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

Abstract

The application discloses development of a novel energy system and application of the novel energy system in conversion of one-carbon substrates, and belongs to the technical field of bioengineering. The application discloses a synthesis of the novel energy system by taking intracellular metabolites nicotinamide and uridine triphosphate as substrates. After fusing a nicotinamide transferase mutant and a phosphite dehydrogenase mutant, the mutant is introduced into escherichia coli together with uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyl transferase and NMN synthetase after RBS optimization. Meanwhile, a formic acid dehydrogenase mutant, a formaldehyde dehydrogenase mutant, a malic enzyme mutant, an ethanol aldehyde synthase, an acetyl phosphate synthase, a phosphoacetyl transferase and a ferredoxin oxidoreductase are introduced, and a high-yield malic acid strain is obtained. The yield of the malic acid can be up to 51.27 mM.

Description

Technical Field

[0001] This 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 Technology

[0002] One-carbon compounds (such as carbon dioxide and formic acid) are abundant renewable carbon resources, and there is an urgent need to develop green conversion technologies to transform them into high-value-added chemicals, such as proteins, esters, alcohols, ethers, and acids. Currently, there are three main methods for utilizing one-carbon compound resources: chemical, physical, and biological methods. Among these, microbial utilization of one-carbon compounds is considered a green, efficient, and sustainable method due to the rapid growth and high productivity of heterotrophic cells. To enable heterotrophic microorganisms to efficiently utilize one-carbon compounds, two key issues need to be addressed: how to design efficient one-carbon compound immobilization pathways to convert them into high-value chemicals; and 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 offers a promising alternative. Regarding energy supply, developing novel energy systems provides a new solution because they can overcome the limitations of traditional energy systems, accumulate at high intracellular concentrations without interfering with the host metabolic network, and promote intracellular one-carbon utilization pathways. However, novel energy systems capable of efficient synthesis and cycling are still lacking. Therefore, establishing efficient novel energy systems to drive novel intracellular one-carbon pathways is an effective strategy to improve the efficient utilization of one-carbon compounds by heterotrophic microorganisms. Summary of the Invention

[0004] This invention provides a novel method for constructing an energy system and has been successfully applied to the synthesis of malic acid from carbon dioxide and formic acid by *E. coli*. Details are as follows:

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

[0006] (b) The nicotinamide transferase Hs Nmnat, whose amino acid sequence is shown in SEQ ID NO.2, was modified by protein engineering to obtain the mutant Hs Nmnat*, which can efficiently utilize NMN and UTP to synthesize the oxidized energy molecule NUD;

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

[0008] (d) Introducing Hs Nmnat* and Ps PTDH* into Escherichia coli enabled the synthesis of intracellular NUD and NUDH; metabolic engineering and optimization promoted the accumulation of intracellular UTP and NMN, and intracellular metabolic balance was achieved through RBS combination optimization, with the final NUDH content reaching 8.73 mM.

[0009] (e) Based on the MetaCyc and ATLAS databases, a six-step FGPM carbon fixation pathway was designed to convert formic acid and carbon dioxide into malic acid. Protein engineering was performed on formic acid dehydrogenase (Ts FDH), formaldehyde dehydrogenase (Bm FADH), and malic acid enzyme (Pc ME) in the metabolic pathway to obtain mutants TsFDH* (V198M / T21E / V256L), Bm FADH* (A193R / M219K / 237R / H268Y / V283L), and Pc ME* (V300Q / Q391Y / L394K) that can efficiently utilize NUDH.

[0010] (f) The FGPM pathway was constructed in the NUDH synthetic strain, which enabled 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 site of the mutant Hs Nmnat* is L27K / V186L / T187K / D220E, and the mutation site of Ps PTDH* is 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*, which 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 gene.

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

[0017] This invention also provides specific sequences used for RBS optimization.

[0018] The present invention also provides gene sequences for enhanced expression through metabolic engineering.

[0019] In one embodiment, the microbial cells use the prokaryotic Escherichia coli Mg1655 as a host.

[0020] This invention also provides a method for obtaining the above-mentioned highly efficient novel energy system for catalytic synthesis of malic acid from carbon dioxide and formic acid, the method comprising the following steps:

[0021] (1) Through iterative mutation and screening, enzymes Hs Nmnat* and Ps PTDH*, which can synthesize NUD and NUDH using UTP and NMN, were obtained;

[0022] (2) Hs Nmnat* and Ps PTDH* were expressed in the p15A vector to obtain the vector p15A-1. p15A-1 was transformed into the host cell Escherichia coli Mg1655. Positive clones were selected to obtain strain K01, and the intracellular NUD and NUDH content was detected.

[0023] (3) Genes SlUMPK and DaNDK, which enhance the UTP synthesis module, were introduced into the p15A-1 vector to obtain the expression vector p15A-2; similarly, genes VpNAMPT and FtNadE, which enhance the NMN synthesis module, were introduced to obtain p15A-3; p15A-3 was transformed into the host cell Escherichia coli Mg1655-01 (by knocking out genes pyrG, purR, pncC, UshA and pnuC related to the UTP and NMN catabolism pathway in E. coli Mg1655), and positive clones were selected to obtain strain K02, which is a strain that synthesizes NUDH efficiently.

[0024] (4) Through iterative mutation and screening, enzymes TsFDH*, BmFADH* and PcME* that can utilize NUDH as an energy molecule were obtained;

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

[0026] (6) Plasmids pETAC-1 and pTET-1 were co-transformed into strain K02 to obtain strain K03, which 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 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: reaction at pH 7.0 and 37°C for 35 hours.

[0031] The present invention also provides the application of the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant Escherichia coli in the development of novel energy systems and the conversion of one-carbon substrates into malic acid.

[0032] This invention provides a nicotinamide transferase mutant, wherein the mutant is a site-directed mutation based on the amino acid sequence of the wild-type nicotinamide transferase, as 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.

[0033] This invention provides a phosphite dehydrogenase mutant, wherein the mutant is a site-directed mutation based on the amino acid sequence of the wild-type phosphite dehydrogenase, the amino acid sequence of which 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.

[0034] This invention provides a formate dehydrogenase mutant, wherein the mutant is based on a site-directed mutation of the amino acid sequence of wild-type formate dehydrogenase, the amino acid sequence of which 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.

[0035] This invention provides a formaldehyde dehydrogenase mutant, wherein the mutant is based on a site-directed mutation of the amino acid sequence of wild-type formaldehyde dehydrogenase, the amino acid sequence of which 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.

[0036] This invention provides a malic acid enzyme mutant, wherein the mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type malic acid enzyme, the amino acid sequence of which 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, and V300L / Q391Y / L394K.

[0037] The present invention also provides genes encoding the above-mentioned nicotinamide transferase mutant, phosphite dehydrogenase mutant, formate dehydrogenase mutant, formaldehyde dehydrogenase mutant and malic acid enzyme mutant, or recombinant vectors carrying said genes.

[0038] The present invention also provides recombinant cells expressing nicotinamide transferase mutants, phosphite dehydrogenase mutants, formate dehydrogenase mutants, formaldehyde dehydrogenase mutants, and malic acid enzyme mutants, or carrying genes of the above mutants or the recombinant vectors thereof.

[0039] In one embodiment, the recombinant cells use bacteria or fungi as expression hosts.

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

[0041] In one embodiment, the genetically engineered bacteria 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, wherein the genetically engineered bacterium is simultaneously overexpressing the above-mentioned nicotinamide transferase mutant and the above-mentioned phosphite dehydrogenase mutant.

[0044] In one embodiment, the genetically engineered bacteria 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, wherein the genetically engineered bacterium is: simultaneously overexpressing 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 together with GGSGSGGSGSG, GGSGSGSGSGSGSGSG or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain a fusion enzyme.

[0046] In one embodiment, the genetically engineered bacteria 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, wherein the genetically engineered bacterium is: simultaneously overexpressing the above-mentioned nicotinamide transferase mutant, the above-mentioned phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, and NMN synthase, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused together with GGSGSGGSGSG, GGSGSGSGSGSGSGSG or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain a fusion enzyme.

[0048] In one embodiment, the genetically engineered bacteria 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, wherein the genetically engineered bacterium is: simultaneously overexpressing the above-mentioned nicotinamide transferase mutant, the above-mentioned phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, and NMN synthase, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GGSGSGGSGSG, GGSGSGSGSGSGSGSG or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain a fusion enzyme;

[0050] Simultaneously, strains were obtained by replacing the original RBS (AAAGAGGAGAAA) before uridine kinase and nucleoside diphosphate kinase with RBS-01, RBS-03, or RBS-05; replacing the original RBS (AAAGAGGAGAAA) before nicotinamide phosphoribosyltransferase and NMN synthase with RBS-01, RBS-03, or RBS-05; and replacing the original RBS (AAAGAGGAGAAA) before fusion enzymes with RBS-01, RBS-03, or RBS-05.

[0051] RBS0-Hs Nmnat mutant-linking peptide-Ps PTDH mutant-RBS0-Sl UMPK-RBS0-Da NDK-RBS0-Vp NAMPT-RBS0-Ft NadE; replace any of the above RBS0 with RBS-01, RBS-03 or RBS-05.

[0052] In one embodiment, the genetically engineered bacteria 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, wherein the genetically engineered bacterium is: simultaneously 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 and the above-mentioned malic enzyme mutant, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GGSGSGGSGSG, GGSGSGSGSGSGSGSG, or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain a fusion enzyme;

[0054] Meanwhile, strains were obtained by replacing the original RBS preceding uridine kinase and nucleoside diphosphate kinase with RBS-01, RBS-03 or RBS-05, replacing the original RBS preceding nicotinamide phosphoribosyltransferase and NMN synthase with RBS-01, RBS-03 or RBS-05, and replacing the original RBS preceding fusion enzyme with RBS-01, RBS-03 or RBS-05.

[0055] RBS0-Hs Nmnat mutant-linking peptide-Ps PTDH mutant-RBS0-Sl UMPK-RBS0-Da NDK-RBS0-Vp NAMPT-RBS0-Ft NadE; replace any of the above RBS0 with RBS-01, RBS-03 or RBS-05.

[0056] In one embodiment, the genetically engineered bacteria 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] This invention also provides a genetically engineered bacterium, which is: simultaneously 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 acid enzyme mutant, alcohol aldehyde synthase, acetyl phosphate synthase, phosphoacetyltransferase, and ferroreductase, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused with GGSGSGGSGSG, GGSGSGSGSGSGSGSG or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain a fusion enzyme;

[0058] Simultaneously, strains were obtained by replacing the original RBS (AAAGAGGAGAAA) before uridine kinase and nucleoside diphosphate kinase with RBS-01, RBS-03, or RBS-05; replacing the original RBS (AAAGAGGAGAAA) before nicotinamide phosphoribosyltransferase and NMN synthase with RBS-01, RBS-03, or RBS-05; and replacing the original RBS (AAAGAGGAGAAA) before fusion enzyme with RBS-01, RBS-03, or RBS-05.

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

[0060] The present invention also provides a method for preparing malic acid, wherein the method comprises fermenting the above-mentioned genetically engineered bacteria to obtain malic acid.

[0061] In one embodiment, the final concentration of the genetically engineered bacteria 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: pH 7.0, 30–38°C, for 30–40 h.

[0065] Preferably, the reaction is carried out at pH 7.0 and 37°C for 35 hours.

[0066] In one implementation, the reaction is carried out in a fermenter, with fermentation conditions for a 7.5L fermenter as follows:

[0067] In a 7.5L fermenter, the initial liquid volume is 4.5L, and the amount of bacterial cells added is 2-4 g / L, preferably 3.5 g / L. A Tris-HCl solution with pH=7.0 is used as the medium for whole-cell transformation. CO2 gas is introduced and the pressure is maintained at 0.05 MPa. At the same time, 50-100 mM formic acid, preferably 75 mM formic acid, and 5-15 mM MgSO4, preferably 10 mM MgSO4, are added, and the phosphite concentration 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 application of the above-mentioned nicotinamide transferase mutant, phosphite dehydrogenase mutant, formate dehydrogenase mutant, formaldehyde dehydrogenase mutant and malic acid enzyme mutant, or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned genetically engineered bacteria in the preparation of malic acid or products containing malic acid.

[0069] Beneficial effects

[0070] (1) This invention provides enzymes HsNmnat* 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, wherein the NUDH concentration accumulates in the cell to 8.73 mM and the NUDH / NUD ratio reaches 58.19.

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

[0073] (4) In this study, the FGPM pathway and NUDH were used together to synthesize 51.27 mM malic acid using carbon dioxide and formic acid as substrates. Attached Figure Description

[0074] Figure 1 Hs Nmnat* and Ps PTDH* were engineered for protein production.

[0075] Figure 2 To enable the intracellular synthesis of a novel energy molecule, NUDH, in Escherichia coli.

[0076] Figure 3 Design of the FGPM pathway and adaptation of the pathway enzyme to NUDH. Detailed Implementation

[0077] In the expression of the strains involved in the following embodiments, the expression vector used can be any conventional Escherichia coli vector suitable for expression to achieve the technical solution of the present invention. In one embodiment, the expression vector can be: pET series vector, preferably pET28a, pET21a, pET32a; pGEX series vector; pMAL series vector; pQE series vector; pTrc series vector; pBAD series vector, etc.

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

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

[0080] It should be noted that the solution of the present invention is not limited to the above-mentioned expression vector. The technical solution of the present invention can be achieved by selecting an expression vector according to the host cell (Escherichia coli).

[0081] The amino acid mutations involved in this invention are all abbreviated. The detailed 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), and valine Val (V). In the expression of amino acid mutations, for example, L151Y indicates that the L at position 151 of the wild-type enzyme is mutated to Y; for example, L151Y / A176R / L208F indicates that the L at position 151 of the wild-type enzyme is mutated to Y, and the A at position 176 is mutated to R, while the L at position 208 is mutated to F. Other mutants of this invention are expressed in the same way as above.

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

[0083] Nicotinamide transferase (Nmnat) activity assay:

[0084] The total reaction volume was 200 μL, containing 0.5 mg of enzyme, 50 mM HEPES buffer (pH 7.5), 5 mM MnCl2, 10 mM MgCl2, 2.0 mM NMN (nicotinamide mononucleotide), and 2.5 mM UTP (uridine triphosphate). The reaction was carried out at 30 °C. The enzyme reaction was terminated with 0.155 mol / L EDTA. Enzyme activity was determined by measuring the consumption of substrate UTP at 450 nm using a kit (Shanghai Kehansheng Biotechnology Co., Ltd.).

[0085] PTDH activity assay:

[0086] The reaction system consisted of 200 μL of 0.5 mg phosphite dehydrogenase, 50 mM HEPES buffer (pH 7.5), 5 mM phosphite, 1.0 mM tetrazolium blue, 0.5 mM phenazine methyl sulfate, and 0.5 mM NUD (nicotinamide uracil dinucleotide). The enzyme solution was added to initiate the reaction at 25 °C. In the enzymatic reaction, phenazine methyl sulfate transferred electrons gained from NUDH to the acceptor tetrazolium blue, reducing it to form blue formalin. Formalin formation was monitored by observing absorbance changes at a specific wavelength (570 nm), and the amount of NUDH produced and enzyme activity were assessed.

[0087] Formate dehydrogenase activity assay:

[0088] The CO2 reducing activity of formate dehydrogenase was determined in 100 mM HEPES buffer (pH 7.0) at 37 °C. The CO2 concentration was expressed as NaHCO3 concentration. The total reaction volume was 400 μL, including 1.0 mg formate dehydrogenase, 200 mM NaHCO3, and 4 mM NUDH. The formic acid product was determined using the following method: Solution A was prepared by dissolving 0.5 g citric acid and 10 g acetamide in 100 mL isopropanol. Solution B was prepared by dissolving 30 g sodium acetate in 100 mL water. During the assay, 100 μL of the sample after catalysis was completed was mixed with 200 μL of solution A, 10 μL of solution B, and 700 μL of pure acetic anhydride. The mixture was then spectrophotometrically measured at 515 nm to determine the catalytic performance and enzyme activity for formic acid production.

[0089] Formaldehyde dehydrogenase activity assay:

[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 30 °C. The total volume of the reaction system was 400 μL, containing 1.0 mg formaldehyde dehydrogenase, 35.0 mM formic acid, and 4 mM NUDH. The formaldehyde product was detected using a colorimetric method. The principle of this method is that formaldehyde reacts with chromic acid in concentrated sulfuric acid solution to form a purple compound with a maximum absorption wavelength of 580 nm. Specifically, 0.1 mL of chromic acid solution (0.1-2% by mass) was added to 0.5 mL of the formaldehyde dehydrogenase reaction product, followed by 0.6 mL of concentrated sulfuric acid. After mixing, the mixture was heated to boiling, and then the formaldehyde yield per unit time was measured at a wavelength of 580 nm.

[0091] Malase activity assay:

[0092] The pyruvate reduction and carboxylation activity of malicase was determined using a standard reaction system. The total volume of the reaction system was 200 μL, containing 100 mM Tris-HCl buffer (pH 7.5), 0.5 mg malicase, 5 mM MgSO4, 4 mM NUDH, 100 mM NaHCO3, and 50 mM pyruvate. The reaction was initiated by adding 1.0 mL of enzyme solution and incubated at 30 °C. After translation was complete, the reaction was terminated by adding 1.0 mL of 10% (w / v) sodium dodecyl sulfate (SDS). The reaction solution was mixed with 1% (w / v) 2,7-dihydroxynaphthalene solution at 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, KH2PO4 2.31 g / L, K2HPO4 16.43 g / L, glycerol 4 g / L;

[0096] Fermentation medium: K2HPO4 5.0g / L, KH2PO4 3.5g / L, 0.35g(NH4)2HPO4 3.5g / L, MgSO4·7H2O 0.25g / L, Vitamin B1 0.5mg / L, CaCl2·2H2O 15mg / L, glucose 5g / L, 10mL trace element solution.

[0097] The formula for the trace element solution is as follows: FeCl3 1.6 g / L, CuCl2 0.1 g / L, NaMnO4 0.2 g / L, CoCl2·6H2O 0.2 g / L, H3BO3 0.05 g / L, ZnCl2·4H2O 0.2 g / L, dissolved in 0.1 M HCl solution.

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

[0099] This invention designs a pathway for synthesizing novel oxidized energy molecules NUD and reduced energy molecules NUDH using UTP and NMN. Figure 1 (A) In this process, NUD is synthesized from UTP and NMN using nicotinamide transferase (Nmnat), and then NUD is catalyzed to NUDH using phosphite dehydrogenase (PTDH).

[0100] 1. Screening of nicotinamide transferase

[0101] To synthesize NUD using UTP and NMN, genes for 27 nicotinamide transferases (Nmnat) from the UniProtKB protein database were synthesized. These genes were 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*, and *Kocuria*. * *Alloscardovia macacae*, *Limosilactobacillus balticus*, *Lactobacillus kefiranofaciens*, *Saccharomyces cerevisiae*, *Homosapiens*, *Mycolicibacterium vanbaalenii*, *Acetothermiabacterium*, *Anaerotignum lactatifermentans*, *Mycobacterium ulcerans*, *Streptomyces coeruleorubidus*, *Bifidobacterium asteroides*, *Xanthomonas translucens*, and *Paraburkholderia solisilvae*.

[0102] The synthesized sequences were ligated into plasmid pET-28. The constructed plasmids were further transformed into E. coli BL21(DE3) competent cells to obtain recombinant bacteria. The recombinant bacteria were inoculated into LB liquid medium and cultured at 37°C and 200 rpm for 8 h to prepare seed culture. The prepared seed culture was then inoculated into TB medium at a 2% (v / v) inoculation rate for protein expression. After culturing at 37°C and 220 rpm for 2 h, when the OD600 reached 0.6-0.8, IPTG (final concentration 0.1 mM) was added, and the culture was induced at 30°C and 220 rpm for 16-18 h. The bacterial culture was then centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was discarded to collect the bacterial cells. After collection, the bacterial cells were subjected to low-temperature disruption and protein purification. The performance of nicotinamide transferases from different sources in catalyzing the generation of NUD using UTP and NMN as substrates was tested according to the previously provided method. Among them, human-derived NMNAT (Hs Nmnat, PDB: 1KQN) exhibited the highest catalytic activity. Figure 1 The gene sequence of B is shown in SEQ ID NO.1.

[0103] 2. Screening for nicotinamide transferase mutants

[0104] To improve the catalytic performance of the human-derived nicotinamide transferase, HsNmnat, obtained in step 1, relevant residues were modified through protein engineering. The specific iterative process and catalytic effects are detailed in [link to relevant documentation]. Figure 1 C in the text. The specific process is as follows:

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

[0106] The 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 consisted of the following components: template DNA (100-150 ng), forward and reverse primers (20 μM, 2 μl each), PrimeSTAR polymerase (1 μl), 5×PrimeSTAR buffer (20 μl), dNTP mixture (8 μl), and sterile water. The PCR amplification conditions were as follows: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 0.5 min; 55℃ annealing for 0.5 min; 72℃ extension for 3 min; for a total of 30-32 cycles; and a final extension at 72℃ for 5 min, followed by pre-storage at 12℃ for 5 min. The PCR amplification products were detected by 1% agarose gel electrophoresis. After confirming the correct product size, 0.5 μL of LpnI digestive enzyme was added to 10 μL of the amplification product, mixed well, and reacted at 37℃ for 40 min, followed by inactivation of the digestive enzyme at 60℃ for 10 min. Subsequently, the amplification products treated with DpnI were transformed into E. coli JM109 competent cells, and the transformation products were plated on cells supplemented with kanamycin (final concentration 50 mg / L). -1 LB solid medium was cultured at 37°C for 8–10 h, and single colonies were grown on the plates.

[0116] Select positive clones and inoculate them with kanamycin (final concentration 50 mg / L). -1Plasmids were extracted and sequenced for verification after culturing in LB liquid medium at 37°C and 220 rpm for 6-8 h. The correctly sequenced pET28a-Hs Nmnat mutant vector was added to E. coli BL21(DE3) strain to prepare genetically engineered bacteria. The prepared genetically engineered bacteria were inoculated into LB medium and cultured at 37°C and 200 rpm for 8 h to prepare seed culture. The seed culture was then transferred to TB medium at an inoculation rate of 10% (v / v) for protein expression induction. After culturing at 37°C and 220 rpm for 2 h until the OD600 reached 0.6-0.8, IPTG (final concentration 0.1 mM) was added, and the culture was inducible at 30°C and 220 rpm for 16-18 h. The bacterial culture was centrifuged at 4℃ and 8000r / min for 10min, the supernatant was discarded, the bacterial cells were collected and the cells were broken up and the protein was purified to obtain pure enzyme solutions of L27K, V186L, V186I and V186M mutants, respectively.

[0117] Following the above method for detecting nicotinamide transferase activity, the enzyme activity of different mutants was detected. 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. A pure enzyme solution of the wild-type enzyme was prepared according to the above method, and its enzyme activity was detected to be 2.25 U / mg.

[0118] (2) The optimal mutant L27K was used for iteration. The construction method was as follows: using one of the single-point mutants as a template, full-plasmid PCR primers for other mutation sites were designed, and double mutant plasmids with correct mutations were obtained through template digestion, product purification, transformation, and selection of clones for sequencing verification. The methods for triple and quadruple mutations were the same as above. Double mutants L27K / V186L and L27K / V186I were generated, and further iterations yielded triple and quad mutants: L27K / V186L / T187K, L27K / V186L / T187Y, L27K / V186L / T187K / D220E, and L27K / V186L / T187K / D220Q. Following the method in step (1), pure enzyme solutions containing mutants L27K / V186L, L27K / V186I, L27K / V186L / T187K, L27K / V186L / T187Y, L27K / V186L / T187K / D220E, and L27K / V186L / T187K / D220Q were prepared respectively.

[0119] Table 2: Primers

[0120] 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] Enzyme activities were measured 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) increased by 49.0 times, reaching 110.25 U / mg. Figure 1 (C in the middle).

[0122] 3. Screening of phosphite dehydrogenases

[0123] To convert 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 aeruginosa*, *Stutzerimonas xanthomarina*, *Pseudomonas stutzeri*, *Klebsiella pneumoniae*, *Pseudomonas gessardii*, *Halopseudomonas bauzanensis*, *Klebsiella pneumoniae*, *Halopseudomonas litoralis*, *Halomonas meridiana*, *Achromobacter pulmonis*, *Halomonas desiderata*, *Brucella intermedia*, and *Dendrosporobacter*. Quercicolus, Arthrobacters audimassiliensis, Synechococcus sp., and Beijerinckiaceaebacterium.

[0124] The synthesized sequences were ligated into plasmid pET-28a, and the constructed plasmids were further transformed into E. coli BL21(DE3) competent cells to obtain recombinant bacteria. The recombinant bacteria were inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 6–10 h to prepare seed culture. This seed culture was then inoculated into TB medium at a 5% (v / v) inoculation rate for protein induction. The protein expression induction conditions were as follows: cultured at 37℃ and 220 rpm for 2 h; after the OD600 reached 0.6–0.8, 0.1 mM IPTG was added, and the culture was continued at 25℃ and 220 rpm for 16–18 h. The bacterial culture was then centrifuged at 4℃ and 8000 rpm for 10 min. The bacterial cells were collected, subjected to low-temperature disruption, and the protein was purified. The performance of 16 different phosphite dehydrogenases catalyzing the conversion of NUD to NUDH was tested according to the previously provided method. The results showed that PTDH from *Pseudomonas stearothermiae* (PsPTDH) exhibited the highest catalytic activity for NUD. Figure 1 The gene sequence of D in the sequence is shown in SEQ ID NO.3.

[0125] 4. Screening for phosphite dehydrogenase mutants

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

[0127] The specific process is as follows:

[0128] (1) Based on the sequence of Ps PTDH (amino acid sequence as shown in SEQ ID NO.4), design mutation primers (as shown in Table 3) to mutate the Ps PTDH enzyme on the pET28a-Ps PTDH plasmid; prepare vectors containing L151Y, L151W, A176R, A207N, and A207M mutants respectively; the PCR reaction and the preparation of pure enzyme solution are 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 the mutants L151Y, L151W, A176R, A207N, and A207M were prepared, and their activities were measured. 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. A pure enzyme solution of the wild-type enzyme was prepared using the same method, and its activity was measured to be 5.14 U / mg.

[0136] (2) The optimal mutant A176R was used for iteration. The construction method was as follows: using one of the single-point mutants as a template, full-plasmid PCR primers for other mutation sites were designed, and double mutant plasmids with correct mutations were obtained through template digestion, product purification, transformation, and selection of clones for sequencing verification. The methods for triple mutation and quadruple mutation are the same as above.

[0137] Double mutants L151Y / A176R, L151W / A176R, A176R / A207N, and A176R / A207M were generated. Further iterations yielded triple mutants: L151Y / A176R / L208F and L151Y / A176R / N211I. Following 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] Enzyme activities were measured 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 also showed that Ps PTDH* (L151Y / A176R / L208F) exhibited 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 the middle).

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

[0143] The specific steps are as follows:

[0144] 1. Preparation of genetically engineered bacteria K01

[0145] (1) Preparation of recombinant vector

[0146] To synthesize NUD and NUDH in *E. coli* using UTP and NMN as substrates, the HsNmnat* (L27K / V186L / T187K / D220E) and PsPTDH* (L151Y / A176R / L208F) and p15A vector prepared in Example 1 were amplified using the primers in Table 4. After determining the fragment and vector sizes by agarose gel electrophoresis, they were digested with DpnI enzyme and recovered by column chromatography. Subsequently, the fragments HsNmnat* and PsPTDH* 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 volume was 10 μL, including 2 μL of multi-fragment homologous recombination enzyme, 100 ng of HsNmnat* fragment, 100 ng of PsPTDH* fragment, and 200 ng of p15A vector. The reaction was carried out at 37°C for 30 min, and then directly transformed into competent Escherichia coli JM109 competent cells and plated on cells supplemented with kanamycin (final concentration 50 mg·L⁻¹). -1 LB agar plates. Incubate at 37°C for 6-8 hours, then select positive clones for inoculation with kanamycin (final concentration 50 mg / L). -1 After culturing in LB liquid medium at 37℃ and 220r / min for 6-8h, the plasmid p15A-HsNmnat*-PsPTDH* was extracted and named p15A-1.

[0147] Table 4: Primers

[0148] 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 competent cells of Escherichia coli Mg1655 to produce strain K01.

[0151] 2. NUD and NUDH were prepared by fermentation using strain K01.

[0152] The prepared strain K01 was added to LB medium and cultured at 37℃ and 220 rpm for 6 h to prepare seed culture. The seed culture was then inoculated into TB medium at a 10% (v / v) inoculation rate and cultured at 37℃ and 220 rpm for 2 h. Once the OD600 reached 0.6-0.8, 0.1 mM IPTG was added, and the culture was continued at 37℃ and 220 rpm for 16-18 h to obtain fermentation broth. The fermentation broth was centrifuged, and the bacterial cells were collected and resuspended in PBS solution. The OD600 was then measured... 600 When the ratio is 1, the number of cells per milliliter of resuspension is approximately 1 × 10⁻⁶. 9 indivual.

[0153] To extract, quantify, and detect intracellular NUD and NUDH concentrations, 2.0 × 10⁻⁶ cells were used. 10 Cells were collected after centrifugation at 12,000 rpm for 5 minutes at 4 °C. The centrifuged cells were washed three times with ice-cold phosphate-buffered saline (PBS). The precipitate was resuspended in 500 μL of extraction buffer (acetonitrile, methanol, and water, v / v 40:40:10) pre-cooled to -20 °C and incubated for 20 minutes. The supernatant was collected after centrifugation at 12,000 rpm for 5 minutes at 4 °C and analyzed using a Waters ACQUITY UPLC system equipped with an Atlantis PREMIER BEH C18 AX column. The mobile phases used for separation included (A) 10 mM ammonium formate (pH 8.9) and (B) 100% acetonitrile. The gradient elution program is 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 NUD and NUDH concentrations of strain K01 were 31.07 μM and 43.22 μM, respectively. Figure 2 (A and B in the text).

[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) Flexible linker peptides (amino acid sequence: GGSSG) were inserted into the C-terminus of Hs Nmnat* and the N-terminus of PsPTDH* in two (GSGSGGSGSG), three (GSGSGGSGSGSGSGSGSG), and four (GSGSGGSGSGSGSGSGSGSGSGSG) copies respectively to prepare fusion enzymes: Hs Nmnat*-GSGSGGSGSGS-Ps PTDH*, Hs Nmnat*-GSGSGGSGSGSGSGSGS-Ps PTDH*, and Hs Nmnat*-GSGSGGSGSGSGSGSGSGSGSGS-Ps PTDH*;

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

[0159] 4. NUD and NUDH were prepared by fermentation using strains K01-1, K01-2, and K01-3.

[0160] Following the method described in step 2, NUD and NUDH were prepared by fermentation. Intracellular NUD and NUDH concentrations were detected by ultra-high performance liquid chromatography. The results showed that strain K01-3 contained four copies of a flexible linker and accumulated the highest level of NUDH, reaching 75.6 μM. Figure 2 (B in the middle).

[0161] 5. Enhanced supply of precursors UTP and NMN in strain K01-3

[0162] To enhance the supply of precursors UTP and NMN, metabolic engineering was performed based on the metabolic pathways of NUD and NUDH. Figure 2 (C) First, the genes pyrG, purR, pncC, UshA, and pnuC related to the UTP and NMN catabolism pathways in E. coli Mg1655 were knocked out 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 amidolytic enzyme encoded by the pncC gene can metabolize NMN into nicotinic acid mononucleotide (NaMN); the 5'-nucleotidase encoded by the UshA gene can metabolize NMN into nicotinamide ribose (NR); and the transporter protein encoded by the pnuC gene can transport NMN out of the cell. Subsequently, the expression of four genes was enhanced: 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), resulting in strain K02.

[0163] The specific method is as follows:

[0164] (1) Blocking the catabolism pathways of UTP and NMN

[0165] To reduce the catabolism of precursors UTP and NMN, the genes pyrG, purR, pncC, UshA, and pnuC in E. coliMg1655 were sequentially knocked out using CRISPR / Cas9 gene editing technology to obtain strain E. coliMg1655-1. The specific steps are as follows: Snap genes were introduced into the target gene fragment, including the gene itself and 500 bp upstream and downstream. The sequence number 'agg' was searched within the target gene fragment, and the 20 bp preceding 'agg' in the uppermost sequence was selected as N20. The N20 sequences of the five genes are shown in Table 5.

[0166] Table 5: N20 sequence of the gene

[0167] pyrG-N20 CATGGAGAACGCCAACTCTA purR-N20 AGAGCCGTTGCTGGCGATGC pncC-N20 CCGTGATGCGGTGCGTCGTC UshA-N20 TCTGGTGGGCTATGACGCGA pnuC-N20 ATCCTGGTACATATACCAAT

[0168] The N20 sequence was copied and pasted into the corresponding N20 position in the pTarget sequence of plasmid. Reverse amplification primers for whole plasmid PCR were designed. The primers used are shown in Table 6.

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

[0170] 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 1000 bp knockout frame was constructed using homologous recombination. The Cas9 plasmid was first electroporated into competent E. coli MG1655 cells, which were then used as the host to prepare competent cells. The knockout frame fragment and the N20-containing plasmid pTargetF were electroporated into the competent cells, and colony PCR was used to verify gene knockout. After knocking out the five genes sequentially in the order of pyrG, purR, pncC, UshA, and pnuC, strain E. coli Mg1655-1 was obtained.

[0172] (2) Preparation of strain K02

[0173] To enhance the synthesis of MNM and UTP, the four genes were ligated into the vector p15A-4 (p15A-Hs Nmnat*-GSGSGGSGSGSGSGSGSGSGSG-Ps PTDH*) via homologous recombination. First, the sequences of uridine kinase (Sl UMPK), nucleoside diphosphate kinase (Da NDK), and vector p15A-4 were amplified using primers listed in Table 7. After determining the fragment and vector sizes by agarose gel electrophoresis, they were digested with DpnI enzyme and recovered by column chromatography. Subsequently, the Sl UMPK and Da NDK fragments were ligated into the amplified p15A-4 vector using a multi-fragment homologous recombination enzyme. The homologous recombination system and method were as follows: the total reaction volume was 10 μL, including 2 μL of multi-fragment homologous recombination enzyme, 150 ng of Sl UMPK fragment, 150 ng of Da NDK fragment, and 350 ng of vector p15A. The reaction was carried out at 37°C for 30 min, and then directly transformed into competent Escherichia coli JM109 competent cells and plated on cells supplemented with kanamycin (final concentration 50 mg·L⁻¹). -1 LB agar plates. Incubate at 37°C for 6-8 hours, then select positive clones for inoculation with kanamycin (final concentration 50 mg / L). -1 In LB liquid medium, after culturing at 37℃ and 220r / min for 6-8h, plasmid p15A-5 (p15A-Hs Nmnat*-GSGSGGSGSGSGSGSGSGSGSG-Ps PTDH*-SlUMPK-Da NDK) was extracted.

[0174] Subsequently, the gene sequences of nicotinamide phosphoribosyltransferase (Vp NAMPT) and NMN synthase (FtNadE) were amplified using a similar method and ligated into vector p15A-5. The amplification primers are shown in Table 7. By culturing bacterial cells and extracting plasmids, vector p15A-6 containing 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*-GSGSGGSGSGSGSGSGSGSGS-Ps PTDH*) was obtained, namely: p15A-UTP synthesis module-NMN synthesis module-NUDH synthesis module.

[0175] Table 7: Primers

[0176] 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 competent cells of strain E.coli Mg1655-1 to produce strain K02.

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

[0179] The prepared strain K02 was added to LB medium and cultured at 37℃ and 220 rpm for 6 h to prepare a seed culture. The seed culture was then inoculated into TB medium at a 10% (v / v) inoculation rate and cultured at 37℃ and 220 rpm for 2 h, until the OD600 reached 0.6-0.8. Then, 0.1 mM IPTG was added, and the culture was continued at 37℃ and 220 rpm for 16-18 h to obtain the fermentation broth. The fermentation broth was centrifuged, and the cells 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), followed by vigorous vortexing for 20 seconds. The mixture was then cooled on ice for 10 minutes and vortexed again for 20 seconds. It was then centrifuged at 4°C and 13,000 rpm for 10 minutes. The resulting clear supernatant was diluted with an equal volume of double-distilled water and vortexed for 60 seconds.

[0180] For UTP detection, these samples could be stored at -80°C or neutralized immediately with 5M potassium carbonate before ultra-high performance liquid chromatography (UPLC) analysis. A Waters ACQUITY UPLC system equipped with a Waters Symmetry C18 (3.5 μm, 150 × 4.6 mm) column was used for supernatant analysis. 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: solvents AB (60:40) at 0 min, (40:60) at 30 min, and (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, dissolved in 30% methanol solution (pH = 7.0).

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

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

[0184] (1) Screening of RBS sequences

[0185] Subsequently, the expression of genes controlled by different strengths of RBS was screened by balancing the relationships between the UTP synthesis modules (Sl UMPK and Da NDK), the NMN synthesis modules (Vp NAMPT and Ft NadE genes), and the NUDH synthesis modules (Hs Nmnat*-GSGSGGSGSGSGSGSGSGSG-PsPTDH*) through ribosome binding site (RBS) engineering.

[0186] The expression of green fluorescent protein (GFP) using the p15A vector and the screening of ribosome binding sites (RBS) of different intensities can be performed according to the following steps: The p15A vector is double-digested with the restriction enzymes BanI and XbaI, and a GFP fragment is inserted between these two restriction sites to obtain the vector p15A-GFP. Subsequently, GFP expression is controlled using six different RBSs (Table 8) (i.e., different RBSs are used to replace the original GFP RBS: AAAGAGGAGAAA), resulting in vectors p15A-GFP-01, p15A-GFP-02, p15A-GFP-03, p15A-GFP-04, p15A-GFP-05, and p15A-GFP-06. These vectors are then transformed into *E. coli* Mg1655 and cultured in LB medium at 37°C and 220 rpm for 7.5 h. The fluorescence intensity of GFP under different RBS control conditions is detected using a microplate reader. Using the final fluorescence intensity of the strain containing the vector p15A-GFP-01 (RBS-01) as a reference value of 100%, the relative intensities of other RBS strains were calculated. The fluorescence intensities of RBS-02, RBS-03, RBS-04, RBS-05, and RBS-06 were 63.71%, 60.12%, 38.03%, 17.06%, and 10.31%, respectively. Figure 2 (E in the text). Therefore, three RBS intensities were selected: high (RBS-01: GGAAACCCTCAGGAGGTAAACCA), medium (RBS-03: ATGGTCTGATCTCCTACGGTTATA), and low (RBS-05: AGGAATGTATCAATTGAGGTTTAA). The RBS sequences involved are shown in Table 8 below:

[0187] Table 8: RBS sequence

[0188] 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-RBS0-Hs Nmnat*-GSGSGGSGSGGSGSGGSGSG-PsPTDH*-RBS0-Sl UMPK-RBS0-Da NDK-RBS0-Vp NAMPT-RBS0-Ft NadE; all RBS0 are AAAGAGGAGAAA;

[0191] Three RBS strengths (high (RBS-01: GGAAACCCTCAGGAGGTAAACCA), medium (RBS-03: ATGGTCTGATCTCCTACGGTTATA), and low (RBS-05: AGGAATGTATCAATTGAGGTTTAA)) were used to replace the original RBS. Homologous recombination was used to replace RBS0 in the UTP synthesis module (RBS0-Sl UMPK-RBS0-DaNDK), NMN synthesis module (RBS0-Vp NAMPT-RBS0-Ft NadE), and NUDH synthesis module (RBS0-Hs Nmnat*-GSGSGGSGSGSGSGSGSGSGS-Ps PTDH*), respectively. The results are shown in Table 9. The serial numbers in Table 9 represent strains. RBS optimization indicates that different RBSs were used to enhance various modules on the strain's genome. For example, the optimization process for strain K02-R1 was as follows:

[0192] The strains obtained by replacing RBS0 in (RBS0-Sl UMPK-RBS0-Da NDK) with RBS-01; replacing RBS0 in (RBS0-Vp NAMPT-RBS0-Ft NadE) with RBS-01; and replacing RBS0 in (RBS0-Hs Nmnat*-GSGSGGSGSGSGSGSGSGSGSG-Ps PTDH*) with RBS-01.

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

[0194]

[0195]

[0196] Strain K02-R11, when controlled by RBS-03 (medium), RBS-01 (high), and RBS-03 (medium), significantly increased intracellular NUDH levels, resulting in the highest NUDH concentration of 8.73 mM and a NUDH / NUD ratio of 58.19, demonstrating the successful construction of the novel energy system.

[0197] Example 3: Design of the FGPM pathway and adaptation of the pathway enzyme to NUDH

[0198] Path design:

[0199] This patent designs a metabolic pathway, the FGPM pathway, that utilizes carbon dioxide and formic acid to synthesize malic acid. Figure 3(A) This pathway consists of four carbon metabolism modules: In module C1, CO2 is converted to formaldehyde by (Ts FDH, gene sequence as shown in SEQ ID NO. 9) and formaldehyde dehydrogenase (Bm FADH, gene sequence as shown in SEQ ID NO. 10). In module C2, formaldehyde is converted to acetyl-CoA by the action of glycolaldehyde synthase (Pp GALS, gene sequence as shown in SEQ ID NO. 11), acetylphosphophosphate synthase (Bm APS, gene sequence as shown in SEQ ID NO. 12), and phosphorylacetyltransferase (Bs PTA, gene sequence as shown in SEQ ID NO. 13). In module C3, acetyl-CoA and CO2 are converted to pyruvate by pyruvate:ferroredoxin oxidoreductase (Da rPFOR, gene sequence as shown in SEQ ID NO. 14). Finally, in module C4, pyruvate is converted to malate by malate enzyme (PcME, gene sequence as shown in SEQ ID NO. 15). The three enzymes in this carbon fixation pathway, Ts FDH, Bm FADH, and PcME, are driven by the natural energy system NADH, characterized by simple steps and independence from ATP. However, the intracellular natural energy system NADH is used in hundreds of catalytic reactions simultaneously, and its concentration is low, only 0.5-1 mM, making it difficult to efficiently drive the carbon fixation pathway. When the FGPM pathway was constructed in *E. coli* strain Mg1655 using CO2 as a substrate, the synthesis of the product malic acid was not detected. Therefore, this patent proposes using a newly developed energy system NUDH (8.73 mM) that can accumulate at high intracellular concentrations to drive the FGPM pathway. To transform the pathway enzymes dependent on the natural energy system NADH into those dependent on the novel energy molecule NUDH, and to achieve compatibility with NUDH, protein engineering was performed through iterative mutagenesis.

[0200] 1. Obtaining the Ts FDH mutant

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

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

[0203] MAKILCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLEANGHTFVVTSDKDGPDSVFEKELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRGITVAEVTYCNSISVAEHVVMMILGLVRNYIPSHDWARKGGWNIADCVEHSYDLEGMTVGSVAA GRIGLAVLRRLAPFDVKLHYTDRHRLPEAVEKELGLVWHDTREDMYPHCDVVTLNVPLHPETEHMINDETLKLFKRGAYIVNTARGKLADRDAIVRAIESGQLAGYAGDVWFPQPAPKDHPWRTMKWEGMTPHISGTSLSAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAG

[0204] Table 10: Primers

[0205] 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) consisted of the following components: template DNA (100-150 ng), forward and reverse primers (20 μM, 2 μl each), PrimeSTAR polymerase (1 μl), 5×PrimeSTAR buffer (20 μl), dNTP mixture (8 μl), and sterile water. The PCR amplification conditions were: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 0.5 min; 55℃ annealing for 0.5 min; 72℃ extension for 3 min; for a total of 30-32 cycles; and a final extension at 72℃ for 5 min, followed by pre-storage at 12℃ for 5 min. The PCR amplification products were detected by 1% agarose gel electrophoresis. After confirming the correct product size, 0.5 μL of DpnI digestive enzyme was added to 10 μL of the amplification product, mixed well, and incubated at 37℃ for 40 min, followed by inactivation of the digestive enzyme at 60℃ for 10 min. Subsequently, the amplification products treated with DpnI were transformed into E. coli JM109 competent cells, and the transformation products were plated on cells supplemented with kanamycin (final concentration 50 mg / L). -1 Incubate the culture medium on LB solids at 37°C for 8–10 h, and then inoculate the single colonies that grow on the plates with ampicillin (final concentration 100 mg·L⁻¹). -1Plasmids were extracted after culturing in LB liquid medium at 37°C and 220 r / min for 6–8 h to obtain vectors containing V198N, V198M, T221E, T221L, T221D and V256L mutants.

[0207] The prepared mutant vectors were added to Escherichia coli BL21(DE3) strain to prepare genetically engineered bacteria. The prepared genetically engineered bacteria were inoculated into TB medium to prepare pure enzyme solutions containing V198N, V198M, T221E, T221L, T221D and V256L mutants. At the same time, pure enzyme solutions of wild type were prepared according to the above method, and the enzyme activity was tested. The results showed that the initial activity of TsFDH using NUDH as an energy molecule was 0.068 U / mg (i.e., the wild-type enzyme activity was 0.068 U / mg). After single-point mutation, six beneficial mutants were obtained: V198N, V198M, T221E, T221L, T221D, and V256L, with enzyme activities of 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) The optimal mutant V256L was used for iteration. The construction method was as follows: using one of the single-point mutants as a template, full-plasmid PCR primers for other mutation sites were designed. The double mutant plasmid with the correct mutation was obtained by template digestion, product purification, transformation, and selection of clones for sequencing verification. The methods for triple mutation and quadruple mutation are the same as above.

[0209] Based on this, iterative mutations were performed to obtain the following mutants: T221D / V256L, T221E / V256L, T221L / V256L, V198N / T221E / V256L, and V198M / T221E / V256L.

[0210] Pure enzyme solutions containing mutants T221D / V256L, T221E / V256L, T221L / V256L, V198N / T221E / V256L, and V198M / T221E / V256L were prepared according to step (1), with enzyme activities of 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) 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 follows: Figure 3 As shown in B in the diagram.

[0211] 2. Screening for Bm FADH mutants

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

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

[0214] MSSNRGVVYLGPGKVEVQKIDYPKMVDPSGRAIGHGVILKVVSTNICGSDQHMVRGRTTAPVGLVLGHEITGEVVEVGRDVETLKIGDLVSVPFNVACGRCAMCKETHTGVCLNVNPSRAGGAYGYVDMGGWIGGQAEYVLVPYADFNLLKFPDRDQAMAKIRDLTCLSDILPTGYHGAVSAGVKPGSTVYIAGAGPVG MAAAASARLLGAAVTIVGDMNAERLAHAKAMGFETVDLSKDATLGEQIAQILGKPEIDCAVDCVGFEAHGHGSSGHAEEAPATVLNSLMEITRPAGAIGIPGLYVTDDPGAQDKAAQHGSLSIRFGLGWAKSHSFFTGQTPVLKYNRNLMQAILYDRLPIAKIVNVTVISLDDAPEGYKKFDGGAPRKFVIDPHGLLAA

[0215] The enzyme activity was determined by purifying the protein and measuring its activity: 0.25 U / mg, 0.49 U / mg, 0.75 U / mg, 0.90 U / mg, 0.49 U / mg, 0.55 U / mg, and 0.64 U / mg. V283L exhibited the highest activity, reaching 0.90 U / mg. Under the same conditions, the wild-type enzyme activity was 0.057 U / mg. Iterative mutagenesis was then performed to generate the optimal double mutant A193R / V283L, increasing the activity to 3.68 U / mg. Finally, the optimal mutant Bm FADH*(A193R / M219K / L237R / H268Y / V283L) was obtained, achieving an activity of 6.13 U / mg. Figure 3 (C in the middle).

[0216] Table 11: Primers

[0217] 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 PcME mutants

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

[0220] The 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] 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 the pure enzyme solutions were measured separately. 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. The enzyme activity of the wild-type enzyme, obtained using the same method, was 0.20 U / mg. These results indicate that protein engineering of PcME using the same method to provide compatibility with NUDH yielded the optimal mutant Pc. ME*(V300Q / Q391Y / L394K) showed an activity against NUDH of 11.12 U / mg. Figure 3 (D in the middle).

[0234] Example 4: NUDH-driven FGPM pathway for the synthesis of malic acid

[0235] The specific steps are as follows:

[0236] 1. Construction of strain K03

[0237] (1) Construction of recombinant vector

[0238] The Ts FDH* (V198M / T21E / V256L), Bm FADH*A193R / M219K / L237R / H268Y / V283L), and Pp GALS genes (gene sequences shown in SEQ ID NO.11) prepared in Example 3 were sequentially ligated into the pETAC vector via homologous recombination to obtain the expression vector: pETAC-Ts FDH*-Bm FADH*-Pp GALS, named pETAC-1. Acetyl phosphate synthase (Bm APS, gene sequence shown in SEQ ID NO.12), Bs PTA (gene sequence shown in SEQ ID NO.13), DarPFOR (gene sequence shown in SEQ ID NO.14), and Pc ME* (V300Q / Q391Y / L394K) were sequentially ligated into the pTET vector via 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] Plasmids pETAC-1 and pTET-1 were co-transformed into strain K02-R11 (E.coli Mg1655-1 / p15A-RBS-03-Hs Nmnat*-GSGSGGSGSGSGSGSGSGSGSG-Ps PTDH*-RBS-03-Sl UMPK-RBS-03-Da NDK-RBS-01-Vp NAMPT-RBS-01-Ft NadE) to obtain strain K03, which can use NUDH as the driving force to convert carbon dioxide and formic acid into malic acid.

[0241] A single colony of K03 was picked from an agar plate and inoculated into LB medium. The culture was incubated overnight (6-8 h) at 37°C and 220 rpm to prepare a seed culture. Subsequently, the prepared seed culture was transferred to 50 mL of TB medium at a 5% (v / v) inoculation rate and cultured at 37°C and 200 rpm until OD reached [the desired growth rate]. 600 The concentration of the solution was 0.6-0.8. IPTG was added to a final concentration of 0.6 mM for induction. After induction at 30°C for 16 h, the bacterial cells were collected by centrifugation.

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

[0243] Conditions for shake-flask fermentation:

[0244] The serum vial contained 150 mL of serum, with a bacterial cell addition of 3.5 g / L. Tris-HCl solution (pH 7.0) was used as the medium for whole-cell transformation. 300 mL of NaHCO3 (to provide CO2) was added, along with 50 mM formic acid and 10 mM MgSO4. The phosphite concentration was maintained at 1-5 mM by adding 8 M phosphite stock solution (pH 7.5). The reaction was catalyzed at 37°C and 50 rpm for 35 hours.

[0245] Fermentation conditions for a 7.5L fermenter:

[0246] In a 7.5L fermenter, the liquid volume was 4.5L, the cell addition was 3.5g / L, and Tris-HCl solution with pH=7.0 was used as the medium for whole cell transformation. CO2 gas was introduced and the pressure was maintained at 0.05MPa. At the same time, 75mM formic acid and 10mM MgSO4 were added, and the phosphite concentration was maintained at 5-10mM. The catalytic reaction was carried out at 37℃ for 35 hours.

[0247] The results showed that malic acid yields reached 37.76 mM in 250 mL serum bottles and 50.61 mM in a 7.5 L fermenter. Figure 3 (E in the middle).

[0248] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A nicotinamide transferase mutant, characterized in that, The nicotinamide transferase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type nicotinamide transferase, as shown in SEQ ID NO.

2. The mutation mode is selected from: L27K, L27K / V186L, L27K / V186I, L27K / V186L / T187K, L27K / V186L / T187Y, L27K / V186L / T187K / D220E or L27K / V186L / T187K / D220Q.

2. The gene encoding the mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. A recombinant cell expressing the mutant of claim 1 or carrying the gene of claim 2 or the recombinant vector of claim 3.

5. The recombinant cell according to claim 4, characterized in that, The recombinant cells use bacteria or fungi as expression hosts.

6. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria expressed the mutant described in claim 1.

7. The genetically engineered bacterium according to claim 6, characterized in that, The genetically engineered bacteria are... E. coli MG1655 E. coli W3110 or E. coli BL21(DE3) is the expression host.

8. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria are those that simultaneously overexpress the nicotinamide transferase mutant and phosphite dehydrogenase mutant described in claim 1; The phosphite dehydrogenase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type phosphite dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

4. 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.

9. The genetically engineered bacterium according to claim 8, characterized in that, The genetically engineered bacteria are... E. coli MG1655 E. coli W3110 or E. coli BL21(DE3) is the expression host.

10. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria are: simultaneously overexpressing the nicotinamide transferase mutant and the phosphite dehydrogenase mutant as described in claim 1, and the nicotinamide transferase mutant and the phosphite dehydrogenase mutant are obtained by fusing GGSGSGGSGSG, GGSGSGSGSGSGSGSG or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain a fusion enzyme. The phosphite dehydrogenase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type phosphite dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

4. 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.

11. The genetically engineered bacterium according to claim 10, characterized in that, The genetically engineered bacteria are... E. coli MG1655 E. coli W3110 or E. coli BL21(DE3) is the expression host.

12. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria are: simultaneously overexpressing the nicotinamide transferase mutant, phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, and NMN synthase as described in claim 1, and the nicotinamide transferase mutant and phosphite dehydrogenase mutant are obtained by fusing GGSGSGGSGSG, GGSGSGSGSGSGSGSG or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain fusion enzymes; The phosphite dehydrogenase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type phosphite dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

4. 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.

13. The genetically engineered bacterium according to claim 12, characterized in that, The genetically engineered bacteria are... E. coli MG1655 E. coli W3110 or E. coli BL21(DE3) is the expression host.

14. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria are those that simultaneously overexpress the nicotinamide transferase mutant, phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, and NMN synthase described in claim 1. The nicotinamide transferase mutant and the phosphite dehydrogenase mutant are fused using GGSGSGGSGSG, GGSGSGSGSGSGSGSG, or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain a fusion enzyme. The phosphite dehydrogenase mutant is based on a site-directed mutation of the amino acid sequence of the wild-type phosphite dehydrogenase, and the amino acid sequence of the wild-type phosphite dehydrogenase is shown in SEQ ID. As shown in NO.4, 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; Simultaneously, strains were obtained by replacing the original RBS (AAAGAGGAGAAA) preceding uridine kinase and nucleoside diphosphate kinase with RBS-01, RBS-03, or RBS-05; replacing the original RBS (AAAGAGGAGAAA) preceding nicotinamide phosphoribosyltransferase and NMN synthase with RBS-01, RBS-03, or RBS-05; and replacing the original RBS (AAAGAGGAGAAA) preceding the fusion enzyme with RBS-01, RBS-03, or RBS-05. The RBS-01 was GGAAACCCTCAGGAGGTAAACCA, the RBS-03 was ATGGTCTGATCTCCTACGGTTATA, and the RBS-05 was AGGAATGTATCAATTGAGGTTTAA.

15. The genetically engineered bacterium according to claim 14, characterized in that, The genetically engineered bacteria are... E. coli MG1655 E. coli W3110 or E. coli BL21(DE3) is the expression host.

16. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria are: simultaneously overexpressing the nicotinamide transferase mutant, phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, NMN synthase, formate dehydrogenase mutant, formaldehyde dehydrogenase mutant, and malic acid oxidase mutant as described in claim 1, and the nicotinamide transferase mutant and phosphite dehydrogenase mutant are obtained by fusing GGSGSGGSGSG, GGSGSGSGSGSGSGSG or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain fusion enzymes; The phosphite dehydrogenase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type phosphite dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

4. 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; The formate dehydrogenase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type formate dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

16. 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. The formaldehyde dehydrogenase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type formaldehyde dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

17. The mutation mode is selected from: L237R, A193R, A193M, V283L, L237Y, H268E, H268Y, A193R / V283L or A193R / M219K / L237R / H268Y / V283L; The malic acid enzyme mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type malic acid enzyme, which is shown in SEQ ID NO.

18. The mutation mode is selected from: V300Q, V300L, G390L, Q391Y, L394K, L394R, Q391Y / L394K, Q391Y / L394R, V300Q / Q391Y / L394K or V300L / Q391Y / L394K; Simultaneously, strains were obtained by replacing the original RBS (AAAGAGGAGAAA) preceding uridine kinase and nucleoside diphosphate kinase with RBS-01, RBS-03, or RBS-05; replacing the original RBS (AAAGAGGAGAAA) preceding nicotinamide phosphoribosyltransferase and NMN synthase with RBS-01, RBS-03, or RBS-05; and replacing the original RBS (AAAGAGGAGAAA) preceding the fusion enzyme with RBS-01, RBS-03, or RBS-05. The RBS-01 was GGAAACCCTCAGGAGGTAAACCA, the RBS-03 was ATGGTCTGATCTCCTACGGTTATA, and the RBS-05 was AGGAATGTATCAATTGAGGTTTAA.

17. The genetically engineered bacterium according to claim 16, characterized in that, The genetically engineered bacteria are... E. coli MG1655 E. coli W3110 or E. coli BL21(DE3) is the expression host.

18. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria are: simultaneously overexpressing the nicotinamide transferase mutant, phosphite dehydrogenase mutant, uridine kinase, nucleoside diphosphate kinase, nicotinamide phosphoribosyltransferase, NMN synthase, formate dehydrogenase mutant, formaldehyde dehydrogenase mutant, malic acid esterase mutant, ethanolaldehyde synthase, acetyl phosphate synthase, phosphoacetyltransferase, and ferric reductase as described in claim 1, and the nicotinamide transferase mutant and phosphite dehydrogenase mutant are fused with GGSGSGGSGSG, GGSGSGSGSGSGSGSG or GGSGSGSGSGSGSGSGSGSGSGSG as linker peptides to obtain a fusion enzyme; The phosphite dehydrogenase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type phosphite dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

4. 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; The formate dehydrogenase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type formate dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

16. 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. The formaldehyde dehydrogenase mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type formaldehyde dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.

17. The mutation mode is selected from: L237R, A193R, A193M, V283L, L237Y, H268E, H268Y, A193R / V283L or A193R / M219K / L237R / H268Y / V283L; The malic acid enzyme mutant is based on site-directed mutagenesis of the amino acid sequence of wild-type malic acid enzyme, which is shown in SEQ ID NO.

18. The mutation mode is selected from: V300Q, V300L, G390L, Q391Y, L394K, L394R, Q391Y / L394K, Q391Y / L394R, V300Q / Q391Y / L394K or V300L / Q391Y / L394K; Simultaneously, strains were obtained by replacing the original RBS (AAAGAGGAGAAA) before uridine kinase and nucleoside diphosphate kinase with RBS-01, RBS-03, or RBS-05; replacing the original RBS (AAAGAGGAGAAA) before nicotinamide phosphoribosyltransferase and NMN synthase with RBS-01, RBS-03, or RBS-05; and replacing the original RBS (AAAGAGGAGAAA) before fusion enzyme with RBS-01, RBS-03, or RBS-05. RBS-01 is GGAAACCCTCAGGAGGTAAACCA, RBS-03 is ATGGTCTGATCTCCTACGGTTATA, and RBS-05 is AGGAATGTATCAATTGAGGTTTAA.

19. The genetically engineered bacterium according to claim 18, characterized in that, The genetically engineered bacteria are... E. coli MG1655 E. coli W3110 or E. coli BL21(DE3) is the expression host.

20. A method for preparing malic acid, characterized in that, The method involves fermenting malic acid using any of the genetically engineered bacteria described in claims 6 to 19.

21. The use of the mutant of claim 1, or the gene of claim 2, or the recombinant vector of claim 3, or the recombinant cell of claim 4 or 5, or the genetically engineered bacteria of any one of claims 6 to 19 in the preparation of malic acid or products containing malic acid.

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