the term "conservative mutation" refers to a mutation that can normally maintain the function of a protein. Representative examples of conservative mutations are conservative substitutions.

[0059] As used herein, the term "conservative substitution" involves replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0060] As used herein, the terms "sequence identity" or "percent identity" in the comparison of two nucleic acids or polypeptides refer to the situation where, when measured using a nucleotide or amino acid residue sequence comparison algorithm or by visual inspection, and compared and aligned with maximum correspondence, they are identical or have a specified percentage of the same sequence. That is, the identity of a nucleotide or amino acid sequence can be defined by the following ratio, which is the ratio of the number of identical nucleotides or amino acids when two or more nucleotide or amino acid sequences are aligned in a manner that maximizes the number of identical nucleotides or amino acids, and adding gaps as needed, to the total number of nucleotides or amino acids in the aligned portion.

[0061] As used herein, the term "codon optimization" means that the nucleotide sequence encoding a polypeptide has been configured to contain codons preferred by a host cell or organism, in order to improve gene expression in the host cell or organism and increase translation efficiency.

[0062] As used herein, the term "recombinant polynucleotide" refers to a polynucleotide having sequences that are not linked together in nature. A recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform a suitable host cell. A host cell containing a recombinant polynucleotide is referred to as a "recombinant host cell". Then the polynucleotide is expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide".

[0063] As used herein, the term "expression" includes any step involved in the production of a polypeptide, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0064] As used herein, the term "expression vector" refers to a DNA construct that contains a DNA sequence operably linked to appropriate control sequences so as to express a gene of interest in a suitable host. A "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired foreign polypeptide. A recombinant expression vector can include, for example, a transcriptional subunit containing i) a set of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and iii) appropriate transcriptional and translational start and stop sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector can be used, including plasmids, viruses, bacteriophages, and transposons.

[0065] As used herein, the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a mutant polypeptide of the present invention, a polynucleotide encoding a mutant polypeptide, or a recombinant expression vector. The term "recombinant host cell" encompasses a host cell that is different from the parental cell after introduction of a polynucleotide encoding a mutant polypeptide or a recombinant expression vector, and the recombinant host cell is specifically achieved by transformation. The host cell of the present invention can be a prokaryotic cell or a eukaryotic cell, as long as it is a cell capable of introducing a polynucleotide encoding a polypeptide having hydrogen-dependent carbon dioxide reductase activity of the present invention. In one embodiment, the host cell refers to a prokaryotic cell. Specifically, the host cell is derived from a microorganism of the genus Escherichia, Bacillus, or Corynebacterium. In some preferred embodiments, the host cell is derived from the genus Escherichia, more preferably Escherichia coli, including Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli Trans T1, Escherichia coli MC1061, etc.

[0066] As used herein, the term "cell culture" refers to a combination of cells and cell culture medium, wherein the cells are cultured in the cell culture medium outside the body.

[0067] The terms "transformation, transfection, transduction" in the present invention have the meanings commonly understood by those skilled in the art, that is, the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acid into a cell, and these methods include but are not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0068] The cultivation of the host cell of the present invention can be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, etc., and various culture conditions such as temperature, time, and pH value of the culture medium can be appropriately adjusted according to the actual situation.

[0069] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0070] The following provides a detailed description of the technical solution of the present invention :

[0071] <The first aspect>

[0072] In a first aspect of the present invention, a hydrogen-dependent carbon dioxide reductase mutant is provided. The hydrogen-dependent carbon dioxide reductase (i.e., the wild-type hydrogen-dependent carbon dioxide reductase) comprises a formate dehydrogenase subunit, a HycB3 subunit, a HycB4 subunit, and a HydA2 subunit;

[0073] Compared with the hydrogen-dependent carbon dioxide reductase, the mutant lacks one or more of the following (a1)-(a3):

[0074] (a1) The HycB3 subunit or a part thereof,

[0075] (a2) The HycB4 subunit or a part thereof,

[0076] (a3) The HydA2 subunit or a part thereof.

[0077] In some optional embodiments, the hydrogen-dependent carbon dioxide reductase is derived from Thermoanaerobacter kivui.

[0078] In some embodiments, the hydrogen-dependent carbon dioxide reductase mutant is a mutant selected from any one of the following (m1)-(m4):

[0079] (m1) Compared with the hydrogen-dependent carbon dioxide reductase, the HydA2 subunit is deleted;

[0080] (m2) Compared with the hydrogen-dependent carbon dioxide reductase, the HycB4 subunit and the HydA2 subunit are deleted;

[0081] (m3) Compared with the hydrogen-dependent carbon dioxide reductase, the HycB3 subunit, the HycB4 subunit, and the HydA2 subunit are deleted;

[0082] (m4) Compared with the hydrogen-dependent carbon dioxide reductase, the HycB4 subunit, the HydA2 subunit, and amino acid residues 159-184 of the HycB3 subunit are deleted.

[0083] In some specific embodiments, the formate dehydrogenase subunit comprises the polypeptide shown in SEQ ID NO:1, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with SEQ ID NO:1.

[0084] In some specific embodiments, the HycB3 subunit comprises the polypeptide shown in SEQ ID NO:2, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with SEQ ID NO:2.

[0085] In some specific embodiments, the HycB4 subunit comprises the polypeptide as shown in SEQ ID NO:3, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with SEQ ID NO:3.

[0086] In some specific embodiments, the HydA2 subunit comprises the polypeptide as shown in SEQ ID NO:4, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with SEQ ID NO:4.

[0087] In some more specific embodiments, the hydrogen-dependent carbon dioxide reductase mutant is any one of the following (n1) to (n4):

[0088] (n1) A mutant composed of the formate dehydrogenase subunit shown in SEQ ID NO:1, the HycB3 subunit shown in SEQ ID NO:2, and the HycB4 subunit shown in SEQ ID NO:3 (i.e., R1(HDCRΔHydA2) in the examples of the present invention);

[0089] (n2) A mutant composed of the formate dehydrogenase subunit shown in SEQ ID NO:1 and the HycB3 subunit shown in SEQ ID NO:2 (i.e., R2(HDCRΔHydA2ΔHycB4) in the examples of the present invention);

[0090] (n3) A mutant composed of the formate dehydrogenase subunit shown in SEQ ID NO:1 (i.e., R3(HDCRΔHydA2ΔHycB4ΔHycB3) in the examples of the present invention);

[0091] (n4) A mutant composed of the formate dehydrogenase subunit shown in SEQ ID NO:1 and the truncated HycB3 subunit shown in SEQ ID NO:5 (i.e., R4(HDCRΔHydA2ΔHycB4Δ159-184) in the examples of the present invention).

[0092] <Second aspect>

[0093] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes the hydrogen-dependent carbon dioxide reductase mutant as described in the first aspect of the present invention.

[0094] The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0095] The polynucleotides encoding the mutants of the present invention include: coding sequences encoding only the mutants; the coding sequences of the mutants and various additional coding sequences; the coding sequences of the mutants (and optional additional coding sequences) and non-coding sequences.

[0096] <The third aspect>

[0097] In the third aspect of the present invention, there is provided a recombinant expression vector, wherein the recombinant expression vector contains the polynucleotide described in the second aspect of the present invention.

[0098] <The fourth aspect>

[0099] In the fourth aspect of the present invention, there is provided a recombinant host cell, wherein the recombinant host cell contains the hydrogen-dependent carbon dioxide reductase mutant described in the first aspect of the present invention, the isolated polynucleotide described in the second aspect of the present invention, or the recombinant expression vector described in the third aspect of the present invention.

[0100] In some alternative embodiments, the recombinant host cell is derived from a microorganism of the genus Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus, or Corynebacterium.

[0101] In some preferred embodiments, the recombinant host cell is derived from Escherichia coli.

[0102] <The fifth aspect>

[0103] In the fifth aspect of the present invention, there is provided a cell culture containing the recombinant host cell described in the fourth aspect of the present invention.

[0104] <The sixth aspect>

[0105] In the sixth aspect of the present invention, there is provided a product comprising the hydrogen-dependent carbon dioxide reductase mutant described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, and the cell culture described in the fifth aspect.

[0106] In some alternative embodiments, the product may include enzyme preparations, bacterial agents, kits, etc.

[0107] <Seventh aspect>

[0108] In the seventh aspect of the present invention, there is provided a method for catalyzing carbon dioxide reduction, which includes the step of using the hydrogen-dependent carbon dioxide reductase mutant described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, the cell culture described in the fifth aspect, or the product described in the sixth aspect.

[0109] In some specific embodiments, carbon dioxide is added to the system containing the hydrogen-dependent carbon dioxide reductase mutant.

[0110] In some exemplary embodiments, the products obtained by catalyzing carbon dioxide reduction include formic acid and / or formate.

[0111] In some alternative embodiments, the system containing the hydrogen-dependent carbon dioxide reductase mutant further contains an electron donor; any reactant (electron donor) capable of providing electrons in an oxidation-reduction reaction is within the scope of the present invention; exemplarily, the electron donor may be methyl viologen.

[0112] In some other alternative embodiments, the method catalyzes carbon dioxide reduction through a three-electrode system.

[0113] The term "three-electrode system" refers to an electrochemical system composed of a working electrode, a counter electrode, and a reference electrode.

[0114] In some specific embodiments, the working electrode is a carbon paper modified with an enzyme solution; specifically, multi-walled carbon nanotubes are coated on the carbon paper, and the enzyme solution containing the hydrogen-dependent carbon dioxide reductase mutant described in the first aspect is coated on the multi-walled carbon nanotubes. More specifically, the concentration of the enzyme solution is not less than 6 U / mL, preferably 6 - 10 U / mL, and more preferably 6 - 7 U / mL.

[0115] In some exemplary embodiments, a platinum electrode is used as the counter electrode and a silver chloride electrode is used as the reference electrode.

[0116] <Eighth aspect>

[0117] In the eighth aspect of the present invention, there is provided the use of the hydrogen-dependent carbon dioxide reductase mutant described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, the cell culture described in the fifth aspect, or the product described in the sixth aspect in catalyzing carbon dioxide reduction.

[0118] In some alternative embodiments, the products obtained by catalyzing carbon dioxide reduction include formic acid and / or formate.

[0119] Examples

[0120] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the described examples are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements all fall within the protection scope of the present invention.

[0121] I. The materials and instruments used in the examples of the present invention are as follows:

[0122] Methyl viologen: purchased from Macklin, product number: M813276;

[0123] pTrcHisA vector: purchased from D&B / Dibo, product number: S201231A;

[0124] Escherichia coli expression strain MC1061: purchased from D&B / Dibo, product number: S209285A;

[0125] Carbon paper: Toray TGP-H-060;

[0126] Electrochemical workstation: purchased from Shanghai Junzhuo Technology, CHI600E;

[0127] II. Method for constructing the vector of the formate dehydrogenase mutant: Using the homologous recombination method, conventional primers were designed (see Table 1), and PCR amplification was carried out using the vector pTrcHisA-hdcr containing wild-type HDCR or the vector of the optimal mutant with partial subunit truncation as the template.

[0128] Table 1

[0129]

[0130] The PCR reaction system was all: 2×PrimerStar mixture 25 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL, and double-distilled water 22 μL was added.

[0131] The PCR reaction amplification conditions were all as follows: pre-denaturation at 98°C for 2 min; subsequently, 25 cycles were carried out with 30 s at 98°C, 15 s at 53°C, and 2 min at 72°C, and finally, incubation at 72°C for 10 min.

[0132] After digesting the PCR product with Dpn I at 37°C for 30 min, it was purified using a PCR product purification kit. The purified PCR product was subjected to homologous recombination ligation using the ClonExpress II One Step Cloning Kit (purchased from Vazyme). After mixing 7 μL of the PCR product evenly with 2 μL of CEⅡ buffer and 1 μL of the recombinase ExnaseⅡ, the reaction was carried out at 37°C for 30 min. The ligation product was directly transformed into competent Escherichia coli MC1061 cells, spread on an LB solid medium plate containing antibiotics, and cultured overnight. Single colonies were picked and cultured overnight in LB liquid medium, and then plasmids were extracted and verified by sequencing.

[0133] III. Expression of HDCR variants: Pick the MC1061 strain containing the mutant gene vector with correct sequencing and inoculate it into 5 mL of LB liquid medium. Culture overnight at 37°C and 200 rpm. Transfer the overnight culture to 1 L of anaerobic fermentation medium (based on LB medium, supplemented with MOPS (3-morpholinopropanesulfonic acid) at a final concentration of 100 mM, glucose at 5 g / L, fumaric acid at 25 mM, pH 7.4) and culture at 37°C in an anaerobic incubator until the OD 600 nm = 0.6 - 0.8, then add IPTG at a final concentration of 0.2 mM, Na2WO4 at 1 mM, FeSO4 at 2 mM, and L-cysteine at 0.3 g / L, and induce anaerobically at 18°C and 150 rpm, and continue to culture for 20 h. Collect the bacteria in an anaerobic glove box, resuspend the bacteria with a buffer (containing 100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM NaNO3), and lyse the bacterial cells by using lysozyme and repeated freezing and thawing methods. After centrifuging to collect the supernatant, it was purified by a nickel column, and the HDCR mutant protein was obtained after buffer replacement.

[0134] IV. Enzyme activity assay method for HDCR mutants: HDCR mutants can use reducing methyl viologen as an electron donor to catalyze the reduction of carbon dioxide to formic acid. In an anaerobic cuvette, add 2 mL of 100 mM HEPES / NaOH (pH 7.0) reaction solution, containing 20 mM NaHCO3, 3 mM methyl viologen, and 0.1 mM sodium dithionite. At a reaction temperature of 30 °C, add 10 μg of the enzyme, and immediately monitor the decrease in the absorbance value of reducing methyl viologen at 604 nm. The molar extinction coefficient ζ = 13.9 mmol / L / cm.

[0135] Definition of enzyme activity: It is expressed by the amount of reducing methyl viologen consumed per unit time at 604 nm. 1 unit of enzyme activity is 1 μmol / min.

[0136] Definition of specific enzyme activity: The number of enzyme activity units per unit weight (mg) of protein.

[0137] Example 1: Truncating HDCR to Obtain HDCR Truncated Mutants

[0138] HDCR is composed of four subunits, FdhF, HycB3, HycB4, and HydA2, to form a nanofiber structure. Among them, HycB3 and HycB4 interact with each other in a non-covalent linkage manner to form the main core of the nanofiber. FdhF and HydA2 then bind to the HycB3 and HycB4 subunits respectively in a non-covalent bond manner to form the final HDCR nanofiber-like complex. Based on this, primers were designed at the C-terminus of HycB4, HycB3, and FdhF to truncate the HydA2 subunit, and at the same time truncate the HydA2 subunit and the HycB4 subunit, and at the same time truncate the HydA2 subunit, the HycB4 subunit, and the HycB3 subunit, so that the remaining subunits interacted with each other to become mutants of heterotrimer, heterodimer, and monomer. The genes of HDCR exist in the form of a gene cluster, and the arrangement order of the genes is fdhf - hycb3 - hycb4 - hyda2 - fdhd. Among them, the fdhd gene expresses the FdhD protein, and the function of this protein is to assist the maturation of the active center of FdhF to express an active FdhF subunit. The expression gene of HDCR was codon-optimized (the obtained sequence is shown in SEQ ID NO:7). Using P1 and P3 as primers, the hdcr gene was amplified by PCR. The amplified hdcr gene was ligated by homologous recombination with the PCR product using P2 and P4 as primers and the pTrcHisA vector as a template to obtain the expression vector of the wild type pTrcHisA-hdcr (pTrcHisA-fdhf - hycb3 - hycb4 - hyda2 - fdhd). Using the vector pTrcHisA-hdcr as a template, with P5 and P6, P5 and P7, and P5 and P8 respectively, expression vectors expressing HDCR mutants R1, R2, and R3 were constructed. Through protein expression, mutant proteins were obtained, and the specific enzyme activity of the mutants catalyzing carbon dioxide reduction was measured. The specific enzyme activity of the wild type HDCR reducing carbon dioxide was 5.2 U / mg, and the specific enzyme activities of the obtained truncated mutants R1 (HDCRΔHydA2), R2 (HDCRΔHydA2ΔHycB4), and R3 (HDCRΔHydA2ΔHycB4ΔHycB3) catalyzing carbon dioxide reduction were 0.9 U / mg, 1.0 U / mg, and 0.2 U / mg( Figure 1)。The specific enzyme activities of all mutants were lower than that of the wild type. Mutant HDCRΔHydA2 contains the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the encoded nucleic acid sequence is shown in SEQ ID NO:8; mutant HDCRΔHydA2ΔHycB4 contains the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2, and the encoded nucleic acid sequence is shown in SEQ ID NO:9; mutant HDCRΔHydA2ΔHycB4ΔHycB3 contains the amino acid sequence shown in SEQ ID NO:1, and the encoded nucleic acid sequence is shown in SEQ ID NO:10; the optimal mutant obtained in this round is HDCRΔHydA2ΔHycB4.

[0139] Example 2: Site-Directed Mutagenesis of HDCR

[0140] (1) Construction and enzyme activity determination of mutant HDCRΔHydA2ΔHycB4Δ159-184

[0141] Using the expression vector of mutant R2 constructed in Example 1 as a template, PCR amplification was performed with primers P9 and P10 to construct an expression vector of mutant R4 (HDCRΔHydA2ΔHycB4Δ159-184) that expresses the deletion of amino acid residues 159-184 of HycB3. Through protein expression, mutant protein R4 was obtained, and the specific enzyme activity of mutant R4 was measured. The results showed that the specific enzyme activity of R4 for catalyzing carbon dioxide was 6.6 U / mg, which was 6.6 times higher than that of mutant R2 and 1.2 times higher than that of the wild type. Mutant R4 contains the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:5, and the encoded nucleic acid sequence is shown in SEQ ID NO:11.

[0142] (2) Construction and specific enzyme activity determination of mutant C83S (HDCRΔHydA2ΔHycB4HycB3-C83S)

[0143] Using the expression vector of mutant R4 constructed in Example 1 as a template, PCR amplification was performed with primers P11 and P12 to construct an expression vector of mutant C83S (HDCRΔHydA2ΔHycB4HycB3-C83S). The expression of mutant C83S was carried out according to the method described above. Mutant C83S contains the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:6, and the encoded nucleic acid sequence is shown in SEQ ID NO:12.

[0144] The enzyme activity was determined and the specific enzyme activity was calculated according to the enzyme activity assay method described above. The results of the specific enzyme activity assay showed that the activity of this mutant was completely lost, indicating that HycB3 is crucial for the activity of FdhF.

[0145] Example 3: Electro-Driven Reduction of Carbon Dioxide by HDCR Mutant R4

[0146] On a 1*1 cm 2 carbon paper, multi-walled carbon nanotubes were coated. The enzyme solution (1 mg / mL) of mutant R4 was dripped onto the multi-walled carbon nanotubes. The carbon paper modified with the enzyme solution was used as the working electrode, the platinum electrode was used as the counter electrode, and the silver chloride electrode was used as the reference electrode to establish a three-electrode system. A solution containing 100 mM HEPES / NaOH (pH 7.0) and 100 mM NaCl was used as the electrolyte, and carbon dioxide gas was introduced as the substrate. Detection was carried out by chronoamperometry, and the production amount of formic acid product was detected by high performance liquid chromatography. The chromatographic column used was Bio-Rad Aminex HPX-87H, 300×7.8 mm, the mobile phase was 5 mM H2SO4, the flow rate was 0.6 ml / min, the column oven temperature was 60 °C, and the collection time was 20 min. The formation of 14.4 mM formic acid product was detected at 20 h of the reaction, and the formation of 14.5 mM formic acid product was detected at 24 h ( Figure 2 ).

[0147] Some sequences used in the examples of the present invention:

[0148] SEQ ID NO:1 (FdhF formate dehydrogenase subunit α)

[0149] MKDGKQEKVLTTCPYCGTGCGLYLKVENEKIVGVEPDKLHPVNQGELCIKGYYGYKYVHDPRRLTSPLIKKNGKFVPVSWDEALNFIANGLKKIKSEYGSDAFAMFCSARATNEDNYAAQKFARAVIGINNVDHCARLCHAPTVAGLAMTLGSGAMTNSIPEISTYSDVIFIIGSNTAECHPLIAAHVIKAKERGAKLIVADPRMNAMVHKADIWLRVPSGYNIPLINGMIHIIIKEGLVKTDFVKNHAVGFEEMAKAVEKYTPEYVEELTGIPKKDLIKAARFYGQAQAAAILYSMGVTQFSHGTGNVVSLANLAVITGNLGRPGAGICPLRGQNNVQGACDVGALPNVLPGYLDVTKEQNRERFEKVWGVKLPSNIGLRVTEVPDAILNKRVRALYIFGENPIMSDPDSDHLRHALEHLDLLIVQDIFLTETARLAHVVLPAACWAEKDGTFTNTERRVQRVRKAVEAPGEAKPDWWIFSQIAERMGYTGMQYNNVQEIWDEVRKIVPEKFGGISYARLEKEKGLAWPCPTEDHTGTPILYLGGKFATPSGKAQMYPVIFYPNTCICDEGAEKQDFNHVIVGSIAELPDEEYPFTLTTGRRVYHYHTATMTRKSPVIDQIAPQELVEINPQDATRLGINDGDFLRVSTRRGYVATRAWVTERVPKGTIFMTFHYWEACCNELTNTASDAICCIPEFKVAAAKVEKISQVEAQAILKEKIEKYQVELEKDVANMLAKEKGGK*

[0150] SEQ ID NO:2 (HycB3 subunit)

[0151] MPNRFVIADPKRCLGCYTCIAACAFVHEEQGLQPFPRLYLTYTSEGIMPIQCRHCEDAPCAEVCPVEAIKKEGNAIIIDEKACIGCKTCLLACSFGAIDFSVQDSLEQSIFKDIKENLMQDQKTQQRIVAVKCDLCNFREEGPACVQFCPTKALKLVDGDEINKMVKNKRTVNVESLLSVYGTK*

[0152] SEQ ID NO:3 (HycB4 subunit)

[0153] MYQKVNCYSILFLKGVDKMKTQLNPFVVANPAKCIGCKACEVACFAVHNRNNHVGATVGTVSIPVIPRLHLIKTEHGTMPIQCRHCEDAPCANVCTVGAIKREGNAIVVDEKLCIGCKSCLLACPFGAIELLPQYEDGREVFQINLKEESESGLVQEPRIIAYKCDLCNDLGEPACVKACPENALTLVMPTEMKKARNKEAALSFLRVVR*

[0154] SEQ ID NO:4 (HydA2 subunit)

[0155] MSANKAIINIDQELCTGCRRCAEVCPVDAIEGEKGKPQKINTEVCVMCGQCVQKCSSYASYFDESITPRNVKLQERGMLDSVKEPLFAAYNLGYARQVKEALENPQLFKVVQCAPAIRVSIAEEFGLDLGDLTPGKLVAALRRLNFDRVYDTNFGADLTIIEEANELVKRIKEGKDLPMFTSCCPAWVKFAEQTYPELLKHISTCKSPQQMTGAIIKTYGAKINNVDPAKIFSVSVMPCTCKSYESDRPEMRSSGYKDVDLVITTRELAHLMKDKGIDFATLPDEEFDSPLGNYTGAATIFGNTGGVMEAALRTAYELITKKPIPNIDIEFVRGGEGIRTATVQVGELELKIAVVSGLKNVIPILEDIKKNKCDLHFVEVMTCPEGCISGGGQPKLLLEEYREVAYKKRKEALYKHDAELELRKSHENPAIKKLYEEFLGEPLGKQSHHLLHTKYTPRKKV*

[0156] SEQ ID NO:5 (Amino acid sequence of HycB3 subunit with the 159th to 184th amino acids at the C-terminus truncated)

[0157] MPNRFVIADPKRCLGCYTCIAACAFVHEEQGLQPFPRLYLTYTSEGIMPIQCRHCEDAPCAEVCPVEAIKKEGNAIIIDEKACIGCKTCLLACSFGAIDFSVQDSLEQSIFKDIKENLMQDQKTQQRIVAVKCDLCNFREEGPACVQFCPTKALKLVD*

[0158] SEQ ID NO:6 (Amino acid sequence of HycB3 subunit with cysteine at position 83 mutated to serine):

[0159] MPNRFVIADPKRCLGCYTCIAACAFVHEEQGLQPFPRLYLTYTSEGIMPIQCRHCEDAPCAEVCPVEAIKKEGNAIIIDEKASIGCKTCLLACSFGAIDFSVQDSLEQSIFKDIKENLMQDQKTQQRIVAVKCDLCNFREEGPACVQFCPTKALKLVD*

[0160] SEQ ID NO:7 (HDCR coding gene, the underlined part is the intron sequence, indicating that the coding sequences of different subunits are connected with these bases as intervals):

[0161] ATGCCGAACCGCTTTGTGATTGCGGATCCGAAACGCTGCCTGGGCTGCTATACCTGCATTGCGGCGTGCGCGTTTGTGCATGAAGAACAAGGCCTGCAGCCGTTTCCGCGCCTGTATCTGACCTATACGAGCGAAGGCATTATGCCGATTCAGTGCCGCCATTGCGAAGATGCGCCGTGCGCGGAAGTGTGCCCGGTGGAAGCGATTAAAAAAGAAGGCAACGCGATTATCATTGATGAAAAAGCGTGCATTGGCTGCAAAACCTGCCTGCTGGCGTGCAGCTTTGGCGCGATTGATTTTAGCGTGCAAGATAGCCTGGAACAGAGCATTTTTAAAGATATTAAAGAAAACCTGATGCAAGATCAGAAAACGCAGCAGCGCATTGTGGCGGTGAAATGCGATCTGTGCAACTTTCGCGAAGAAGGCCCGGCGTGCGTGCAGTTTTGCCCGACCAAAGCGCTGAAACTGGTGGATGGCGATGAAATTAACAAAATGGTGAAAAACAAACGCACCGTGAACGTGGAAAGCCTGCTGAGCGTGTATGGCACCAAATAA TGATTTTTGCTAATAT TTCTATCTATTTGAGATTTTCATGTATCAGAAAGTGAACTGCTATAGCATTCTGTTTCTGAAAGGCGTGGATAAAATGAAAACGCAGCTGAACCCGTTTGTGGTGGCGAACCCGGCGAAATGCATTGGCTGCAAAGCCTGCGAAGTGGCGTGCTTTGCGGTGCATAACCGCAACAACCATGTGGGCGCGACCGTGGGCACCGTGAGCATTCCGGTGATTCCGCGCCTGCATCTGATTAAAACCGAACATGGCACCATGCCGATTCAGTGCCGCCATTGCGAAGATGCGCCGTGCGCGAACGTGTGCACCGTGGGCGCGATTAAACGCGAAGGCAACGCGATTGTGGTGGATGAAAAACTGTGCATTGGCTGTAAGAGCTGCCTGCTGGCGTGCCCGTTTGGCGCGATTGAACTGCTGCCGCAGTATGAAGATGGCCGCGAAGTGTTTCAGATTAACCTGAAAGAAGAAAGCGAAAGCGGCCTGGTGCAAGAACCGCGCATTATTGCGTATAAATGCGATCTGTGCAACGATCTGGGCGAACCGGCGTGCGTGAAAGCGTGCCCGGAAAACGCGCTGACCCTGGTGATGCCGACCGAAATGAAAAAAGCGCGCAACAAAGAAGCGGCGCTGAGCTTTCTGCGCGTGGTGCGCTAA TTTATTAATTAATAATATTGAAAAGAAAGG GATGTGAACAAATCAAAAATTTTGTTGGTAGTGGGTTGTAAACAATCCTGTGAGAATA AGGCATGGCGTAAAGCTACCAAACTTCTCACGAAGTCTCAACCTTTGAAGGTGGGAGTAGTTCACGAAATCTCTTA AGGAGGAAGTGA atgGAAAGTTTTAAAAGTATCAACATCCTGAAATATAAAAACGGCCTGGTTGAAAGTTTTAGCGATAGCGTGATTGTGGAATATATTCTGAAACTGTATGTGAATGCGATTGAATTTGCGAGTTTCTTCTGCACCCCGCTGGCCCTGGATTGTCTGGTAGTTGGTTATCTGCAATCTCAGGGTATTATTGAAAAGAAGGAAGATATTAAACGTATCTTTATCGAAGAACGTGAAGGTAAAGCCCATGTGGAAATTCTGAAAAGTATTGATGCGAGTAGTGTTAAAAACCTGTTTATCATGAGCAGCGGTGAAAAGAATATTTGTTTTCATGATCAGCTGAAAATCGGTAAAACATGTTGTCATTGTAACTTTACCTGTAATCCGTGTATTTGTTTTCCGGAACAGCTGAATATTGGTCCGATTACAAGCAATATCAAATATAAACTGAAAGATATTATCAACCTGAGCGAACGTTTTAACAATGGTAGCGGCCTGTTTAAAATCACCGGCGGTGTTCATTCATGTGCAATCGCTGATGATAAAGATTTTATTATCTTTCATGAAGATATTGGTCGCCATAATGCCTTTGATAAAGCATTTGGTCAGGCACTGCTGGATGGTATTGATCTGCAAGATAAAGCTGTATTTACCAGCGGTCGTATTTCTGTTGAAATGCTGCTGAAAGCAGCAAAACGTAAAGTACCTGTTGTGGTTAGCATTAGCGCACCTACCGCACTGGCAGTTGAAGTAGGTCGCAAACTGAATATTACCATTGCTGGTTTTGCGCGTGGTGATCGTCTGAATATTTATAGCTGTCCGGAACGTTTTGTTTGGTAA

[0162] SEQ ID NO:8 (HDCRΔHydA2 encoding gene, the underlined part is the intron sequence, indicating that the coding sequences of different subunits are connected with this base as the interval):

[0163] ATGCCGAACCGCTTTGTGATTGCGGATCCGAAACGCTGCCTGGGCTGCTATACCTGCATTGCGGCGTGCGCGTTTGTGCATGAAGAACAAGGCCTGCAGCCGTTTCCGCGCCTGTATCTGACCTATACGAGCGAAGGCATTATGCCGATTCAGTGCCGCCATTGCGAAGATGCGCCGTGCGCGGAAGTGTGCCCGGTGGAAGCGATTAAAAAAGAAGGCAACGCGATTATCATTGATGAAAAAGCGTGCATTGGCTGCAAAACCTGCCTGCTGGCGTGCAGCTTTGGCGCGATTGATTTTAGCGTGCAAGATAGCCTGGAACAGAGCATTTTTAAAGATATTAAAGAAAACCTGATGCAAGATCAGAAAACGCAGCAGCGCATTGTGGCGGTGAAATGCGATCTGTGCAACTTTCGCGAAGAAGGCCCGGCGTGCGTGCAGTTTTGCCCGACCAAAGCGCTGAAACTGGTGGATGGCGATGAAATTAACAAAATGGTGAAAAACAAACGCACCGTGAACGTGGAAAGCCTGCTGAGCGTGTATGGCACCAAATAA TGATTTTTGCTAATAT TTCTATCTATTTGAGATTTTCATGTATCAGAAAGTGAACTGCTATAGCATTCTGTTTCTGAAAGGCGTGGATAAAATGAAAACGCAGCTGAACCCGTTTGTGGTGGCGAACCCGGCGAAATGCATTGGCTGCAAAGCCTGCGAAGTGGCGTGCTTTGCGGTGCATAACCGCAACAACCATGTGGGCGCGACCGTGGGCACCGTGAGCATTCCGGTGATTCCGCGCCTGCATCTGATTAAAACCGAACATGGCACCATGCCGATTCAGTGCCGCCATTGCGAAGATGCGCCGTGCGCGAACGTGTGCACCGTGGGCGCGATTAAACGCGAAGGCAACGCGATTGTGGTGGATGAAAAACTGTGCATTGGCTGTAAGAGCTGCCTGCTGGCGTGCCCGTTTGGCGCGATTGAACTGCTGCCGCAGTATGAAGATGGCCGCGAAGTGTTTCAGATTAACCTGAAAGAAGAAAGCGAAAGCGGCCTGGTGCAAGAACCGCGCATTATTGCGTATAAATGCGATCTGTGCAACGATCTGGGCGAACCGGCGTGCGTGAAAGCGTGCCCGGAAAACGCGCTGACCCTGGTGATGCCGACCGAAATGAAAAAAGCGCGCAACAAAGAAGCGGCGCTGAGCTTTCTGCGCGTGGTGCGCTAA AAATCAAAAATTTTGTTGGTAGTGGGTTGT AAACAATCCTGTGAGAATAAGGCATGGCGTAAAGCTACCAAACTTCTCACGAAGTCTCAACCTTTGAAGGTGGGAG TAGTTCACGAAATCTCTTAAGGAGGAAGTGAatgGAAAGTTTTAAAAGTATCAACATCCTGAAATATAAAAACGGCCTGGTTGAAAGTTTTAGCGATAGCGTGATTGTGGAATATATTCTGAAACTGTATGTGAATGCGATTGAATTTGCGAGTTTCTTCTGCACCCCGCTGGCCCTGGATTGTCTGGTAGTTGGTTATCTGCAATCTCAGGGTATTATTGAAAAGAAGGAAGATATTAAACGTATCTTTATCGAAGAACGTGAAGGTAAAGCCCATGTGGAAATTCTGAAAAGTATTGATGCGAGTAGTGTTAAAAACCTGTTTATCATGAGCAGCGGTGAAAAGAATATTTGTTTTCATGATCAGCTGAAAATCGGTAAAACATGTTGTCATTGTAACTTTACCTGTAATCCGTGTATTTGTTTTCCGGAACAGCTGAATATTGGTCCGATTACAAGCAATATCAAATATAAACTGAAAGATATTATCAACCTGAGCGAACGTTTTAACAATGGTAGCGGCCTGTTTAAAATCACCGGCGGTGTTCATTCATGTGCAATCGCTGATGATAAAGATTTTATTATCTTTCATGAAGATATTGGTCGCCATAATGCCTTTGATAAAGCATTTGGTCAGGCACTGCTGGATGGTATTGATCTGCAAGATAAAGCTGTATTTACCAGCGGTCGTATTTCTGTTGAAATGCTGCTGAAAGCAGCAAAACGTAAAGTACCTGTTGTGGTTAGCATTAGCGCACCTACCGCACTGGCAGTTGAAGTAGGTCGCAAACTGAATATTACCATTGCTGGTTTTGCGCGTGGTGATCGTCTGAATATTTATAGCTGTCCGGAACGTTTTGTTTGGTAA

[0164] SEQ ID NO:9 (HDCRΔHydA2ΔHycB4 encoding gene, the underlined part is the intron sequence, indicating that the coding sequences of different subunits are connected with this base as the interval):

[0165] ATGCCGAACCGCTTTGTGATTGCGGATCCGAAACGCTGCCTGGGCTGCTATACCTGCATTGCGGCGTGCGCGTTTGTGCATGAAGAACAAGGCCTGCAGCCGTTTCCGCGCCTGTATCTGACCTATACGAGCGAAGGCATTATGCCGATTCAGTGCCGCCATTGCGAAGATGCGCCGTGCGCGGAAGTGTGCCCGGTGGAAGCGATTAAAAAAGAAGGCAACGCGATTATCATTGATGAAAAAGCGTGCATTGGCTGCAAAACCTGCCTGCTGGCGTGCAGCTTTGGCGCGATTGATTTTAGCGTGCAAGATAGCCTGGAACAGAGCATTTTTAAAGATATTAAAGAAAACCTGATGCAAGATCAGAAAACGCAGCAGCGCATTGTGGCGGTGAAATGCGATCTGTGCAACTTTCGCGAAGAAGGCCCGGCGTGCGTGCAGTTTTGCCCGACCAAAGCGCTGAAACTGGTGGATGGCGATGAAATTAACAAAATGGTGAAAAACAAACGCACCGTGAACGTGGAAAGCCTGCTGAGCGTGTATGGCACCAAATAA AAATCAAAAATTTTGT TGGTAGTGGGTTGTAAACAATCCTGTGAGAATAAGGCATGGCGTAAAGCTACCAAACTTCTCACGAAGTCTCAACC TTTGAAGGTGGGAGTAGTTCACGAAATCTCTTAAGGAGGAAGTGAatgGAAAGTTTTAAAAGTATCAACATCCTGAAATATAAAAACGGCCTGGTTGAAAGTTTTAGCGATAGCGTGATTGTGGAATATATTCTGAAACTGTATGTGAATGCGATTGAATTTGCGAGTTTCTTCTGCACCCCGCTGGCCCTGGATTGTCTGGTAGTTGGTTATCTGCAATCTCAGGGTATTATTGAAAAGAAGGAAGATATTAAACGTATCTTTATCGAAGAACGTGAAGGTAAAGCCCATGTGGAAATTCTGAAAAGTATTGATGCGAGTAGTGTTAAAAACCTGTTTATCATGAGCAGCGGTGAAAAGAATATTTGTTTTCATGATCAGCTGAAAATCGGTAAAACATGTTGTCATTGTAACTTTACCTGTAATCCGTGTATTTGTTTTCCGGAACAGCTGAATATTGGTCCGATTACAAGCAATATCAAATATAAACTGAAAGATATTATCAACCTGAGCGAACGTTTTAACAATGGTAGCGGCCTGTTTAAAATCACCGGCGGTGTTCATTCATGTGCAATCGCTGATGATAAAGATTTTATTATCTTTCATGAAGATATTGGTCGCCATAATGCCTTTGATAAAGCATTTGGTCAGGCACTGCTGGATGGTATTGATCTGCAAGATAAAGCTGTATTTACCAGCGGTCGTATTTCTGTTGAAATGCTGCTGAAAGCAGCAAAACGTAAAGTACCTGTTGTGGTTAGCATTAGCGCACCTACCGCACTGGCAGTTGAAGTAGGTCGCAAACTGAATATTACCATTGCTGGTTTTGCGCGTGGTGATCGTCTGAATATTTATAGCTGTCCGGAACGTTTTGTTTGGTAA

[0166] SEQ ID NO:10 (HDCRΔHydA2ΔHycB4ΔHycB3 encoding gene, the underlined part is the intron sequence):

[0167] AAATCAAAAATTTTGTTGGTAGTGGGTTGTAAACAATCCT GTGAGAATAAGGCATGGCGTAAAGCTACCAAACTTCTCACGAAGTCTCAACCTTTGAAGGTGGGAGTAGTTCACGA AATCTCTTAAGGAGGAAGTGA atgGAAAGTTTTAAAAGTATCAACATCCTGAAATATAAAAACGGCCTGGTTGAAAGTTTTAGCGATAGCGTGATTGTGGAATATATTCTGAAACTGTATGTGAATGCGATTGAATTTGCGAGTTTCTTCTGCACCCCGCTGGCCCTGGATTGTCTGGTAGTTGGTTATCTGCAATCTCAGGGTATTATTGAAAAGAAGGAAGATATTAAACGTATCTTTATCGAAGAACGTGAAGGTAAAGCCCATGTGGAAATTCTGAAAAGTATTGATGCGAGTAGTGTTAAAAACCTGTTTATCATGAGCAGCGGTGAAAAGAATATTTGTTTTCATGATCAGCTGAAAATCGGTAAAACATGTTGTCATTGTAACTTTACCTGTAATCCGTGTATTTGTTTTCCGGAACAGCTGAATATTGGTCCGATTACAAGCAATATCAAATATAAACTGAAAGATATTATCAACCTGAGCGAACGTTTTAACAATGGTAGCGGCCTGTTTAAAATCACCGGCGGTGTTCATTCATGTGCAATCGCTGATGATAAAGATTTTATTATCTTTCATGAAGATATTGGTCGCCATAATGCCTTTGATAAAGCATTTGGTCAGGCACTGCTGGATGGTATTGATCTGCAAGATAAAGCTGTATTTACCAGCGGTCGTATTTCTGTTGAAATGCTGCTGAAAGCAGCAAAACGTAAAGTACCTGTTGTGGTTAGCATTAGCGCACCTACCGCACTGGCAGTTGAAGTAGGTCGCAAACTGAATATTACCATTGCTGGTTTTGCGCGTGGTGATCGTCTGAATATTTATAGCTGTCCGGAACGTTTTGTTTGGTAA

[0168] SEQ ID NO:11 (HDCRΔHydA2ΔHycB4Δ159 - 184 coding gene, the underlined part is the intron sequence, indicating that the coding sequences of different subunits are connected with this base as the interval):

[0169] ATGCCGAACCGCTTTGTGATTGCGGATCCGAAACGCTGCCTGGGCTGCTATACCTGCATTGCGGCGTGCGCGTTTGTGCATGAAGAACAAGGCCTGCAGCCGTTTCCGCGCCTGTATCTGACCTATACGAGCGAAGGCATTATGCCGATTCAGTGCCGCCATTGCGAAGATGCGCCGTGCGCGGAAGTGTGCCCGGTGGAAGCGATTAAAAAAGAAGGCAACGCGATTATCATTGATGAAAAAGCGAGCATTGGCTGCAAAACCTGCCTGCTGGCGTGCAGCTTTGGCGCGATTGATTTTAGCGTGCAAGATAGCCTGGAACAGAGCATTTTTAAAGATATTAAAGAAAACCTGATGCAAGATCAGAAAACGCAGCAGCGCATTGTGGCGGTGAAATGCGATCTGTGCAACTTTCGCGAAGAAGGCCCGGCGTGCGTGCAGTTTTGCCCGACCAAAGCGCTGAAACTGGTGGATTAA AAATCAAAAATTTTGTTG GTAGTGGGTTGTAAACAATCCTGTGAGAATAAGGCATGGCGTAAAGCTACCAAACTTCTCACGAAGTCTCAACCTT TGAAGGTGGGAGTAGTTCACGAAATCTCTTAAGGAGGAAGTGAatgGAAAGTTTTAAAAGTATCAACATCCTGAAATATAAAAACGGCCTGGTTGAAAGTTTTAGCGATAGCGTGATTGTGGAATATATTCTGAAACTGTATGTGAATGCGATTGAATTTGCGAGTTTCTTCTGCACCCCGCTGGCCCTGGATTGTCTGGTAGTTGGTTATCTGCAATCTCAGGGTATTATTGAAAAGAAGGAAGATATTAAACGTATCTTTATCGAAGAACGTGAAGGTAAAGCCCATGTGGAAATTCTGAAAAGTATTGATGCGAGTAGTGTTAAAAACCTGTTTATCATGAGCAGCGGTGAAAAGAATATTTGTTTTCATGATCAGCTGAAAATCGGTAAAACATGTTGTCATTGTAACTTTACCTGTAATCCGTGTATTTGTTTTCCGGAACAGCTGAATATTGGTCCGATTACAAGCAATATCAAATATAAACTGAAAGATATTATCAACCTGAGCGAACGTTTTAACAATGGTAGCGGCCTGTTTAAAATCACCGGCGGTGTTCATTCATGTGCAATCGCTGATGATAAAGATTTTATTATCTTTCATGAAGATATTGGTCGCCATAATGCCTTTGATAAAGCATTTGGTCAGGCACTGCTGGATGGTATTGATCTGCAAGATAAAGCTGTATTTACCAGCGGTCGTATTTCTGTTGAAATGCTGCTGAAAGCAGCAAAACGTAAAGTACCTGTTGTGGTTAGCATTAGCGCACCTACCGCACTGGCAGTTGAAGTAGGTCGCAAACTGAATATTACCATTGCTGGTTTTGCGCGTGGTGATCGTCTGAATATTTATAGCTGTCCGGAACGTTTTGTTTGGTAA

[0170] SEQ ID NO:12 (HDCRΔHydA2ΔHycB4HycB3-C83S encoding gene, the underlined part is the intron sequence, indicating that the coding sequences of different subunits are connected at this base interval):

[0171] ATGCCGAACCGCTTTGTGATTGCGGATCCGAAACGCTGCCTGGGCTGCTATACCTGCATTGCGGCGTGCGCGTTTGTGCATGAAGAACAAGGCCTGCAGCCGTTTCCGCGCCTGTATCTGACCTATACGAGCGAAGGCATTATGCCGATTCAGTGCCGCCATTGCGAAGATGCGCCGTGCGCGGAAGTGTGCCCGGTGGAAGCGATTAAAAAAGAAGGCAACGCGATTATCATTGATGAAAAAGCGAGCATTGGCTGCAAAACCTGCCTGCTGGCGTGCAGCTTTGGCGCGATTGATTTTAGCGTGCAAGATAGCCTGGAACAGAGCATTTTTAAAGATATTAAAGAAAACCTGATGCAAGATCAGAAAACGCAGCAGCGCATTGTGGCGGTGAAATGCGATCTGTGCAACTTTCGCGAAGAAGGCCCGGCGTGCGTGCAGTTTTGCCCGACCAAAGCGCTGAAACTGGTGGATGGCGATGAAATTAACAAAATGGTGAAAAACAAACGCACCGTGAACGTGGAAAGCCTGCTGAGCGTGTATGGCACCAAATAA AAATCAAAAATTTTGT TGGTAGTGGGTTGTAAACAATCCTGTGAGAATAAGGCATGGCGTAAAGCTACCAAACTTCTCACGAAGTCTCAACC TTTGAAGGTGGGAGTAGTTCACGAAATCTCTTAAGGAGGAAGTGAatgGAAAGTTTTAAAAGTATCAACATCCTGAAATATAAAAACGGCCTGGTTGAAAGTTTTAGCGATAGCGTGATTGTGGAATATATTCTGAAACTGTATGTGAATGCGATTGAATTTGCGAGTTTCTTCTGCACCCCGCTGGCCCTGGATTGTCTGGTAGTTGGTTATCTGCAATCTCAGGGTATTATTGAAAAGAAGGAAGATATTAAACGTATCTTTATCGAAGAACGTGAAGGTAAAGCCCATGTGGAAATTCTGAAAAGTATTGATGCGAGTAGTGTTAAAAACCTGTTTATCATGAGCAGCGGTGAAAAGAATATTTGTTTTCATGATCAGCTGAAAATCGGTAAAACATGTTGTCATTGTAACTTTACCTGTAATCCGTGTATTTGTTTTCCGGAACAGCTGAATATTGGTCCGATTACAAGCAATATCAAATATAAACTGAAAGATATTATCAACCTGAGCGAACGTTTTAACAATGGTAGCGGCCTGTTTAAAATCACCGGCGGTGTTCATTCATGTGCAATCGCTGATGATAAAGATTTTATTATCTTTCATGAAGATATTGGTCGCCATAATGCCTTTGATAAAGCATTTGGTCAGGCACTGCTGGATGGTATTGATCTGCAAGATAAAGCTGTATTTACCAGCGGTCGTATTTCTGTTGAAATGCTGCTGAAAGCAGCAAAACGTAAAGTACCTGTTGTGGTTAGCATTAGCGCACCTACCGCACTGGCAGTTGAAGTAGGTCGCAAACTGAATATTACCATTGCTGGTTTTGCGCGTGGTGATCGTCTGAATATTTATAGCTGTCCGGAACGTTTTGTTTGGTAA

[0172] It should be noted that although the technical solutions of the present invention are introduced by specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

[0173] The embodiments of the present invention have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, the practical application, or the improvement of technologies in the market, or to enable other ordinary skilled persons in the art to understand the embodiments disclosed herein.

Claims

1. A hydrogen-dependent carbon dioxide reductase mutant, wherein, The hydrogen-dependent carbon dioxide reductase comprises a formate dehydrogenase subunit, a HycB3 subunit, a HycB4 subunit, and a HydA2 subunit; Compared with the hydrogen-dependent carbon dioxide reductase, the mutant lacks one or more of the following (a1)-(a3): (a1) The HycB3 subunit or a part thereof, (a2) The HycB4 subunit or a part thereof, (a3) The HydA2 subunit or a part thereof; Optionally, the hydrogen-dependent carbon dioxide reductase is derived from Thermoanaerobacter kivui.

2. The hydrogen-dependent carbon dioxide reductase mutant according to claim 1, wherein, The hydrogen-dependent carbon dioxide reductase mutant is a mutant selected from any one of the following (m1)-(m4): (m1) Compared with the hydrogen-dependent carbon dioxide reductase, the HydA2 subunit is absent; (m2) Compared with the hydrogen-dependent carbon dioxide reductase, the HycB4 subunit and the HydA2 subunit are absent; (m3) Compared with the hydrogen-dependent carbon dioxide reductase, the HycB3 subunit, the HycB4 subunit, and the HydA2 subunit are absent; (m4) Compared with the hydrogen-dependent carbon dioxide reductase, the HycB4 subunit, the HydA2 subunit, and amino acid residues 159-184 of the HycB3 subunit are absent.

3. The hydrogen-dependent carbon dioxide reductase mutant according to claim 1 or 2, wherein the formate dehydrogenase subunit comprises the polypeptide shown in SEQ ID NO:1, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID NO:1; the HycB3 subunit comprises the polypeptide shown in SEQ ID NO:2, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID NO:2; the HycB4 subunit comprises the polypeptide shown in SEQ ID NO:3, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID NO:3; the HydA2 subunit comprises the polypeptide shown in SEQ ID NO:4, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with SEQ ID NO:

4.

4. An isolated polynucleotide, wherein, The polynucleotide encodes the hydrogen-dependent carbon dioxide reductase mutant according to any one of claims 1-3.

5. A recombinant expression vector, wherein, The recombinant expression vector comprises the polynucleotide according to claim 4.

6. A recombinant host cell, wherein, The recombinant host cell comprises the hydrogen-dependent carbon dioxide reductase mutant according to any one of claims 1-3, the isolated polynucleotide according to claim 3, or the recombinant expression vector according to claim 4; Optionally, the recombinant host cell is derived from a microorganism of the genus Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus or Corynebacterium; Preferably, the recombinant host cell is derived from Escherichia coli.

7. A cell culture comprising the recombinant host cell as claimed in claim 6.

8. A product comprising the hydrogen-dependent carbon dioxide reductase mutant as claimed in any one of claims 1 to 3, the polynucleotide as claimed in claim 4, the recombinant expression vector as claimed in claim 5, the recombinant host cell as claimed in claim 6 or the cell culture as claimed in claim 7.

9. A method for catalyzing carbon dioxide reduction, wherein, Comprising the steps of using the hydrogen-dependent carbon dioxide reductase mutant as claimed in any one of claims 1 to 3, the polynucleotide as claimed in claim 4, the recombinant expression vector as claimed in claim 5, the recombinant host cell as claimed in claim 6, the cell culture as claimed in claim 7, or the product as claimed in claim 8; Optionally, carbon dioxide is added to the system comprising the hydrogen-dependent carbon dioxide reductase mutant.

10. The method according to claim 9, wherein, The system comprising the hydrogen-dependent carbon dioxide reductase mutant further comprises an electron donor; Optionally, the products obtained by catalyzing the reduction of carbon dioxide include formic acid and / or formate.

11. The method according to claim 9, wherein, The method catalyzes the reduction of carbon dioxide through a three-electrode system; Preferably, the working electrode in the three-electrode system is a carbon paper containing the hydrogen-dependent carbon dioxide reductase mutant as claimed in any one of claims 1 to 3; Optionally, the products obtained by catalyzing the reduction of carbon dioxide include formic acid and / or formate.

12. Use of the hydrogen-dependent carbon dioxide reductase mutant as claimed in any one of claims 1 to 3, the polynucleotide as claimed in claim 4, the recombinant expression vector as claimed in claim 5, the recombinant host cell as claimed in claim 6, the cell culture as claimed in claim 7, or the product as claimed in claim 8 in catalyzing the reduction of carbon dioxide; Optionally, the products obtained by catalyzing the reduction of carbon dioxide include formic acid and / or formate.

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