Development of novel energy system and application of novel energy system in one-carbon substrate conversion

Abstract

The invention discloses development of a novel energy system and application of the novel energy system in one-carbon substrate conversion, and belongs to the technical field of bioengineering. The invention discloses a novel energy system synthesized by using intracellular metabolites nicotinamide and uridine triphosphate as substrates, a nicotinamide transferase mutant and a phosphite dehydrogenase mutant are fused, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase and NMN synthase are subjected to RBS optimization and then introduced into escherichia coli, and the novel energy system is obtained. Meanwhile, a formate dehydrogenase mutant, a formaldehyde dehydrogenase mutant, a malic enzyme mutant, glycolaldehyde synthase, acetyl phosphate synthase, acetyl phosphate transferase and ferredoxin oxidoreductase are introduced to obtain a strain with high malic acid yield, and the malic acid yield can reach 51.27 mM.

Description

Technical Field

[0001] The present invention relates to the development of a novel energy system and its application in the conversion of one-carbon substrates, belonging to the field of bioengineering technology. Background Art

[0002] One-carbon compounds (such as carbon dioxide and formic acid) are abundant renewable carbon resources. There is an urgent need to develop green conversion technologies to convert one-carbon compounds into high-value chemicals, such as proteins, esters, alcohols, ethers, and acids. Currently, there are mainly three methods for utilizing one-carbon compound resources: chemical method, physical method, and biological method. Among them, the utilization of one-carbon compounds by microorganisms is considered a green, efficient, and sustainable method due to the rapid growth of heterotrophic cells and high production intensity. In order to enable heterotrophic microorganisms to efficiently utilize one-carbon compounds, two key problems need to be solved: how to design an efficient one-carbon compound fixation pathway to convert one-carbon compounds into high-value chemicals? How to provide sufficient energy and driving force for the utilization of one-carbon compounds?

[0003] Complex steps and low carbon fixation efficiency limit the utilization of natural one-carbon compounds. Redesigning artificial one-carbon substrate utilization pathways in microorganisms provides a promising alternative. In terms of energy supply, the development of a novel energy system provides a new solution because the novel energy system can break the limitations of traditional energy systems, can accumulate intracellularly at high concentrations without interfering with the host metabolic network, and can promote the one-carbon utilization pathway in cells. However, there is still a lack of a novel energy system that can be efficiently synthesized and recycled. Therefore, establishing an efficient novel energy system to drive the intracellular novel one-carbon pathway is an effective strategy to improve the efficient utilization of one-carbon compounds by heterotrophic microorganisms. Summary of the Invention

[0004] The present invention provides a method for constructing a novel energy system and successfully uses it for Escherichia coli to synthesize malic acid using carbon dioxide and formic acid. The specific content is as follows:

[0005] (a) Designed a novel energy system named NUD / NUDH using intracellular metabolites nicotinamide (NMN) and uridine triphosphate (UTP) as substrates, where NUD is an oxidized energy molecule and NUDH is a reduced energy molecule;

[0006] (b) Protein engineering modification was performed on nicotinamide transferase Hs Nmnat with the amino acid sequence shown in SEQ ID NO.2 to obtain a mutant Hs Nmnat*, which can efficiently use NMN and UTP to synthesize the oxidized energy molecule NUD;

[0007] (c) The phosphite dehydrogenase PsPTDH with the amino acid sequence shown in SEQ ID NO. 4 was subjected to protein engineering to obtain a mutant PsPTDH*, which can efficiently convert NUD into the reduced energy molecule NUDH.

[0008] (d) HsNmnat* and PsPTDH* were introduced into Escherichia coli to achieve the intracellular synthesis of NUD and NUDH; through metabolic engineering transformation and optimization, the accumulation of intracellular UTP and NMN was promoted, and intracellular metabolic balance was achieved through the optimization of the RBS combination. Finally, the NUDH content reached 8.73 mM.

[0009] (e) Based on the mining of the MetaCyc and ATLAS databases, a FGPM carbon fixation pathway consisting of six steps was designed, which can convert formic acid and carbon dioxide into malic acid; the formate dehydrogenase (TsFDH), formaldehyde dehydrogenase (BmFADH), and malic enzyme (PcME) in the metabolic pathway were subjected to protein engineering to obtain mutants TsFDH*(V198M / T21E / V256L), BmFADH*(A193R / M219K / 237R / H268Y / V283L), and PcME*(V300Q / Q391Y / L394K) that can efficiently utilize NUDH.

[0010] (f) The FGPM pathway was constructed in the NUDH synthesis strain, achieving the efficient conversion of carbon dioxide and formic acid into malic acid, with a final yield of 50.61 mM.

[0011] In one embodiment, the novel energy system NUD / NUDH is synthesized using nicotinamide (NMN) and uridine triphosphate (UTP) as substrates.

[0012] In one embodiment, the mutation sites of the mutant HsNmnat* are L27K / V186L / T187K / D220E, and the mutation sites of PsPTDH* are L151Y / A176R / L208F.

[0013] In one embodiment, the FGPM carbon fixation pathway uses carbon dioxide and formic acid as common substrates, and this carbon fixation pathway can utilize the natural energy molecule NADH and the non-natural energy molecule NUDH as energy supply and driving force.

[0014] In one embodiment, the mutation sites of the mutant Ts FDH* that efficiently utilizes the non-natural energy molecule NUDH are V198M / T21E / V256L, the mutation sites of Bm FADH* are A193R / M219K / 237R / H268Y / V283L, and the mutation sites of PcME* are V300Q / Q391Y / L394K.

[0015] The present invention also provides a recombinant vector carrying the said gene.

[0016] In one embodiment, the recombinant vector uses p15A as the expression vector.

[0017] The present invention also provides the specific sequences used for RBS optimization.

[0018] The present invention also provides the gene sequences expressed by metabolic engineering enhancement.

[0019] In one embodiment, the microbial cell uses the prokaryote Escherichia coli Mg1655 as the host.

[0020] The present invention also provides a method for obtaining the above-mentioned method for efficiently synthesizing malic acid from carbon dioxide and formic acid using a novel energy system, and the method includes the following steps:

[0021] (1) Through iterative mutation and screening, obtain the enzymes Hs Nmnat* and Ps PTDH* that can utilize UTP and NMN to synthesize NUD and NUDH;

[0022] (2) Express Hs Nmnat* and Ps PTDH* in the p15A vector to obtain the vector p15A-1, transform p15A-1 into the host cell Escherichia coli Mg1655, select positive clones to obtain the strain K01, and detect the intracellular contents of NUD and NUDH.

[0023] (3) Introduce the genes Sl UMPK and Da NDK that strengthen the UTP synthesis module into the p15A-1 vector to obtain the expression vector p15A-2; similarly, continue to introduce the genes Vp NAMPT and Ft NadE that strengthen the NMN synthesis module to obtain p15A-3; transform p15A-3 into the host cell Escherichia coli Mg1655-01 (knock out the related genes pyrG, purR, pncC, UshA, and pnuC in the UTP and NMN catabolic pathways of E. coli Mg1655), select positive clones to obtain the strain K02, and obtain the strain K02 that efficiently synthesizes NUDH;

[0024] (4) Through iterative mutation and screening, obtain the enzymes Ts FDH*, BmFADH* and Pc ME* that can utilize NUDH as an energy molecule;

[0025] (5) The FGPM pathway genes Ts FDH*, Bm FADH* and Pp GALS gene are ligated to the vector pETAC to obtain the expression vector pETAC-1, and the Bs PTA, Da rPFOR and Pc ME* genes are ligated to the vector pTET to obtain the expression vector pTET-1;

[0026] (6) The plasmids pETAC-1 and pTET-1 are co-transformed into the strain K02 to obtain the strain K03 that can use NUDH as a driving force to convert carbon dioxide and formic acid into malic acid;

[0027] The present invention also provides a method for converting carbon dioxide and formic acid into malic acid.

[0028] In one embodiment, the final concentration of the microbial cells, or the above-mentioned recombinant Escherichia coli added to the reaction system is: 3.5 g / L.

[0029] In one embodiment, the carbon dioxide concentration is 300 mM and the formic acid concentration is 50 mM.

[0030] In one embodiment, the reaction conditions are: reacting at pH 7.0 and 37 °C for 35 h.

[0031] The present invention also provides the use of the above-mentioned genes, or the above-mentioned recombinant vectors, or the above-mentioned recombinant cells, or the above-mentioned recombinant Escherichia coli in the development of a novel energy system and the conversion of one-carbon substrates into malic acid.

[0032] The present invention provides a nicotinamide transferase mutant, the mutant is a nicotinamide transferase mutant obtained by site-directed mutagenesis based on the amino acid sequence of wild-type nicotinamide transferase, the amino acid sequence of the wild-type nicotinamide transferase is shown in SEQ ID NO.2, and the mutation modes are selected from: L27K, V186L, V186I, V186M, L27K / V186L, L27K / V186I, L27K / V186L / T187K, L27K / V186L / T187Y, L27K / V186L / T187K / D220E or L27K / V186L / T187K / D220Q.

[0033] The present invention provides a phosphite dehydrogenase mutant, which is a phosphite dehydrogenase mutant obtained by site-directed mutagenesis based on the amino acid sequence of wild-type phosphite dehydrogenase. The amino acid sequence of the wild-type phosphite dehydrogenase is shown as SEQ ID NO.4, and the mutagenesis modes include: L151Y, L151W, A176R, A207N, A207M, L151Y / A176R, L151W / A176R, A176R / A207N, A176R / A207M, L151Y / A176R / L208F or L151Y / A176R / N211I.

[0034] The present invention provides a formate dehydrogenase mutant, which is a formate dehydrogenase mutant obtained by site-directed mutagenesis based on the amino acid sequence of wild-type formate dehydrogenase. The amino acid sequence of the wild-type formate dehydrogenase is shown as SEQ ID NO.16, and the mutagenesis modes include: V198N, V198M, T221E, T221L, T221D, V256L, T221D / V256L, T221E / V256L, T221L / V256L, V198N / T221E / V256L or V198M / T221E / V256L.

[0035] The present invention provides a formaldehyde dehydrogenase mutant, which is a formaldehyde dehydrogenase mutant obtained by site-directed mutagenesis based on the amino acid sequence of wild-type formaldehyde dehydrogenase. The amino acid sequence of the wild-type formaldehyde dehydrogenase is shown as SEQ ID NO.17, and the mutagenesis modes include: L237R, A193R, A193M, V283L, L237Y, H268E, H268Y, A193R / V283L or A193R / M219K / L237R / H268Y / V283L.

[0036] The present invention provides a malic enzyme mutant, which is a malic enzyme mutant obtained by site-directed mutagenesis based on the amino acid sequence of wild-type malic enzyme. The amino acid sequence of the wild-type malic enzyme is shown as SEQ ID NO.18, and the mutagenesis modes include: V300Q, V300L, G390L, Q391Y, L394K, L394R, Q391Y / L394K, Q391Y / L394R, V300Q / Q391Y / L394K, V300L / Q391Y / L394K.

[0037] The present invention further provides a gene encoding the above-mentioned nicotinamide transferase mutant, phosphite dehydrogenase mutant, formate dehydrogenase mutant, formaldehyde dehydrogenase mutant and malic enzyme mutant, or a recombinant vector carrying the gene.

[0038] The present invention also provides a recombinant cell expressing a nicotinamide transferase mutant, a phosphite dehydrogenase mutant, a formate dehydrogenase mutant, a formaldehyde dehydrogenase mutant, and a malic enzyme mutant, or a gene or the recombinant vector carrying the above mutants.

[0039] In one embodiment, the recombinant cell uses bacteria or fungi as the expression host.

[0040] The present invention also provides a genetically engineered bacterium that expresses one or more of a nicotinamide transferase mutant, a phosphite dehydrogenase mutant, a formate dehydrogenase mutant, a formaldehyde dehydrogenase mutant, and a malic enzyme mutant.

[0041] In one embodiment, the genetically engineered bacterium uses E. coli MG1655(DE3), E. coli W3110(DE3), E. coli JM109, E. coli DH5α, E. coli Top10, or E. coli BL21(DE3) as the expression host.

[0042] In one embodiment, the expression vector is: pET28a.

[0043] The present invention also provides a genetically engineered bacterium that simultaneously overexpresses the above-mentioned nicotinamide transferase mutant and the above-mentioned phosphite dehydrogenase mutant.

[0044] In one embodiment, the genetically engineered bacterium uses E. coli MG1655(DE3), E. coli W3110(DE3), E. coli JM109, E. coli DH5α, E. coli Top10, or E. coli BL21(DE3) as the expression host.

[0045] The present invention also provides a genetically engineered bacterium that simultaneously overexpresses the above-mentioned nicotinamide transferase mutant and the above-mentioned phosphite dehydrogenase mutant, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with a linker peptide of GSGSGGSGSG, GSGSGGSGSGGSGSG, or GSGSGGSGSGGSGSGGSGSG to obtain a fusion enzyme.

[0046] In one embodiment, the genetically engineered bacterium uses E. coli MG1655(DE3), E. coli W3110(DE3), E. coli JM109, E. coli DH5α, E. coli Top10, or E. coli BL21(DE3) as the expression host.

[0047] The present invention also provides a genetically engineered bacterium, which is: overexpressing the above-mentioned nicotinamide transferase mutant, the above-mentioned phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, and NMN synthase at the same time, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with a linker peptide of GSGSGGSGSG, GSGSGGSGSGGSGSG or GSGSGGSGSGGSGSGGSGSG to obtain a fusion enzyme.

[0048] In one embodiment, the genetically engineered bacterium uses E. coli MG1655(DE3), E. coli W3110(DE3), E. coli JM109, E. coli DH5α, E. coli Top10 or E. coli BL21(DE3) as the expression host.

[0049] The present invention also provides a genetically engineered bacterium, which is: overexpressing the above-mentioned nicotinamide transferase mutant, the above-mentioned phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, and NMN synthase at the same time, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with a linker peptide of GSGSGGSGSG, GSGSGGSGSGGSGSG or GSGSGGSGSGGSGSGGSGSG to obtain a fusion enzyme;

[0050] At the same time, the original RBS in front of uridine kinase and nucleoside diphosphate kinase: AAAGAGGAGAAA is replaced with RBS-01, RBS-03 or RBS-05, the original RBS in front of nicotinamide phosphoribosyltransferase and NMN synthase: AAAGAGGAGAAA is replaced with RBS-01, RBS-03 or RBS-05, and the original RBS in front of the fusion enzyme: AAAGAGGAGAAA is replaced with RBS-01, RBS-03 or RBS-05 to obtain a strain.

[0051] RBS 0 -Hs Nmnat mutant - linker peptide - Ps PTDH mutant - RBS 0 -Sl UMPK - RBS 0 -Da NDK - RBS 0 -Vp NAMPT - RBS 0 -Ft NadE; Replace any one of the above RBSs with RBS-01, RBS-03 or RBS-05 0 。

[0052] In one embodiment, the genetically engineered bacterium uses E. coli MG1655(DE3), E. coli W3110(DE3), E. coli JM109, E. coli DH5α, E. coli Top10 or E. coli BL21(DE3) as the expression host.

[0053] The present invention also provides a genetically engineered bacterium, which overexpresses the above-mentioned nicotinamide transferase mutant, the above-mentioned phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, NMN synthase, the above-mentioned formate dehydrogenase mutant, the above-mentioned formaldehyde dehydrogenase mutant and the above-mentioned malic enzyme mutant at the same time, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GSGSGGSGSG, GSGSGGSGSGGSGSG, or GSGSGGSGSGGSGSGGSGSG as the linker peptide to obtain a fusion enzyme;

[0054] At the same time, the original RBS in front of uridine kinase and nucleoside diphosphate kinase is replaced with RBS-01, RBS-03 or RBS-05, the original RBS in front of nicotinamide phosphoribosyltransferase and NMN synthase is replaced with RBS-01, RBS-03 or RBS-05, and the original RBS in front of the fusion enzyme is replaced with RBS-01, RBS-03 or RBS-05 to obtain the strain;

[0055] RBS 0 -Hs Nmnat mutant - linker peptide - Ps PTDH mutant - RBS 0 -Sl UMPK - RBS 0 -Da NDK - RBS 0 -Vp NAMPT - RBS 0 -Ft NadE; Replace any one of the above RBSs with RBS-01, RBS-03 or RBS-05 0 。

[0056] In one embodiment, the genetically engineered bacterium uses E. coli MG1655(DE3), E. coli W3110(DE3), E. coli JM109, E. coli DH5α, E. coli Top10 or E. coli BL21(DE3) as the expression host.

[0057] The present invention also provides a genetically engineered bacterium, which is: overexpressing the above-mentioned nicotinamide transferase mutant, the above-mentioned phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, NMN synthase, the above-mentioned formate dehydrogenase mutant, the above-mentioned formaldehyde dehydrogenase mutant, the above-mentioned malic enzyme mutant, glycolaldehyde synthase, acetyl phosphate synthase, phosphoacetyltransferase, ferredoxin oxidoreductase at the same time, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GSGSGGSGSG, GSGSGGSGSGGSGSG or GSGSGGSGSGGSGSGGSGSG as a linker peptide to obtain a fusion enzyme;

[0058] Meanwhile, the original RBS in front of uridine kinase and nucleoside diphosphate kinase: AAAGAGGAGAAA is replaced with RBS-01, RBS-03 or RBS-05; the original RBS in front of nicotinamide phosphoribosyltransferase and NMN synthase: AAAGAGGAGAAA is replaced with RBS-01, RBS-03 or RBS-05; the original RBS in front of the fusion enzyme: AAAGAGGAGAAA is replaced with RBS-01, RBS-03 or RBS-05 to obtain the strain;

[0059] The RBS-01 is GGAAACCCTCAGGAGGTAAACCA, the RBS-03 is ATGGTCTGATCTCCTACGGTTATA, and the RBS-05 is AGGAATGTATCAATTGAGGTTTAA.

[0060] The present invention also provides a method for preparing malic acid, which is to ferment with the above-mentioned genetically engineered bacterium to obtain malic acid.

[0061] In one embodiment, the final concentration of the genetically engineered bacterium added to the reaction system is: 2 - 4 g / L; preferably 3.5 g / L.

[0062] In one embodiment, the carbon dioxide concentration is 100 - 400 mM, preferably 300 mM;

[0063] In one embodiment, the formic acid concentration is 30 - 70 mM, preferably 50 mM.

[0064] In one embodiment, the reaction conditions are: reacting at pH 7.0 and 30 - 38 °C for 30 - 40 h.

[0065] Preferably, reacting at pH 7.0 and 37 °C for 35 h.

[0066] In one embodiment, the reaction is carried out in a fermenter. The fermentation conditions of a 7.5 L fermenter are as follows:

[0067] In a 7.5 L fermenter, the initial liquid loading is 4.5 L, the cell addition amount is 2 - 4 g / L, preferably 3.5 g / L; Tris-HCl solution with pH = 7.0 is used as the medium for whole-cell conversion, and CO 2 gas is introduced and the pressure is maintained at 0.05 MPa. Meanwhile, 50 - 100 mM formic acid, preferably 75 mM formic acid, is added; 5 - 15 mM MgSO 4 , preferably 10 mM MgSO 4 is added, and the concentration of phosphite is maintained at 5 - 10 mM. The catalytic reaction is carried out at 37 °C for 30 - 40 hours, preferably 35 hours.

[0068] The present invention also provides the use of the above-mentioned nicotinamide transferase mutant, phosphite dehydrogenase mutant, formate dehydrogenase mutant, formaldehyde dehydrogenase mutant and malic enzyme mutant, or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned genetically engineered bacterium in the preparation of malic acid or a product containing malic acid.

[0069] Beneficial effects

[0070] (1) The present invention provides the enzymes Hs Nmnat* and PsPTDH* that can efficiently utilize UTP and NMN to synthesize novel energy molecules, with activities reaching 110.25 U / mg and 102.69 U / mg respectively.

[0071] (2) The present invention provides a method for synthesizing a novel energy molecule NUDH in Escherichia coli, and the accumulation amount of NUDH concentration in the cells reaches 8.73 mM, and NUDH / NUD reaches 58.19.

[0072] (3) This study provides three enzymes, Ts FDH*, Bm FADH* and Pc ME*, that can efficiently utilize NUDH as an energy drive, realizing the use of NUDH as a driving force.

[0073] (4) This study uses the FGPM pathway in combination with NUDH to achieve the synthesis of 51.27 mM malic acid using carbon dioxide and formic acid as substrates. Description of the drawings

[0074] Figure 1 For protein engineering modification of Hs Nmnat* and Ps PTDH*.

[0075] Figure 2 For intracellular synthesis of a novel energy molecule NUDH in Escherichia coli.

[0076] Figure 3For the design of the FGPM path and the adaptation of path enzymes to NUDH. Detailed implementation

[0077] In the expression of the strains involved in the following embodiments, for the expression vectors used, as long as they are conventional vectors suitable for the expression of Escherichia coli, the technical solutions of the present invention can be achieved. In one solution, the expression vectors can be: pET series vectors, preferably pET28a, pET21a, pET32a; the expression vectors can be: pGEX series vectors; the expression vectors can be pMAL series vectors; the expression vectors can be pQE series vectors; the expression vectors can be pTrc series vectors; the expression vectors can be pBAD series vectors, etc.

[0078] In one solution, the pETAC vector and pTET vector are used in the present invention. The pETAC vector is described in the paper "Designing autonomous biological switches to rewire carbon flux for chemical production in Escherichia coli[J].ACS Sustainable Chemistry & Engineering,2024,12,39,14492–14504." by Guangjie Liang et al., and the pTET vector is described in the paper "Programmable biomolecular switches for rewiring flux in Escherichia coli[J].Nature Communications,2019,10:3751-3763.(#Authors contributed equally)" by Cong Gao et al.

[0079] The p15A vector involved in the following embodiments is the pACYC184 commercial plasmid, which is named p15A vector in this article.

[0080] It should be noted that the solutions of the present invention are not limited to the above expression vectors, and the technical solutions of the present invention can be achieved by conventionally selecting expression vectors according to the host cell (Escherichia coli).

[0081] In the present invention, the mutations of amino acids are all expressed in abbreviated forms. The abbreviations of various amino acids are as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V). In the expression of amino acid mutations, for example, L151Y means that the L at position 151 of the wild-type enzyme is mutated to Y; for example, L151Y / A176R / L208F means that the L at position 151 of the wild-type enzyme is mutated to Y, the A at position 176 is mutated to R, and at the same time the L at position 208 is mutated to F. The expression methods of other mutants in the present invention are the same as above.

[0082] The detection methods involved in the following examples are as follows:

[0083] Detection of nicotinamide transferase (Nmnat) activity:

[0084] The total volume of the reaction system is 200 μL, containing 0.5 mg of enzyme, 50 mM HEPES buffer (pH 7.5), 5 mM MnCl 2 、10 mM MgCl 2 、2.0 mM NMN (nicotinamide mononucleotide), and 2.5 mM UTP (uridine triphosphate). The reaction is carried out at 30 °C. The enzyme reaction is terminated with 0.155 mol / L EDTA, and the activity of the enzyme is determined by measuring the consumption of the substrate UTP at a wavelength of 450 nm using a kit (Shanghai Kesenghong Biotechnology Co., Ltd.).

[0085] Detection of phosphite dehydrogenase (PTDH) activity:

[0086] The reaction system is 200 μL, containing 0.5 mg of phosphite dehydrogenase, 50 mM HEPES buffer (pH 7.5), 5 mM phosphite, 1.0 mM tetrazolium blue, 0.5 mM phenazine methosulfate, and 0.5 mM NUD (nicotinamide uracil dinucleotide). The reaction is initiated by adding the enzyme solution at 25 °C. In the enzymatic reaction, phenazine methosulfate transfers the electrons obtained from NUDH to the acceptor tetrazolium blue, reducing it to form blue formazan. The formation of formazan is monitored by measuring the change in absorbance at a specific wavelength (570 nm) to evaluate the production of the product NUDH and the enzyme activity.

[0087] Detection of formate dehydrogenase activity:

[0088] The CO of formate dehydrogenase 2 The reduction activity assay was carried out in 100 mM HEPES buffer (pH 7.0) at a reaction temperature of 37 °C. The concentration of CO 2 was expressed as the concentration of NaHCO 3 . The total volume of the reaction system was 400 μL, including 1.0 mg of formate dehydrogenase, 200 mM NaHCO 3 and 4 mM NUDH. The determination of the product formic acid was carried out by the following method: Solution A was prepared by dissolving 0.5 g of citric acid and 10 g of acetamide in 100 mL of isopropanol. Solution B was prepared by dissolving 30 g of sodium acetate in 100 mL of water. During the determination, 100 μL of the sample after catalysis was taken, mixed with 200 μL of Solution A, 10 μL of Solution B, and 700 μL of pure acetic anhydride, and photometric determination was carried out at a wavelength of 515 nm to determine the performance of catalyzing the production of formic acid and the enzyme activity.

[0089] Detection of formaldehyde dehydrogenase activity:

[0090] The reaction of formaldehyde dehydrogenase catalyzing the reduction of formic acid to formaldehyde was carried out in 100 mM sodium phosphate buffer (pH = 7.5) at a reaction temperature of 30 °C. The total volume of the reaction system was 400 μL, containing 1.0 mg of formaldehyde dehydrogenase, 35.0 mM formic acid, and 4 mM NUDH. The detection method of the product formaldehyde was the colorimetric method. The principle of this method is that formaldehyde reacts with chromotropic acid in concentrated sulfuric acid solution to form a purple compound, and its maximum absorption wavelength is 580 nm. The specific operation is as follows: Add 0.1 mL of chromotropic acid solution (mass fraction 0.1 - 2%) to 0.5 mL of the reaction product of formaldehyde dehydrogenase, then add 0.6 mL of concentrated sulfuric acid, mix and heat to boiling, and then measure the production of formaldehyde per unit time at a wavelength of 580 nm.

[0091] Detection of malic enzyme activity:

[0092] The pyruvate reductase carboxylation activity of malic enzyme was measured using a standard reaction system. The total volume of this reaction system was 200 μL, containing 100 mM Tris-HCl buffer (pH 7.5), 0.5 mg of malic enzyme, 5 mM MgSO 4 , 4 mM NUDH, 100 mM NaHCO 3 and 50 mM pyruvate. The reaction was initiated by adding 1.0 mL of the enzyme solution and incubated at 30 °C. After the reaction ended, 1.0 mL of 10% (w / v) sodium dodecyl sulfate (SDS) was added to terminate the reaction. The reaction solution was mixed with 1% (w / v) 2,7-dihydroxynaphthalene solution in a ratio of 1:6, and the amount of malic acid produced per unit time was measured at a wavelength of 390 nm.

[0093] The culture media involved in the following examples are as follows:

[0094] LB medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 10 g / L;

[0095] TB medium: Yeast extract 24 g / L, Tryptone 12 g / L, KH 2 PO 4 2.31 g / L, K 2 HPO 4 16.43 g / L, Glycerol 4 g / L;

[0096] Fermentation medium: K 2 HPO 4 5.0 g / L, KH 2 PO 4 3.5 g / L, 0.35 g (NH 4 ) 2 HPO 4 3.5 g / L, MgSO 4 ·7H 2 O 0.25 g / L, Vitamin B1 0.5 mg / L, CaCl 2 ·2H 2 O 15 mg / L, Glucose 5 g / L, 10 mL trace element solution.

[0097] Trace element solution formula: FeCl 3 1.6 g / L, CuCl 2 0.1 g / L, NaMnO 4 0.2 g / L, CoCl 2 ·6H2O 0.2 g / L, H 3 BO 3 0.05 g / L, ZnCl2·4H2O 0.2 g / L, dissolved in 0.1 M HCl solution.

[0098] Example 1: Protein engineering modification of Hs Nmnat* and Ps PTDH* to synthesize novel energy molecules NUD and NUDH

[0099] The present invention designs a method for synthesizing novel oxidized energy molecule NUD and reduced energy molecule NUDH using UTP and NMN ( Figure 1 A in). Among them, nicotinamide transferase (Nmnat) is used to synthesize NUD using UTP and NMN, and then NUD is catalyzed into NUDH by phosphite dehydrogenase (PTDH).

[0100] 1. Screening of nicotinamide transferase

[0101] To synthesize NUD using UTP and NMN, genes of 27 nicotinamide transferases (Nmnat) from the UniProtKB protein database were synthesized, respectively derived from: Pseudobutyrivibrio ruminis, Candidatus Angelobacter, Elusimicrobia bacterium, Holdemanella biformis, Stenotrophomonas sp, Flavonifractor plautii, Chlorobium chlorochromatii, Pseudomonas aeruginosa, Staphylococcus aureus, Desmonostoc muscorum, Salmonella arizonae, Oryza sativa, Geodermatophilus dictyosporus, Kocuria varians, Alloscardovia macacae, Limosilactobacillus balticus, Lactobacillus kefiranofaciens, Saccharomyces cerevisiae, Homo sapiens, Mycolicibacterium vanbaalenii, Acetothermiabacterium, Anaerotignum lactatifermentans, Mycobacterium ulcerans, Streptomyces coeruleorubidus, Bifidobacterium asteroides, Xanthomonas translucens, Paraburkholderia solisilvae.

[0102] The above synthesized sequences were respectively ligated to the plasmid pET-28; the constructed plasmids were further transformed into E. coli BL21(DE3) competent cells to obtain recombinant bacteria respectively; the recombinant bacteria were respectively inoculated into LB liquid medium and cultured at 37 °C and 200 r / min for 8 h to prepare seed solutions, and the prepared seed solutions were respectively inoculated into TB medium for protein expression at an inoculation amount of 2% (v / v). After culturing at 37 °C and 220 r / min for 2 h until the OD600 reached 0.6 - 0.8, IPTG (final concentration 0.1 mM) was added, and then the culture was induced at 30 °C and 220 r / min for 16 - 18 h. The bacterial solution was centrifuged at 4 °C and 8000 r / min for 10 min, and the supernatant was discarded to collect the bacterial cells. After collecting the bacterial cells, low-temperature disruption and protein purification were carried out, and the performance of nicotinamide transferases from different sources in catalyzing the formation of NUD using UTP and NMN as substrates was detected according to the method provided previously. Among them, human-derived NMNAT (Hs Nmnat, PDB: 1KQN) showed the highest catalytic activity ( Figure 1 B in

[0103] 2. Screening of nicotinamide transferase mutants

[0104] To improve the catalytic performance of the human-derived nicotinamide transferase, namely Hs Nmnat, screened in step 1, relevant residues were modified by protein engineering. The specific iterative process and catalytic effect are shown in Figure 1 C in

[0105] (1) According to the sequence of Hs Nmnat (the amino acid sequence is shown in SEQ ID NO.2), mutant primers were respectively designed (as shown in Table 1), and site-directed mutagenesis was carried out on the Hs Nmnat enzyme on the pET28a plasmid (obtained in step 1); vectors containing L27K, V186L, V186I, and V186M mutants were respectively prepared.

[0106] Sequence of Hs Nmnat (SEQ ID NO.2)

[0107] MENSEKTEVVLLACGSFNPITNMHLRLFELAKDYMNGTGRYTVVKGIISPVGDAYKKKGLIPAYHRVIM

[0108] AELATKNSKWVEVDTWESLQKEWKETLKVLRHHQEKLEASDCDHQQNSPTLERPGRKRKWTETQDSS

[0109] QKKSLEPKTKAVPKVKLLCGADLLESFAVPNLWKSEDITQIVANYGLICVTRAGNDAQKFIYESDVLWK

[0110] HRSNIHVVNEWIANDISSTKIRRALRRGQSIRYLVPDLVQEYIEKHNLYSSESEDRNAGVILAPLQRNTAE

[0111] AKT

[0112] Table 1: Primers

[0113]

[0114]

[0115] The PCR reaction system is as follows: The PCR system (100 μl) consists of the following components: template DNA (100 - 150 ng), upstream and downstream primers (20 μM, 2 μl each), PrimeSTAR polymerase (1 μl), 5×PrimeSTAR buffer (20 μl), dNTP mixture (8 μl), and sterile water. The PCR product amplification conditions are: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 0.5 min; annealing at 55°C for 0.5 min; extension at 72°C for 3 min; a total of 30 - 32 cycles are carried out; finally, extension at 72°C for 5 min, and the temperature is reduced to 12°C for pre-preservation for 5 min. The PCR amplification product is detected by 1% agarose gel electrophoresis. After determining that the size of the amplification product is correct, 0.5 μl of DpnI digestion enzyme is added to 10 μL of the amplification product, mixed well, and reacted at 37°C for 40 min, and then reacted at 60°C for 10 min to inactivate the digestion enzyme. Subsequently, the DpnI-treated amplification product is transformed into competent Escherichia coli JM109, and the transformed product is spread on LB solid medium supplemented with kanamycin (final concentration 50 mg·L -1 ) and cultured at 37°C for 8 - 10 h, and the single colonies grown on the plate.

[0116] Positive clones are selected and inoculated into LB medium supplemented with kanamycin (final concentration 50 mg·L -1) in LB liquid medium, cultured at 37 °C and 220 r / min for 6 - 8 h, then the plasmid was extracted and verified by sequencing. The correctly sequenced pET28a-Hs Nmnat mutant vector was added to E. coli BL21(DE3) strain to prepare the genetically engineered bacteria. The prepared genetically engineered bacteria were inoculated into LB medium, cultured at 37 °C and 200 r / min for 8 h to prepare the seed solution, and then transferred to TB medium for protein induction expression at an inoculation amount of 10% (v / v). Cultured at 37 °C and 220 r / min for 2 h until OD600 reached 0.6 - 0.8, after adding IPTG (final concentration 0.1 mM), the culture was switched to 30 °C and 220 r / min for induction culture for 16 - 18 h. The bacterial solution was centrifuged at 4 °C and 8000 r / min for 10 min, the supernatant was discarded, the bacteria were collected and broken, and protein purification was carried out to obtain pure enzyme solutions of L27K, V186L, V186I and V186M mutants respectively;

[0117] According to the above detection method of nicotinamide transferase enzyme activity, the enzyme activities of different mutants were detected respectively. The results showed that the enzyme activities of L27K, V186L, V186I and V186M were 24.35 U / mg, 19.55 U / mg, 11.25 U / mg and 13.45 U / mg respectively; According to the above method, a pure enzyme solution of wild-type enzyme was prepared, and its enzyme activity was detected to be 2.25 U / mg.

[0118] (2) Using the optimal mutant L27K for iteration, the construction method was as follows: Using one of the single-point mutants as a template, by designing full-plasmid PCR primers for other mutation sites, the correctly mutated double-mutant plasmid was obtained through template digestion, product purification, transformation and picking clone for sequencing verification. The methods for triple mutation and quadruple mutation were the same. Double mutants L27K / V186L and L27K / V186I were generated, and further iteration yielded triple and quadruple mutants: L27K / V186L / T187K, L27K / V186L / T187Y, L27K / V186L / T187K / D220E, L27K / V186L / T187K / D220Q. According to the method in step (1), pure enzyme solutions containing mutants L27K / V186L, L27K / V186I, L27K / V186L / T187K, L27K / V186L / T187Y, L27K / V186L / T187K / D220E, L27K / V186L / T187K / D220Q were prepared respectively;

[0119] Table 2: Primers

[0120] Primers for mutagenesis Sequence(5’-3’) T187K-F TGCGTAAAACGCGCAGGTAAT T187K-R TGCGCGTTTTACGCAGATCAA T187Y-F TGCGTATATCGCGCAGGTAAT T187Y-R TGCGCGATATACGCAGATCAA D220E-F GCCAACGAAATTAGTTCAACG D220E-R ACTAATTTCGTTGGCAATCCA D220Q-F GCCAACCAGATTAGTTCAACG D220Q-R ACTAATCTGGTTGGCAATCCA

[0121] The enzyme activities were detected separately, and the results showed that the enzyme activities of L27K / V186L, L27K / V186I, L27K / V186L / T187K, L27K / V186L / T187Y, L27K / V186L / T187K / D220E, and L27K / V186L / T187K / D220Q were 41.96 U / mg, 40.36 U / mg, 77.67 U / mg, 65.86 U / mg, 110.25 U / mg, and 91.98 U / mg, respectively. Compared with the wild type (enzyme activity: 2.25 U / mg), the activity of Hs Nmnat* (L27K / V186L / T187K / D220E) was increased by 49.0-fold, and the enzyme activity reached 110.25 U / mg( Figure 1 C) in

[0122] 3. Screening of Phosphite Dehydrogenase

[0123] To reduce NUD to NUDH, 16 phosphite dehydrogenases from different sources in the UniProt protein database were synthesized and screened. The specific sources are as follows: Pseudomonas fulva, Stutzerimonas xanthomarina, Pseudomonas stutzeri, Klebsiella quasipneumoniae, Pseudomonas gessardii, Halopseudomonas bauzanensis, Klebsiella pneumoniae, Halopseudomonas litoralis, Halomonas meridiana, Achromobacter pulmonis, Halomonas desiderata, Brucella intermedia, Dendrosporobacter quercicolus, Arthrobacter saudimassiliensis, Synechococcus sp, and Beijerinckiaceae bacterium.

[0124] The synthesized sequences mentioned above were respectively ligated onto the plasmid pET-28a, and the constructed plasmids were further transformed into E. coli BL21(DE3) competent cells to obtain recombinant bacteria respectively; the recombinant bacteria were respectively inoculated into LB liquid medium and cultured at 37 °C and 200 r / min for 6 - 10 h to prepare seed solutions, which were respectively inoculated into TB medium at an inoculation amount of 5% (v / v) for protein induction. The protein expression induction conditions were: culturing at 37 °C and 220 r / min for 2 h, after the OD600 reached 0.6 - 0.8, adding IPTG with a final concentration of 0.1 mM, and then culturing at 25 °C and 220 r / min for 16 - 18 h. The bacterial solution was centrifuged at high speed at 4 °C and 8000 r / min for 10 min. After collecting the bacterial cells, low-temperature disruption and protein purification were carried out, and the performance of 16 different sources of phosphite dehydrogenases in catalyzing NUD to generate NUDH was detected respectively according to the method provided previously. The results showed that PTDH (Ps PTDH) from Pseudomonas stutzeri exhibited the highest catalytic activity towards NUD ( Figure 1 D in

[0125] 4. Screening of phosphite dehydrogenase mutants

[0126] To improve the catalytic performance of Ps PTDH from Pseudomonas stutzeri, relevant residues were modified through protein engineering. The specific iterative process and catalytic effects are shown in Figure 1 E.

[0127] The specific process is as follows:

[0128] (1) According to the sequence of Ps PTDH (the amino acid sequence is shown in SEQ ID NO.4), mutant primers were respectively designed (as shown in Table 3) to mutate the Ps PTDH enzyme on the pET28a-Ps PTDH plasmid; vectors containing L151Y, L151W, A176R, A207N, and A207M mutants were respectively prepared; the methods for PCR reaction and preparation of pure enzyme solution were the same as in step 3.

[0129] The sequence of Ps PTDH (SEQ ID NO.4)

[0130] MLPKLVITHRVHDEILQLLAPHCELMTNQTDSTLTREEILRRCRDAQAMMAFMPDRVDADFLQACPELRV

[0131] VGCALKGFDNFDVDACTARGVWLTFVPDLLTVPTAELAIGLAVGLGRHLRAADAFVRSGEFQGWQPQFY

[0132] GTGLDNATVGILGMGAIGLAMADRLQGWGATLQYHEAKALDTQTEQRLGLRQVACSELFASSDFILLAL

[0133] PLNADTQHLVNAELLALVRPGALLVNPCRGSVVDEAAVLAALERGQLGGYAADVFEMEDWARADRPRL

[0134] IDPALLAHPNTLFTPHIGSAVRAVRLEIERCAAQNIIQVLAGARPINAANRLPKAEPAAC

[0135] Pure enzyme solutions containing L151Y, L151W, A176R, A207N, and A207M mutants were prepared respectively, and the enzyme activities were detected respectively. The results showed that the enzyme activities of L151Y, L151W, A176R, A207N, and A207M were 34.62 U / mg, 24.37 U / mg, 43.01 U / mg, 18.72 U / mg, and 15.08 U / mg respectively; according to the above method, a pure enzyme solution of wild-type enzyme was prepared, and its enzyme activity was detected to be 5.14 U / mg.

[0136] (2) Iteration was carried out with the optimal mutant A176R. The construction method was as follows: using one of the single-point mutants as a template, through designing whole-plasmid PCR primers for other mutation sites, and obtaining the correctly mutated double-mutant plasmid through template digestion, product purification, transformation, and picking clone colonies for sequencing verification. The methods for triple mutants and quadruple mutants were the same as above.

[0137] Double mutants L151Y / A176R, L151W / A176R, A176R / A207N, and A176R / A207M were generated. Further iteration resulted in triple mutants: L151Y / A176R / L208F and L151Y / A176R / N211I. According to the method in step 3, pure enzyme solutions containing mutants L151Y / A176R, L151W / A176R, A176R / A207N, A176R / A207M, L151Y / A176R / L208F, and L151Y / A176R / N211I were prepared respectively;

[0138] Table 3: Primers

[0139]

[0140]

[0141] The enzyme activities were detected separately, and the results showed that the enzyme activities of L151Y / A176R, L151W / A176R, A176R / A207N, A176R / A207M, L151Y / A176R / L208F, and L151Y / A176R / N211I were 80.65 U / mg, 73.28 U / mg, 56.42 U / mg, 50.77 U / mg, 102.69 U / mg, and 97.08 U / mg, respectively. The results showed that Ps PTDH*(L151Y / A176R / L208F) had the highest activity (102.69 U / mg) in catalyzing the synthesis of NUDH from NUD, which was 19.98 times higher than that of the wild type (5.14 U / mg). Figure 1 D) in

[0142] Example 2: Intracellular synthesis of a novel energy molecule NUDH in Escherichia coli

[0143] The specific steps are as follows:

[0144] 1. Preparation of genetically engineered bacterium K01

[0145] (1) Preparation of recombinant vector

[0146] To synthesize NUD and NUDH using UTP and NMN as substrates in Escherichia coli, HsNmnat*(L27K / V186L / T187K / D220E) and Ps PTDH*(L151Y / A176R / L208F) prepared in Example 1 and the p15A vector were amplified using the primers in Table 4 respectively. After determining the fragment and vector sizes by agarose gel electrophoresis, they were digested with DpnI enzyme and recovered by column. Subsequently, the fragments Hs Nmnat* and Ps PTDH* were ligated to the amplified p15A vector using a multi-fragment homologous recombination enzyme. The homologous recombination system and method are as follows: The total reaction system is 10 μL, including 2 μL of multi-fragment homologous recombination enzyme, 100 ng of fragment Hs Nmnat* in the system, 100 ng of fragment Ps PTDH*, and the addition amount of vector p15A is 200 ng. React at 37 °C for 30 min, then directly transform it into competent Escherichia coli JM109 cells and spread it on an LB solid plate supplemented with kanamycin (final concentration 50 mg·L -1 ) and incubate at 37 °C for 6 - 8 h. Select positive clones and inoculate them into an LB liquid medium supplemented with kanamycin (final concentration 50 mg·L -1 ) and culture at 37 °C and 220 r / min for 6 - 8 h, then extract the plasmid p15A-HsNmnat*-Ps PTDH*, named p15A-1.

[0147] Table 4: Primers

[0148] Primers for mutagenesis Sequence(5’-3’) Hs Nmnat*-F GAGGAGAAAAAGCTTGGGCCCATGGAAAATTCAGAAAAGACAGAGGTC Hs Nmnat*-R GCATTTTAGCGGCAGCCTCTGTTTTGGCTTCAGCAGTATTGCG Ps PTDH*-F AAACAGAGGCTGCCGCTAAAATGCTGCCGAAGCTGGTCATCA Ps PTDH*-R CTTTTTACGGTTCCTGGCCTTAACACGCCGCTGGTTCAGCTT p15A-F GGGCCCAAGCTTTTTCTCCTCTTTAC p15A-R GGCCAGGAACCGTAAAAAGGC

[0149] (2) Preparation of genetically engineered bacteria

[0150] The prepared p15A-1 was introduced into the competent cells of Escherichia coli Mg1655 to generate strain K01.

[0151] 2. Fermentation of strain K01 to prepare NUD and NUDH

[0152] The prepared strain K01 was added to LB medium and cultured at 37 °C and 220 r / min for 6 h to obtain a seed solution; the prepared seed solution was inoculated into TB medium at an inoculation amount of 10% (v / v) and cultured at 37 °C and 220 r / min. After culturing at 37 °C and 220 r / min for 2 h, when OD600 reached 0.6 - 0.8, IPTG with a final concentration of 0.1 mM was added, and then cultured at 37 °C and 220 r / min for 16 - 18 h to obtain a fermentation broth; the fermentation broth was centrifuged to collect the cells, and then resuspended with PBS solution. At OD 600 = 1, the number of cells per milliliter of resuspended solution was approximately 1×10 9 cells.

[0153] To extract, quantify, and detect the intracellular concentrations of NUD and NUDH, 2.0×10 10 cells were collected by centrifugation at 4 °C and 12,000 r / min for 5 minutes. The centrifuged cells were washed three times with ice-cold phosphate-buffered saline (PBS). The pellet was resuspended in 500 μL of extraction solution (acetonitrile, methanol, and water, volume ratio 40:40:10) pre-cooled to -20 °C and incubated for 20 minutes. The supernatant was collected by centrifugation at 4 °C and 12,000 r / min for 5 minutes, and the supernatant was analyzed using a Waters ACQUITY UPLC high-performance liquid chromatograph equipped with an Atlantis PREMIER BEH C18 AX chromatographic column. The mobile phase for separation included (A) 10 mM ammonium formate (pH = 8.9) and (B) 100% acetonitrile. The gradient elution program was as follows: 0 - 2 min, 99% A; 2 - 4 min, 99% - 88% A; 4 - 5 min, 88% - 10% A; 5 - 6 min, 10% A; 6 - 7 min, 10% - 99% A; 7 - 12 min, 99% A.

[0154] The results showed that the intracellular concentrations of NUD and NUDH in strain K01 were 31.07 μM and 43.22 μM, respectively ( Figure 2 A and B in

[0155] 3. Modification of genetically engineered bacteria

[0156] To enhance NUDH accumulation, Hs Nmnat*(L27K / V186L / T187K / D220E) and Ps PTDH*(L151Y / A176R / L208F) were fused to generate NUDH synthase (NUDHS).

[0157] (1) The flexible linker peptide (amino acid sequence: GSGSG) was inserted between the C-terminus of Hs Nmnat* and the N-terminus of PsPTDH* in two (GSGSGGSGSG), three (GSGSGGSGSGGSGSG), and four copies (GSGSGGSGSGGSGSGGSGSG), respectively, to prepare the fusion enzymes: Hs Nmnat*-GSGSGGSGSG-Ps PTDH*, Hs Nmnat*-GSGSGGSGSGGSGSG-Ps PTDH*, Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-Ps PTDH*;

[0158] (2) The fusion enzymes Hs Nmnat*-GSGSGGSGSG-Ps PTDH*, Hs Nmnat*-GSGSGGSGSGGSGSG-Ps PTDH*, and Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-Ps PTDH* obtained in step (1) were successively ligated to the p15A vector by the homologous recombination method of step 2. Then, the prepared recombinant vectors p15A-2 (containing the fusion enzyme HsNmnat*-GSGSGGSGSG-Ps PTDH*), p15A-3 (containing Hs Nmnat*-GSGSGGSGSGGSGSG-Ps PTDH*), and p15A-4 (containing Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-Ps PTDH*) were respectively introduced into the strain Mg1655 to prepare the strains K01-1, K01-2, and K01-3.

[0159] 4. Strains K01-1, K01-2, and K01-3 were fermented to prepare NUD and NUDH

[0160] According to the method described in step 2, NUD and NUDH were fermented and prepared. The intracellular concentrations of NUD and NUDH in the strains were detected by ultra-high performance liquid chromatography. The results showed that strain K01-3, which contained four copies of the flexible linker, accumulated the highest level of NUDH up to 75.6 μM ( Figure 2 B in

[0161] 5. Supply of precursor UTP and NMN was enhanced in strain K01-3

[0162] To enhance the supply of precursor UTP and NMN, metabolic engineering was carried out according to the metabolic pathways of NUD and NUDH ( Figure 2 C) in []. First, the related genes pyrG, purR, pncC, UshA, and pnuC in the catabolic pathways of UTP and NMN in E. coli Mg1655 were knocked out in sequence to generate strain E. coli Mg1655-01. Among them, the CTP synthase encoded by the pyrG gene can convert UTP into cytidine triphosphate (CTP); the negative regulatory protein encoded by the purR gene can inhibit the supply of NMN precursors; the nicotinamide nucleotide amidohydrolase encoded by the pncC gene can metabolize NMN into nicotinic acid mononucleotide (NaMN); the 5'-nucleotidase encoded by the UshA gene metabolizes NMN into nicotinamide riboside (NR); the transporter encoded by the pnuC gene can transport NMN out of the cell. Subsequently, the expression of four genes was co-enhanced, namely uridine kinase (Sl UMPK, gene sequence as SEQ ID NO.5), nucleoside diphosphate kinase (Da NDK, gene sequence as SEQ ID NO.6), nicotinamide phosphoribosyltransferase (Vp NAMPT, gene sequence as SEQ ID NO.7), and NMN synthase (Ft NadE, gene sequence as SEQ ID NO.8), to generate strain K02.

[0163] The specific method is as follows:

[0164] (1) Block the catabolic pathways of UTP and NMN

[0165] To reduce the catabolism of precursor UTP and NMN, based on the CRISPR / Cas9 gene editing technology, the genes pyrG, purR, pncC, UshA, and pnuC in E. coli Mg1655 were iteratively knocked out in sequence to obtain strain E. coli Mg1655-1. The specific steps are as follows: For the gene and 500 bp upstream and downstream, import it into Snap gene, search for agg within the target gene fragment, and select the 20 bp in front of agg of the upper sequence as N20. The N20 sequences of the above five genes are shown in Table 5.

[0166] Table 5: N20 sequences of genes

[0167] Name Sequence(5’-3’) pyrG-N20 CATGGAGAACGCCAACTCTA purR-N20 AGAGCCGTTGCTGGCGATGC pncC-N20 CCGTGATGCGGTGCGTCGTC UshA-N20 TCTGGTGGGCTATGACGCGA pnuC-N20 ATCCTGGTACATATACCAAT

[0168] Copy and paste the N20 sequence to the corresponding N20 in the plasmid pTarget sequence, and design the reverse amplification primer for whole plasmid PCR. The primers used are shown in Table 6.

[0169] Table 6: Primer sequences for constructing gene N20

[0170] Name Sequence(5’-3’) pyrG-N20-F CATGGAGAACGCCAACTCTAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT pyrG-N20-R TAGAGTTGGCGTTCTCCATGACTAGTATTATACCTAGGACTGAGC purR-N20-F AGAGCCGTTGCTGGCGATGCGTTTTAGAGCTAGAAATAGCAAGTTAAAAT purR-N20-R GCATCGCCAGCAACGGCTCTACTAGTATTATACCTAGGACTGAG pncC-N20-F CCGTGATGCGGTGCGTCGTCGTTTTAGAGCTAGAAATAGCAAGTTAAAATA pncC-N20-R GACGACGCACCGCATCACGGACTAGTATTATACCTAGGACTGA UshA-N20-F TCTGGTGGGCTATGACGCGAGTTTTAGAGCTAGAAATAGCAAGTTAAAAT UshA-N20-R TCGCGTCATAGCCCACCAGAACTAGTATTATACCTAGGACTGAG pnuC-N20-F ATCCTGGTACATATACCAATGTTTTAGAGCTAGAAATAGCAAGTTAAAAT pnuC-N20-R ATTGGTATATGTACCAGGATACTAGTATTATACCTAGGACTGA

[0171] Subsequently, a knockout cassette with a length of 1000 bp was constructed by homologous recombination. First, the Cas9 plasmid was electrotransformed into competent E. coli MG1655 cells, and then competent cells were prepared using these cells as the host. The knockout cassette fragment and the plasmid pTargetF containing N20 were electrotransformed into the competent cells, and colony PCR was used to verify whether the gene was knocked out. After sequentially knocking out the five genes in the order of pyrG, purR, pncC, UshA, and pnuC, the strain E. coli Mg1655-1 could be obtained.

[0172] (2) Preparation of Strain K02

[0173] To enhance the synthesis of MNM and UTP, the above four genes were respectively ligated to the vector p15A-4 (p15A-Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-Ps PTDH*) by homologous recombination. First, the uridine kinase (Sl UMPK), nucleoside diphosphate kinase (Da NDK), and the gene sequence of the vector p15A-4 were amplified using the primers in Table 7. After determining the fragment and vector sizes by agarose gel electrophoresis, DpnI enzyme digestion and column recovery were carried out respectively. Subsequently, the fragments Sl UMPK and Da NDK were ligated to the amplified p15A-4 vector using a multi-fragment homologous recombination enzyme. The homologous recombination system and method are as follows: The total reaction system was 10 μL, including 2 μL of the multi-fragment homologous recombination enzyme. In the system, there were 150 ng of the fragment Sl UMPK, 150 ng of the fragment Da NDK, and 350 ng of the vector p15A. The reaction was carried out at 37 °C for 30 min, and then directly transformed into competent Escherichia coli JM109 cells and spread on an LB solid plate supplemented with kanamycin (final concentration 50 mg·L -1 )). The cells were cultured at 37 °C for 6 - 8 h, and positive clones were selected and inoculated into an LB liquid medium supplemented with kanamycin (final concentration 50 mg·L -1 ). After culturing at 37 °C and 220 r / min for 6 - 8 h, the plasmid p15A-5 (p15A-Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-Ps PTDH*-SlUMPK-Da NDK) was extracted.

[0174] Subsequently, a similar method was used to amplify the gene sequences of nicotinamide phosphoribosyltransferase (Vp NAMPT) and NMN synthase (Ft NadE) and ligate them into the vector p15A-5. The amplification primers are shown in Table 7. By culturing the bacteria and extracting the plasmid, the vector p15A-6 containing the UTP synthesis module (Sl UMPK and Da NDK), NMN synthesis module (Vp NAMPT and Ft NadE genes), and NUDH synthesis module (Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-Ps PTDH*) was obtained, i.e., p15A-UTP synthesis module-NMN synthesis module-NUDH synthesis module.

[0175] Table 7: Primers

[0176] Primer Name Sequence (5'-3') Sl UMPK-F ATGACGAAACGCGTGCTGG Sl UMPK-R ATCACCTGAAGTCAGCCCTCATTTCACAATGCTACACAGGCTCATATC Da NDK-F AAATGAGGGCTGACTTCAGGTGATGTTAGAAAAAACACTATCAATTAT Da NDK-R TTACAGGATCTCTAACGCGCTGAAGAAAT p15A-3-F GACCAGCACGCGTTTCGTCATGGGCCCAAGCTTTTTCTCCTCTTTACTA p15A-3-R AGATTTCAGTGCAATTTATCTCTTCAAATGTAGTTACAGGATCTCTAACGCGCTGAA Vp NAMPT-F ATGCTGAATCTGAATCAGAACATTGCCA Vp NAMPT-R TAATCAGATAAAATATTACGCGGTCTGGATTTTTTTGGCGA Ft NadE-F CAGACCGCGTAATATTTTATCTGATTATGAAAATCGTCAAGGACTTCAGT Ft NadE-R TAGAAATTAGGGGTTAATGCAAGTTTGCG p15A-4-F GTTCTGATTCAGATTCAGCATGGGCCCAAGCTTTTTCTCCTCTTTACTAGT p15A-4-R TGCATTAACCCCTAATTTCTAAAATAAGATGATCTTCTTGAGATCGTTTTGGTCTG

[0177] The prepared p15A-6 was introduced into the competent cells of strain E. coli Mg1655-1 to generate strain K02.

[0178] (3) Detection of intracellular NMN and UTP

[0179] The prepared strain K02 was added to LB medium and cultured at 37 °C and 220 r / min for 6 h to obtain a seed solution; the prepared seed solution was inoculated into TB medium at an inoculation amount of 10% (v / v) and cultured at 37 °C and 220 r / min for 2 h. When OD600 reached 0.6 - 0.8, IPTG with a final concentration of 0.1 mM was added, and then cultured at 37 °C and 220 r / min for 16 - 18 h to obtain a fermentation broth; the fermentation broth was centrifuged and the bacteria were collected. The centrifuged cells were washed three times with ice-cold phosphate-buffered saline (PBS) and then collected. Protein removal was performed by adding an equal volume of 6% trichloroacetic acid (TCA), and the mixture was vigorously mixed with a vortex oscillator for 20 seconds. Then the mixture was cooled on ice for 10 minutes and vortexed again for 20 seconds. After centrifugation at 4 °C and 13,000 rpm for 10 minutes, the obtained clear supernatant was diluted with an equal volume of double-distilled water and vortexed for 60 seconds.

[0180] For the detection of UTP, these samples can be stored at -80 °C or neutralized immediately with 5 M potassium carbonate before ultra-high performance liquid chromatography (UPLC) analysis. The supernatant was analyzed using a Waters ACQUITY UPLC high performance liquid chromatograph equipped with a Waters SymmetryC18 (3.5 μm, 150×4.6 mm) column. The UPLC column temperature was maintained at 25 °C, the injection volume was 25 μL, and the flow rate was 0.8 mL / min. A stepwise gradient elution program was used: solvent A-B (60:40) at 0 min, (40:60) at 30 min, (40:60) at 60 min. Solvent A consisted of 10 mM tetrabutylammonium hydroxide and 10 mM potassium phosphate and contained 0.25% methanol (pH = 6.9). Solvent B consisted of 5.6 mM tetrabutylammonium hydroxide and 50 mM potassium phosphate and was dissolved in a 30% methanol solution (pH = 7.0).

[0181] The detection of NMN was performed using a detection kit from AO RUIDA BIOLOGY (Guangdong, China). This kit used a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) method. Samples, standards, and HRP-labeled detection antibodies were added sequentially to the wells pre-coated with NMN antibodies, followed by incubation and thorough washing. Then, the substrate TMB was added for color development; under the catalysis of peroxidase, TMB was converted to blue and changed to yellow under acidic conditions. The intensity of the color was proportional to the concentration of NMN in the sample. The absorbance (OD value) was measured at a wavelength of 450 nm using a spectrophotometer to calculate the sample concentration.

[0182] The results showed that the intracellular NMN and UTP concentrations of strain K02 reached 5.47 mM and 7.10 mM, respectively ( Figure 2 D in).

[0183] 6. Optimize the RBS sequence in strain K02

[0184] (1) Screening of the RBS sequence

[0185] Subsequently, the relationship between the UTP synthesis module (Sl UMPK and Da NDK), the NMN synthesis module (Vp NAMPT and Ft NadE genes), and the NUDH synthesis module (Hs Nmnat*-GSGSGGSGSGGSGSGSGGSGSG-PsPTDH*) was balanced through ribosome binding site (RBS) engineering, and different strengths of RBS were screened to control gene expression.

[0186] To express green fluorescent protein (GFP) using a p15A vector and screen ribosome binding sites (RBSs) of different strengths, the following steps can be taken: Double-digest the p15A vector with restriction enzymes BanI and XbaI, insert the green fluorescent protein (GFP) fragment between these two digestion sites to obtain the vector p15A-GFP. Subsequently, control the expression of GFP using 6 different RBSs (Table 8) (i.e., replace the original RBS of GFP: AAAGAGGAGAAA with different RBSs) to obtain the vectors p15A-GFP-01, p15A-GFP-02, p15A-GFP-03, p15A-GFP-04, p15A-GFP-05, and p15A-GFP-06. Transform the above vectors into Escherichia coli Mg1655 respectively, and culture them in LB medium at 37 °C and 220 r / min for 7.5 h. Use a microplate reader to detect the GFP fluorescence intensity under different RBS control conditions. Take the final fluorescence intensity of the strain containing the vector p15A-GFP-01 (RBS-01) as the reference value of 100%, and calculate the relative strengths of other RBSs respectively. The fluorescence intensities of RBS-02, RBS-03, RBS-04, RBS-05, and RBS-06 are 63.71%, 60.12%, 38.03%, 17.06, and 10.31% ( Figure 2 E in

[0187] Table 8: RBS sequences

[0188] RBS Name Sequence RBS-01 GGAAACCCTCAGGAGGTAAACCA RBS-02 TCACACAGGAAACC RBS-03 ATGGTCTGATCTCCTACGGTTATA RBS-04 TTCACACAGGAAACC RBS-05 AGGAATGTATCAATTGAGGTTTAA RBS-06 TCACACAGGAC

[0189] (2) Construction of strains K02-R1 to K02-R27

[0190] K02: E.coli Mg1655-1 / p15A-RBS 0 -Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-PsPTDH*-RBS 0 -Sl UMPK-RBS 0 -Da NDK-RBS 0 -Vp NAMPT-RBS 0 -Ft NadE; all RBSs 0 are all AAAGAGGAGAAA;

[0191] Three ribosome binding sites (RBSs) with high (RBS-01: GGAAACCCTCAGGAGGTAAACCA), medium (RBS-03: ATGGTCTGATCTCCTACGGTTATA), and low (RBS-05: AGGAATGTATCAATTGAGGTTTAA) strengths were used to replace the original RBS. The replacement was carried out by homologous recombination for the RBSs in the UTP synthesis module (RBS 0 -Sl UMPK-RBS 0 -DaNDK), NMN synthesis module (RBS 0 -Vp NAMPT-RBS 0 -Ft NadE), and NUDH synthesis module (RBS 0 -Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-Ps PTDH*), respectively. The results are shown in Table 9. The serial numbers in Table 9 represent the strains. RBS optimization means that different RBSs were used to strengthen each module on the strain genome. For example, the optimization process of strain K02-R1 was as follows: 0

[0192] The RBS in (RBS 0 -Sl UMPK-RBS 0 -Da NDK) was replaced with RBS-01 0 ; the RBS in (RBS 0 -Vp NAMPT-RBS 0 -Ft NadE) was replaced with RBS-01 0 ; the RBS in (RBS 0 -Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-Ps PTDH*) was replaced with RBS-01 0 to obtain the strain.

[0193] Table 9: Detection of RBS optimization control and intracellular NUDH level

[0194]

[0195]

[0196] For strain K02-R11, using RBS-03 (medium), RBS-01 (high), and RBS-03 (medium) to control the UTP, NMN, and NUDH synthesis modules could significantly increase the intracellular NUDH level, resulting in the highest NUDH concentration of 8.73 mM and an NUDH / NUD ratio of 58.19, proving the successful construction of the new energy system.

[0197] ​Example 3: Design of the FGPM Pathway and Adaptation of Pathway Enzymes to NUDH

[0198] Pathway design:

[0199] This patent designs a metabolic pathway FGPM pathway for synthesizing malic acid from carbon dioxide and formic acid (A in Figure 3 ). This pathway consists of four carbon metabolism modules: In the C1 module, CO 2 is converted to formaldehyde by (Ts FDH, gene sequence as SEQ ID NO.9) and formaldehyde dehydrogenase (Bm FADH, gene sequence as SEQ ID NO.10). The C2 module converts formaldehyde to acetyl-CoA through the action of glycolaldehyde synthase (Pp GALS, gene sequence as SEQ ID NO.11), acetyl-phosphate synthase (Bm APS, gene sequence as SEQ ID NO.12), and phosphotransacetylase (Bs PTA, gene sequence as SEQ ID NO.13). In the C3 module, acetyl-CoA and CO 2 convert acetyl-CoA to pyruvate through pyruvate:ferredoxin oxidoreductase (Da rPFOR, gene sequence as SEQ ID NO.14). Finally, in the C4 module, pyruvate is converted to malic acid by malic enzyme (PcME, gene sequence as SEQ ID NO.15). Three enzymes Ts FDH, Bm FADH, and PcME in this carbon fixation pathway are driven by the natural energy system NADH in the cell, with the characteristics of simple steps and independence from ATP. However, the natural energy system NADH in the cell is simultaneously used in hundreds of catalytic reactions, and its concentration is relatively low, only 0.5 - 1 mM, making it difficult to efficiently drive the operation of the carbon fixation pathway. The FGPM pathway was constructed in Escherichia coli strain Mg1655, and using CO 2 as a substrate, no synthesis of the product malic acid was detected. Therefore, this patent proposes to use the newly developed energy system NUDH (8.73 mM) that can accumulate at a high concentration in the cell to drive the FGPM pathway. In order to convert the pathway enzymes that depend on the natural energy system NADH into those that depend on the new energy molecule NUDH and achieve adaptation to the new energy molecule NUDH, protein engineering modifications were carried out through iterative mutations respectively.

[0200] 1. Obtaining of Ts FDH mutants

[0201] (1) According to the sequence of Ts FDH (amino acid sequence as shown in SEQ ID NO.16), mutation primers were designed respectively (as shown in Table 10), and site-directed mutagenesis was carried out on the Ts FDH enzyme on the pET28a plasmid; vectors containing V198N, V198M, T221E, T221L, T221D, and V256L mutants were prepared respectively.

[0202] Sequence of Ts FDH (SEQ ID NO.16)

[0203] MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFEKELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVTYCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAAGRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPETEHMINDETLKLFKRGAYIVNTARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAG

[0204] Table 10: Primers

[0205] Primers for mutation sites Sequence (5’-3’) V198N-F GGTAGCAACGCGGCGGGCCGT V198N-R CGCCGCGTTGCTACCCACGGT V198M-F GGTAGCATGGCGGCGGGCCGT V198M-R CGCCGCCATGCTACCCACGGT T221E-F CATTATGAAGATCGCCATCGC T221E-R GCGATCTTCATAATGCAGTTT T221L-F CTGCATTATCTGGATCGCCATCGC T221L-R GCGATCCAGATAATGCAGTTT T221D-F CATTATGATGATCGCCATCGC T221D-R GCGATCATCATAATGCAGTTT V256L-F CTGAACCTGCCGCTGCATCCG V256L-R CAGCGGCAGGTTCAGGGTCAC

[0206] The PCR system (100 μl) consists of the following components: template DNA (100 - 150 ng), upstream and downstream primers (20 μM, 2 μl each), PrimeSTAR polymerase (1 μl), 5×PrimeSTAR buffer (20 μl), dNTP mixture (8 μl), and sterile water. The amplification conditions for the PCR product are: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 0.5 min; annealing at 55°C for 0.5 min; extension at 72°C for 3 min; a total of 30 - 32 cycles; finally, extension at 72°C for 5 min, and cooling to 12°C for pre-preservation for 5 min. The PCR amplification product is detected by 1% agarose gel electrophoresis. After determining that the size of the amplification product is correct, 0.5 μl of DpnI digestion enzyme is added to 10 μL of the amplification product, mixed well, and reacted at 37°C for 40 min, and then reacted at 60°C for 10 min to inactivate the digestion enzyme. Subsequently, the DpnI-treated amplification product is transformed into competent Escherichia coli JM109, and the transformed product is spread on LB solid medium supplemented with kanamycin (final concentration 50 mg·L -1 ) and cultured at 37°C for 8 - 10 h to obtain the single colonies grown on the plate. The single colonies grown on the plate are inoculated into LB medium supplemented with ampicillin (final concentration 100 mg·L-1 ) in LB liquid medium at 37 °C and 220 r / min for 6 - 8 h, then extract the plasmid to obtain the vector containing V198N, V198M, T221E, T221L, T221D and V256L mutants.

[0207] Add the prepared mutant vectors to Escherichia coli BL21(DE3) strain respectively to prepare genetically engineered bacteria. Inoculate the prepared genetically engineered bacteria into TB medium to prepare pure enzyme solutions containing V198N, V198M, T221E, T221L, T221D and V256L mutants. At the same time, prepare the wild-type pure enzyme solution according to the above method and detect the enzyme activity respectively. The results show that the initial activity of TsFDH using NUDH as the energy molecule is 0.068 U / mg (i.e., the enzyme activity of the wild type is 0.068 U / mg). Six beneficial mutants were obtained by single-point mutation, namely V198N, V198M, T221E, T221L, T221D and V256L, and their enzyme activities are 0.23 U / mg, 0.51 U / mg, 0.68 U / mg, 0.38 U / mg, 0.55 U / mg and 0.87 U / mg respectively;

[0208] (2) Iterate with the optimal mutant V256L. The construction method is as follows: Use one of the single-point mutants as the template, design the whole plasmid PCR primers for other mutation sites, and obtain the correctly mutated double-mutant plasmid through template digestion, product purification, transformation and picking clone for sequencing verification. The methods for triple mutation and quadruple mutation are the same as above.

[0209] Based on this, iterative mutation is carried out to obtain mutants: T221D / V256L, T221E / V256L, T221L / V256L, V198N / T221E / V256L, V198M / T221E / V256L;

[0210] Prepare pure enzyme solutions containing mutants T221D / V256L, T221E / V256L, T221L / V256L, V198N / T221E / V256L, V198M / T221E / V256L according to the method in step (1). The enzyme activities are 2.08 U / mg, 3.79 U / mg, 2.76 U / mg, 4.36 U / mg and 5.69 U / mg respectively. The activity of Ts FDH*(V198M / T21E / V256L) is increased to 5.69 U / mg, which is 83.68 times that of the wild type (enzyme activity: 0.068 U / mg). The iterative process is as shown in Figure 3 B in

[0211] 2. Screening of Bm FADH mutants

[0212] According to the sequence of Bm FADH (the amino acid sequence is shown in SEQ ID NO.17), mutation primers were designed respectively (as shown in Table 11), and site-directed mutagenesis was carried out on Bm FADH on the pET28a plasmid; vectors containing L237R, A193R, A193M, V283L, L237Y, H268E and H268Y mutants were prepared respectively; the above vectors were introduced into Escherichia coli BL21(DE3), and cell culture and protein induction expression were carried out according to the previous method.

[0213] Sequence of Bm FADH (SEQ ID NO.17)

[0214] MSSNRGVVYLGPGKVEVQKIDYPKMVDPSGRAIGHGVILKVVSTNICGSDQHMVRGRTTAPVGLVLGHEITGEVVEVGRDVETLKIGDLVSVPFNVACGRCAMCKETHTGVCLNVNPSRAGGAYGYVDMGGWIGGQAEYVLVPYADFNLLKFPDRDQAMAKIRDLTCLSDILPTGYHGAVSAGVKPGSTVYIAGAGPVGMAAAASARLLGAAVTIVGDMNAERLAHAKAMGFETVDLSKDATLGEQIAQILGKPEIDCAVDCVGFEAHGHGSSGHAEEAPATVLNSLMEITRPAGAIGIPGLYVTDDPGAQDKAAQHGSLSIRFGLGWAKSHSFFTGQTPVLKYNRNLMQAILYDRLPIAKIVNVTVISLDDAPEGYKKFDGGAPRKFVIDPHGLLAA

[0215] By purifying the protein and measuring the enzyme activity, they were respectively: 0.25 U / mg, 0.49 U / mg, 0.75 U / mg, 0.90 U / mg, 0.49 U / mg, 0.55 U / mg, 0.64 U / mg. Among them, the activity of V283L was the highest, reaching 0.90 U / mg. Under the same conditions, the enzyme activity of the wild type was: 0.057 U / mg; on this basis, iterative mutagenesis was carried out to generate the optimal double mutant A193R / V283L, and the activity was increased to 3.68 U / mg. Finally, the optimal mutant Bm FADH* (A193R / M219K / L237R / H268Y / V283L) was obtained, and the activity reached 6.13 U / mg ( Figure 3 C) in

[0216] Table 11: Primers

[0217] Primers for mutagenesis Sequence(5’-3’) L237R-F GTGGATCGCTCGAAAGATGCA L237R-R TTTCGAGCGATCCACGGTTTC A193R-F TACATCCGTGGTGCAGGTCCC A193R-R TGCACCACGGATGTAAACCGT A193M-F TACATCATGGGTGCAGGTCCC A193M-R TGCACCCATGATGTAAACCGT M219K-F GGCGACAAAAACGCCGAACGT M219K-R GGCGTTTTTGTCGCCAACGAT V283L-F GCTACCTTACTTAACAGTTTG V283L-R GTTAAGTAAGGTAGCCGGAGC L237Y-F GTGGATTACTCGAAAGATGCA L237Y-R TTTCGAGTAATCCACGGTTTC H268E-F GAAGCGGAAGGGCACGGATCT H268E-R GTGCCCTTCCGCTTCAAAACC H268Y-F GAAGCGTACGGGCACGGATCT H268Y-R GTGCCCGTACGCTTCAAAACC

[0218] 3. Screening of Pc ME mutants

[0219] According to the sequence of Pc ME (the amino acid sequence is shown in SEQ ID NO.18), mutation primers were designed respectively (as shown in Table 12), and site-directed mutagenesis was performed on Pc ME on the plasmid; according to the method in step 1, pure enzyme solutions containing mutants V300Q, V300L, G390L, Q391Y, L394K, L394R, Q391Y / L394K, Q391Y / L394R, V300Q / Q391Y / L394K, and V300L / Q391Y / L394K were prepared respectively;

[0220] Sequence of Pc ME (SEQ ID NO.18)

[0221] MTERKSNYPL YTHYSGPALL EMPLLNKGSA FSPQERIDFN LIGLLPQTVE TIEEQAERVY 60

[0222] QQYLQCASDL DRHVYLRSIQ DNNETLFFRL LEGHLEEMLP IIYTPTVGQA CQDFSKIYRT 120

[0223] HRGLFVSWPD RERIDDVLRS ATKDKVKIIV VTDSERILGL GDQGIGGMGIPIGKLSLYTA 180

[0224] CGGISPAYTL PIVLDVGTNN QALLDDPLYM GWRHPRISGK DYDDFVELFI RAVQRRWPGV 240

[0225] LLQFEDFAQT NAMPLLARYR DQLCCFNDDIQGTASVAVGT LLAACKVKQQ KLSEQVITFV 300

[0226] GAGSAGCGIA EHIIAAMQLE GLSEAQARQR IFMVDRHGLL VEGMDGLLDF QRKLAQKAVD 360

[0227] VADWQRGEDG IGLLEVVSRA KPTVMIGVSG QRGLFSEAVI REMYKHCPLP LVMPLSNPTS 420

[0228] RVEATPAEIL QWTEGRALVA TGSPFQPVEH GGRSIHIAQC NNSYIFPGIG LGVIAAGARR 480

[0229] VTEAMLMAAS NALAECSPVV TGEGDAVLPP LRDIQQVSRR IAFAVAKQAQ AEGVALETPD 540

[0230] DMLQAAIERN FWVPRYREYR RRSLLE 566

[0231] Table 12: Primers

[0232] Primers for mutagenesis Sequence(5’-3’) V300Q-F ACATTTCAAGGAGCAGGGTCC V300Q-R TGCTCCTTGAAATGTAATGAC V300L-F ACATTTTTGGGAGCAGGGTCC V300L-R TGCTCCCAAAAATGTAATGAC G390L-F GTGTCCTTGCAGCGCGGGCTG G390L-R GCGCTGCAAGGACACTCCAAT Q391Y-F GTGTCCGGATATCGCGGGCTGTTT Q391Y-R GCGATATCCGGACACTCC L394K-F CGCGGGAAGTTTAGCGAGGCC L394K-R GCTAAACTTCCCGCGCTGTCC L394R-F CGCGGGCGTTTTAGCGAGGCC L394R-R GCTAAAACGCCCGCGCTGTCC

[0233] The enzyme activities of pure enzyme solutions were detected respectively. The results showed that the enzyme activities of V300Q, V300L, G390L, Q391Y, L394K, L394R, Q391Y / L394K, Q391Y / L394R, V300Q / Q391Y / L394K, and V300L / Q391Y / L394K were 1.08 U / mg, 1.25 U / mg, 1.06 U / mg, 1.49 U / mg, 1.25 U / mg, 1.18 U / mg, 5.02 U / mg, 3.43 U / mg, 11.12 U / mg, and 8.43 U / mg respectively. Among them, according to the same method, the enzyme activity of the wild-type enzyme was 0.20 U / mg. The results showed that protein engineering modification of Pc ME by the same method provided adaptability to NUDH, and the optimal mutant Pc ME*(V300Q / Q391Y / L394K) was obtained, with an activity of 11.12 U / mg towards NUDH( Figure 3 in D).

[0234] Example 4: Efficient driving of the FGPM pathway by NUDH to synthesize malic acid

[0235] The specific steps are as follows:

[0236] 1. Construction of strain K03

[0237] (1) Construction of the recombinant vector

[0238] The Ts FDH*(V198M / T21E / V256L), Bm FADH*A193R / M219K / L237R / H268Y / V283L) and Pp GALS gene (the gene sequence is shown in SEQ ID NO.11) prepared in Example 3 were respectively ligated to the vector pETAC in turn by homologous recombination to obtain the expression vector: pETAC-Ts FDH*-Bm FADH*-Pp GALS, named: pETAC-1; The acetyl phosphate synthase (Bm APS, the gene sequence is shown in SEQ ID NO.12), Bs PTA (the gene sequence is shown in SEQ ID NO.13), Da rPFOR (the gene sequence is shown in SEQ ID NO.14) and Pc ME*(V300Q / Q391Y / L394K) were respectively ligated to the vector pTET in turn by homologous recombination to obtain the expression vector pTET-Bm APS-Bs PTA-DarPFOR-Pc ME*, named pTET-1.

[0239] (2) Construction and evaluation of recombinant strains

[0240] The plasmids pETAC-1 and pTET-1 were co-transformed into the strain K02-R11 (E. coli Mg1655-1 / p15A-RBS-03-Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-Ps PTDH*-RBS-03-Sl UMPK-RBS-03-Da NDK-RBS-01-Vp NAMPT-RBS-01-Ft NadE) to obtain the strain K03 that can use NUDH as the driving force to convert carbon dioxide and formic acid into malic acid.

[0241] Single colonies K03 were picked from the plate and inoculated into LB medium, and cultured overnight (6-8 h) at 37 °C and 220 r / min to prepare the seed solution. Subsequently, the prepared seed solution was transferred to 50 mL of TB medium for culture at an inoculation amount of 5% (v / v), and cultured at 37 °C and 200 rpm until the OD 600 reached 0.6-0.8, IPTG with a final concentration of 0.6 mM was added for induction, and the cells were collected by centrifugation after induction at 30 °C for 16 h.

[0242] The cells collected under the above conditions were used for whole-cell transformation and evaluation in 250 mL serum bottles and 7.5 L fermenters respectively.

[0243] Conditions for shake-flask fermentation:

[0244] The liquid loading of the serum bottle is 150 mL, the addition amount of the bacterial cells is 3.5 g / L, and the Tris-HCl solution with pH = 7.0 is used as the medium for whole-cell transformation. 300 NaHCO 3 (providing CO 2 ), and at the same time, 50 mM formic acid and 10 mM MgSO 4 are added. And by adding the phosphite mother liquor with a concentration of 8 M (pH 7.5), the phosphite concentration is maintained at 1 - 5 mM. The catalytic reaction is carried out for 35 hours at 37 °C and 50 rpm.

[0245] Fermentation conditions of the 7.5 L fermenter:

[0246] In the 7.5 L fermenter, the liquid loading is 4.5 L, the addition amount of the bacterial cells is 3.5 g / L, and the Tris-HCl solution with pH = 7.0 is used as the medium for whole-cell transformation. CO 2 gas is introduced and the pressure is maintained at 0.05 MPa. At the same time, 75 mM formic acid and 10 mM MgSO 4 are added, and the phosphite concentration is maintained at 5 - 10 mM. The catalytic reaction is carried out for 35 hours at 37 °C.

[0247] The results show that the malic acid yields in the 250 mL serum bottle and the 7.5 L fermenter reach 37.76 mM and 50.61 mM respectively ( Figure 3 E in).

[0248] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A nicotinamide transferase mutant, characterized in that: The nicotinamide transferase mutant is subjected to site-directed mutagenesis based on the amino acid sequence of the wild-type nicotinamide transferase, the amino acid sequence of the wild-type nicotinamide transferase is shown in SEQ ID NO.2, and the mutation mode is selected from: L27K, V186L, V186I, V186M, L27K / V186L, L27K / V186I, L27K / V186L / T187K, L27K / V186L / T187Y, L27K / V186L / T187K / D220E or L27K / V186L / T187K / D220Q.

2. A phosphite dehydrogenase mutant, characterized in that: The phosphite dehydrogenase mutant is subjected to site-directed mutagenesis based on the amino acid sequence of the wild-type phosphite dehydrogenase, the amino acid sequence of the wild-type phosphite dehydrogenase is shown in SEQ ID NO.4, and the mutation mode is selected from: L151Y, L151W, A176R, A207N, A207M, L151Y / A176R, L151W / A176R, A176R / A207N, A176R / A207M, L151Y / A176R / L208F or L151Y / A176R / N211I.

3. A formate dehydrogenase mutant, characterized in that The formate dehydrogenase mutant is subjected to site-directed mutagenesis based on the amino acid sequence of the wild-type formate dehydrogenase, the amino acid sequence of the wild-type formate dehydrogenase is shown in SEQ ID NO.16, and the mutation mode is selected from: V198N, V198M, T221E, T221L, T221D, V256L, T221D / V256L, T221E / V256L, T221L / V256L, V198N / T221E / V256L or V198M / T221E / V256L.

4. A formaldehyde dehydrogenase mutant, characterized in that The formaldehyde dehydrogenase mutant is subjected to site-directed mutagenesis based on the amino acid sequence of the wild-type formaldehyde dehydrogenase, the amino acid sequence of the wild-type formaldehyde dehydrogenase is shown in SEQ ID NO.17, and the mutation mode is selected from: L237R, A193R, A193M, V283L, L237Y, H268E, H268Y, A193R / V283L or A193R / M219K / L237R / H268Y / V283L.

5. A malic enzyme mutant, characterized in that The malic enzyme mutant is subjected to site-directed mutagenesis based on the amino acid sequence of the wild-type malic enzyme, the amino acid sequence of the wild-type malic enzyme is shown in SEQ ID NO.18, and the mutation mode is selected from: V300Q, V300L, G390L, Q391Y, L394K, L394R, Q391Y / L394K, Q391Y / L394R, V300Q / Q391Y / L394K or V300L / Q391Y / L394K.

6. A gene encoding the mutant according to any one of claims 1 to 5 or a recombinant vector carrying the gene.

7. A recombinant cell expressing the mutant according to any one of claims 1 to 5 or carrying the gene or the recombinant vector according to claim 6; preferably, the recombinant cell uses bacteria or fungi as expression hosts.

8. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria express one or more of the mutants described in any one of claims 1 to 5; Or the genetically engineered bacteria is: simultaneously overexpressing the nicotinamide transferase mutant according to claim 1 and the phosphite dehydrogenase mutant according to claim 2; Or the genetically engineered bacteria is: simultaneously overexpressing the nicotinamide transferase mutant according to claim 1 and the phosphite dehydrogenase mutant according to claim 2, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GSGSGGSGSG, GSGSGGSGSGGSGSG or GSGSGGSGSGGSGSGGSGSG as the connecting peptide to obtain a fusion enzyme; Or the genetically engineered bacteria is: simultaneously overexpressing the nicotinamide transferase mutant of claim 1, the phosphite dehydrogenase mutant of claim 2, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, and NMN synthase, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GSGSGGSGSG, GSGSGGSGSGGSGSG or GSGSGGSGSGSGSGSGGSGSG as the connecting peptide to obtain a fusion enzyme; Or the genetically engineered bacteria is: simultaneously overexpressing the nicotinamide transferase mutant of claim 1, the phosphite dehydrogenase mutant of claim 2, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, and NMN synthase, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GSGSGGSGSG, GSGSGGSGSGGSGSG or GSGSGGSGSGSGSGSGGSGSG as the connecting peptide to obtain a fusion enzyme; At the same time, RBS-01, RBS-03 or RBS-05 is used to replace the original RBS in front of uridine kinase and nucleoside diphosphate kinase: AAAGAGGAGAAA, RBS-01, RBS-03 or RBS-05 is used to replace the original RBS in front of nicotinamide phosphoribosyltransferase and NMN synthase: AAAGAGGAGAAA, and RBS-01, RBS-03 or RBS-05 is used to replace the original RBS in front of fusion enzyme: AAAGAGGAGAAA to obtain the strain; Or the genetically engineered bacteria is: simultaneously overexpressing the nicotinamide transferase mutant of claim 1, the phosphite dehydrogenase mutant of claim 2, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, NMN synthase, the formate dehydrogenase mutant of claim 3, the formaldehyde dehydrogenase mutant of claim 4 and the malic enzyme mutant of claim 5, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GSGSGGSGSG, GSGSGGSGSGGSGSG or GSGSGGSGSGSGSGGSGSG as the connecting peptide to obtain a fusion enzyme; At the same time, RBS-01, RBS-03 or RBS-05 is used to replace the original RBS in front of uridine kinase and nucleoside diphosphate kinase: AAAGAGGAGAAA, RBS-01, RBS-03 or RBS-05 is used to replace the original RBS in front of nicotinamide phosphoribosyltransferase and NMN synthase: AAAGAGGAGAAA, and RBS-01, RBS-03 or RBS-05 is used to replace the original RBS in front of fusion enzyme: AAAGAGGAGAAA to obtain the strain; Or the genetically engineered bacteria simultaneously overexpress the nicotinamide transferase mutant according to claim 1, the phosphite dehydrogenase mutant according to claim 2, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, NMN synthase, the formate dehydrogenase mutant according to claim 3, the formaldehyde dehydrogenase mutant according to claim 4, the malic enzyme mutant according to claim 5, glycolaldehyde synthase, acetyl phosphate synthase, phosphate acetyltransferase, and ferredoxin oxidoreductase, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GSGSGGSGSG, GSGSGGSGSGGSGSG or GSGSGGSGSGSGGSGSGGSG as the connecting peptide to obtain a fusion enzyme; At the same time, RBS-01, RBS-03 or RBS-05 is used to replace the original RBS in front of uridine kinase and nucleoside diphosphate kinase: AAAGAGGAGAAA, RBS-01, RBS-03 or RBS-05 is used to replace the original RBS in front of nicotinamide phosphoribosyltransferase and NMN synthase: AAAGAGGAGAAA, and RBS-01, RBS-03 or RBS-05 is used to replace the original RBS in front of fusion enzyme: AAAGAGGAGAAA to obtain the strain; The RBS-01 is GGAAACCCTCAGGAGGTAAACCA, the RBS-03 is ATGGTCTGATCTCCTACGGTTATA, and the RBS-05 is AGGAATGTATCAATTGAGGTTTAA; Preferably, the genetically engineered bacteria uses E. coli MG1655, E. coli W3110 or E. coli BL21 (DE3) as the expression host.

9. A method for preparing malic acid, characterized in that: The method comprises the following steps: using the genetically engineered bacteria as claimed in claim 8 to carry out fermentation to prepare malic acid.

10. Use of the mutant according to any one of claims 1 to 5, or the gene or recombinant vector according to claim 6, or the recombinant cell according to claim 7, or the genetically engineered bacteria according to claim 8 in the preparation of malic acid or products containing malic acid.

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