Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase mutants with preference for nad analogues and uses thereof

Abstract

The application discloses a non-phosphorylated glycerinaldehyde-3-phosphate dehydrogenase mutant with preference for NAD analogues and application thereof, and belongs to the technical field of synthetic biology. The application provides the non-phosphorylated glycerinaldehyde-3-phosphate dehydrogenase mutant, which is obtained by site-directed mutagenesis of non-phosphorylated glycerinaldehyde-3-phosphate dehydrogenase. The mutant has significant preference for NAD analogues, can catalyze irreversible catalytic conversion of glycerinaldehyde-3-phosphate into 3-phosphoglycerate, and convert NAD analogues into their reduced states. The mutant provided by the application can be used for producing reduced-state NAD analogues, and can also provide reducing power for enzymatic reactions consuming reduced-state analogues. The reduced-state NAD analogues can act as coenzymes, and under the catalysis of other enzymes with NAD analogue activity, corresponding products are generated. The mutant provided by the application can also be applied to NAD analogue-mediated growth-dependent screening or production platform, and provides a new model and unique solution for precise regulation of sugar substance conversion and energy transmission.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant with a preference for NAD analogs and its applications. Background Technology

[0002] There are thousands of redox reactions in cells involving nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), and their corresponding reduced forms as cofactors, involving the decomposition and synthesis of most compounds. When regulating metabolic pathways involving redox reactions, simply overexpressing or knocking out certain key enzyme genes is insufficient; it is often necessary to enhance the supply of reducing power. Simply increasing the level of a single intracellular cofactor often leads to metabolic overflow and reducing power leakage, thereby reducing substrate conversion and affecting atom economy. Furthermore, since NAD(P)(H) also participates in regulating cell proliferation, growth, differentiation, and apoptosis, regulating the concentration and ratio of intracellular NAD(P)(H) may significantly perturb the entire metabolic network and cellular physiological state.

[0003] To achieve precise regulation of redox reactions involving NAD(P)(H), the creation of bioorthogonal redox systems is crucial. Through bioorthogonal non-natural cofactors (NAD analogues), target synthetic pathways can be isolated from complex natural metabolic pathways, enabling selective regulation at the energy level.

[0004] The carbohydrate pathway plays a crucial role in bioenergy production and biomass formation across all biological domains. Glycolysis, which breaks down glucose into pyruvate, is key in the central carbon metabolism of most prokaryotes, particularly heterotrophs. In nature, besides the two-step enzymatically coupled pathway that catalyzes the conversion of the central metabolic precursor glyceraldehyde-3-phosphate (GAP) to 3-PG, an atypical one-step catalytic conversion pathway exists, including non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPN) and ferroredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase (GAPOR). GAPN, in particular, largely depends on the cofactor NADP, produces no ATP, and its catalytic reaction is irreversible.

[0005] Because intracellular redox reactions are continuous, complex, and precise, applications often disrupt intracellular metabolic pathways, posing a challenge to achieving complete intracellular production. Currently, there are no reports on directed evolutionary modification of non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase mutants to further reduce NAD analogues. Summary of the Invention

[0006] Based on the above-mentioned technological needs, the purpose of this invention is to provide a non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant with a preference for NAD analogs and its applications. The non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant provided by this invention exhibits a significant preference for NAD analogs, capable of catalyzing the irreversible catalytic conversion of glyceraldehyde triphosphate to 3-phosphoglycerate, while simultaneously converting NAD analogs to their reduced state. The reduced NAD analog can serve as a coenzyme for other oxidoreductases in reduction reactions; therefore, the method of this invention has significant value in the fields of biocatalysis and biotransformation.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a nonphosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant with a preference for NAD analogs. This nonphosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant is obtained by mutating an amino acid sequence as shown in SEQ ID NO. 1. The mutated site and the corresponding amino acid sequence of the mutant are as follows:

[0009] (1) Mutant 1-F-AQVPR: is obtained by changing amino acid I to R, amino acid I to F, amino acid P to A, amino acid T to Q, amino acid R to V, and amino acid G to P in the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence of mutant 1-F-AQVPR is shown in SEQ ID NO.3.

[0010] (2) Mutant 2-F-AQWPR: is obtained by changing amino acid I to R, amino acid I to F, amino acid P to A, amino acid T to Q, amino acid W to W, and amino acid G to P in the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence of mutant 2-F-AQWPR is shown in SEQ ID NO.4.

[0011] (3) Mutant 3-F-SERR: is obtained by changing amino acid I to R, amino acid I to F, amino acid P to S, amino acid T to E, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence of mutant 3-F-SERR is shown in SEQ ID NO.5.

[0012] (4) Mutant 4-F-SEPRR: is obtained by changing amino acid I to R, amino acid I to F, amino acid P to S, amino acid T to E, amino acid R to P, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence of mutant 4-F-SEPRR is shown in SEQ ID NO.6.

[0013] (5) Mutant 5-FS-SEPRR: is obtained by changing amino acid I to R, amino acid I to F, amino acid A to S, amino acid P to S, amino acid T to E, amino acid R to P, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence of mutant 5-FS-SEPRR is shown in SEQ ID NO.7.

[0014] (6) Mutant 6-MV-SERR: is obtained by changing amino acid I to R, amino acid I to M, amino acid A to V, amino acid P to S, amino acid T to E, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence of mutant 6-MV-SERR is shown in SEQ ID NO.8.

[0015] (7) Mutant 7-MV-SEKRR: is obtained by changing amino acid I to R, amino acid I to M, amino acid A to V, amino acid P to S, amino acid T to E, amino acid K to K, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.1. The amino acid sequence of mutant 7-MV-SEKRR is shown in SEQ ID NO.9.

[0016] (8) Mutant 8-MI-SERR: is obtained by changing amino acid I to R, amino acid I to M, amino acid A to I, amino acid P to S, amino acid T to E, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.10;

[0017] (9) Mutant 9-LT-SELRR: is obtained by changing amino acid 234 from I to R, amino acid 20 from I to L, amino acid 36 from A to T, amino acid 179 from P to S, amino acid 180 from T to E, amino acid 209 from R to L, and amino acid 210 from G to R in the amino acid sequence shown in SEQ ID NO.11.

[0018] (10) Mutant 10-AQR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to A, the 180th amino acid to Q, and the 234th amino acid to R. The amino acid sequence of mutant 10-AQR is shown in SEQ ID NO.12.

[0019] (11) Mutant 11-SQR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to S, the 180th amino acid to Q, and the 234th amino acid to R. The amino acid sequence of mutant 11-SQR is shown in SEQ ID NO.13.

[0020] (12) Mutant 12-SER: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 from P to S, the 180th amino acid from T to E, and the 234th amino acid from I to R. The amino acid sequence of mutant 12-SER is shown in SEQ ID NO.14.

[0021] (13) Mutant 13-AQVR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to A, the 180th amino acid to Q, the 210th amino acid to V, and the 234th amino acid to R. The amino acid sequence of mutant 13-AQVR is shown in SEQ ID NO.15.

[0022] (14) Mutant 14-AQTR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to A, the 180th amino acid to Q, the 210th amino acid to T, and the 234th amino acid to R. The amino acid sequence of mutant 14-AQTR is shown in SEQ ID NO.16.

[0023] (15) Mutant 15-SQVR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to S, the 180th amino acid to Q, the 210th amino acid to V, and the 234th amino acid to R. The amino acid sequence of mutant 15-SQVR is shown in SEQ ID NO.17.

[0024] (16) Mutant 16-SERR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to S, the 180th amino acid to E, the 210th amino acid to R, and the 234th amino acid to R. The amino acid sequence of mutant 16-SERR is shown in SEQ ID NO.18.

[0025] (17) Mutant 17-SELRR: is obtained by changing the amino acid position 179 from P to S, the amino acid position 180 from T to E, the amino acid position 209 from R to L, the amino acid position 210 from G to R, and the amino acid position 234 from I to R in the amino acid sequence shown in SEQ ID NO.19.

[0026] (18) Mutant 18-M-SERR: is obtained by changing the 20th amino acid of the amino acid sequence shown in SEQ ID NO.1 from I to M, the 179th amino acid from P to S, the 180th amino acid from T to E, the 210th amino acid from G to R, and the 234th amino acid from I to R. The amino acid sequence of mutant 18-M-SERR is shown in SEQ ID NO.20.

[0027] The present invention also provides the application of the aforementioned nonphosphorylated glyceraldehyde triphosphate dehydrogenase mutant in the preparation of formulations containing multi-enzyme-driven NAD analogues.

[0028] Furthermore, the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant can catalyze the conversion of glyceraldehyde triphosphate to 3-phosphoglycerate and can convert NAD analogs to their reduced form.

[0029] Furthermore, the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant can catalyze the reductive amination of pyruvate to prepare alanine, the reductive reduction of pyruvate to prepare lactic acid, and the reductive carboxylation of pyruvate to prepare malic acid.

[0030] Furthermore, the multienzyme includes at least one of malic acid oxidase, lactate dehydrogenase, alanine dehydrogenase, and fructose-1,5-bisphosphate aldolase.

[0031] Furthermore, the reaction system for the coupled multi-enzyme-driven reduction of NAD analogues includes: a buffer system with pH 3-9, a substrate concentration of 1-500 mM, an NAD analogue concentration of 0.05-20 mM, an enzyme concentration of 0.01-5 mg / mL, and a reaction temperature of 10-40℃ for 5 min-24 h.

[0032] Furthermore, the substrate used for catalysis by the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant is glyceraldehyde triphosphate, which can be added directly to the system or indirectly generated by adding an indirect substrate and its corresponding enzyme.

[0033] The present invention also provides the application of the aforementioned non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant in screening NAD analogue-mediated growth-dependent strains.

[0034] The present invention also provides the application of the aforementioned nonphosphorylated glyceraldehyde triphosphate dehydrogenase mutant in promoting NAD analogue-mediated bacterial growth.

[0035] Furthermore, the specific methods of application include:

[0036] (1) As a screening platform for enzymes utilizing reduced NAD analogues: The non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant is expressed in an engineered strain with the gapA and / or mgsA gene knocked out to serve as a screening platform strain. The mutant library of the enzyme to be screened is induced to be expressed in the platform strain and cultured at 18-37°C for 2-15 days in a liquid or solid medium with glucose as the sole carbon source. Only mutants of enzymes in the library that can utilize NAD analogues can grow, so as to screen for enzymes that can utilize reduced NAD analogues.

[0037] (2) Provide a growth-dependent production platform: By expressing the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant in the engineered strain as a production platform, the enzyme utilizing the reduced NAD analog is further expressed. In a medium with glucose as the sole carbon source, the strain is cultured at 18-37℃ for 2-15 days. The strain accumulates products while growing, so as to achieve the coupling of the production of the target product and the growth of the strain.

[0038] Furthermore, by adding fructose 1,6-bisphosphate and fructose diphosphate aldolase FbaA to the system, dihydroxyacetone phosphate and glyceraldehyde triphosphate are generated, among which glyceraldehyde triphosphate can be utilized by the catalytic system.

[0039] Furthermore, the NAD analogue is at least one of NCD, NUD, and NpUD, and its chemical structure is as follows:

[0040]

[0041] The NADH analogues include at least one of NCDH, NUDH, or NpUDH, and their chemical structures are as follows:

[0042]

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] 1. The non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant provided by this invention has a significant preference for NAD analogs. By using the NAD analog-dependent GAPN pathway to replace the endogenous NAD-dependent glyceraldehyde triphosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) pathways in E. coli, the reaction steps can be simplified, intracellular cofactor interference and oxygen consumption can be reduced, and the process is thermodynamically easier. It is expected to selectively transfer the carbon source and reducing power provided by glucose to the target metabolic pathway based on NAD analogs, reduce crosstalk problems caused by natural cofactors, and provide a new model and unique solution for the precise regulation of carbohydrate conversion and energy transfer.

[0045] 2. The non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant provided by this invention has the following applications in growth-coupled screening or production: (1) As a growth-dependent screening platform, by transferring mutant libraries (including semi-rational design, rational design, and random mutant libraries) into engineered strains, only enzymes expressing the ability to utilize reduced NAD analogs can grow in a culture medium with glucose as the sole carbon source, thus playing the role of screening enzymes that utilize reduced NAD analogs. (2) As a growth-dependent production platform, by expressing enzymes that utilize reduced NAD analogs in engineered strains, the production of the target product and the growth of the strain can be coupled.

[0046] 3. Compared with traditional chemical reduction methods, this method has milder reaction conditions, stronger reaction specificity, and no over-reduction. Compared with other enzymatic reduction methods, the enzyme-catalyzed reaction is irreversible, which is conducive to the formation of reduced products. Attached Figure Description

[0047] Figure 1 The principle of determining the cofactor kinetics of mutants.

[0048] Figure 2 This is a schematic diagram of a growth platform dependent on non-phosphorylated glyceraldehyde triphosphate dehydrogenase, where GAP: D-glyceraldehyde triphosphate; DHAP: dihydroxyacetone phosphate; 1,3-BPG: 1,3-diphosphoglycerate; 3-PG: 3-phosphoglycerate; PYR: pyruvate; MG: methylglyoxal; Lald: lactal; Malate: malate; Lactate: lactate; mgsA: E. coli methylglyoxal synthase; gapA: E. coli glyceraldehyde triphosphate dehydrogenase; GAPN*: NCD-dependent non-phosphorylated glyceraldehyde triphosphate dehydrogenase; ME*: NCD-dependent malate enzyme; NCDS: NCD synthesis module integrated into the genome.

[0049] Figure 3 The results are from the growth curve measurement.

[0050] Figure 4 This diagram illustrates a non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase-dependent production platform. GAP: D-glyceraldehyde-3-phosphate; DHAP: dihydroxyacetone phosphate; 1,3-BPG: 1,3-diphosphoglycerate; 3-PG: 3-phosphoglycerate; PYR: pyruvate; MG: methylglyoxal; Lald: lactal; Malate: malate; Lactate: lactate; L-Ala: L-alanine; mgsA: E. coli methylglyoxal synthase; gapA: E. coli glyceraldehyde-3-phosphate dehydrogenase; GAPN*: NCD-dependent non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase; AlaDH*: NCD-dependent alanine dehydrogenase; NCDS: NCD synthesis module integrated into the genome.

[0051] Figure 5 The crystal structure diagram of the mutant 6-MV-SERR is shown.

[0052] Figure 6 The crystal structure diagram of the mutant 6-MV-SERR monomer. Detailed Implementation

[0053] The technical solution of the present invention will be further described in detail with reference to the following specific examples, but the scope of protection claimed by the present invention is not limited to the scope described in the examples.

[0054] The sequence of the non-phosphorylated glyceraldehyde triphosphate dehydrogenase GAPN used in this invention is publicly available at Uniprot Primary accession: Q59931.

[0055] The sequence of the alanine dehydrogenase used in this invention is publicly available under the accession number UniProt Primaryaccession: A8QVZ6. The mutant A225P / S219E has a mutation site where amino acid A at position 225 is changed to P and amino acid S at position 219 is changed to E. The mutant A225P / V165A / S219E has a mutation site where amino acid A at position 225 is changed to P, amino acid V at position 165 is changed to A, and amino acid S at position 219 is changed to E.

[0056] The sequence of the fructose diphosphate aldolase FbaA used in this invention is publicly available at Uniprot Primaryaccession: P0AB71.

[0057] The sequence of the malicase ME used in this invention is publicly available under UniProt Primary Accession: P26616. The mutant ME-L310R / Q401V has a mutation site where amino acid L changes to R at position 310 and amino acid Q changes to V at position 401. The mutant ME-L310R / Q401C has a mutation site where amino acid L changes to R at position 310 and amino acid Q changes to C at position 401.

[0058] The sequence of D-lactate dehydrogenase DLDH used in this invention is publicly available under the accession number UniProt Primaryaccession: P30901. The mutant DLDH-V152R has a mutation site where amino acid 152 is changed from V to R.

[0059] The purified enzymes used in this invention were expressed and purified according to the methods described in the literature (Protein Expression and Purification, 2007, 53, 97-103). Unless otherwise specified, the reagents and biological materials used in the specific embodiments are commercially available.

[0060] Obtaining NAD analogues: The preparation of nicotinamide cytosine dinucleotide (NCD) and nicotinamide uracil dinucleotide (NUD) was synthesized and purified according to the methods in the reference (Tetrahedron Letters 2022, 88, 153568.); the preparation of nicotinamide pseudouracil dinucleotide (NpUD) was synthesized and purified according to the methods in the reference (Tetrahedron Letters 2025, 155, 155432.).

[0061] Example 1: Construction of an NAD analog-dependent nonphosphorylated glyceraldehyde triphosphate dehydrogenase mutant

[0062] The non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase (NCBI database Accession No. AAN58410, containing the complete amino acid sequence from 1 to 475) from *Streptococcus mutans* was selected. Based on its amino acid sequence (SEQ ID NO. 1), the sequence was optimized according to the species *E. coli*, and the gene was synthesized (the synthesized gene sequence is shown in SEQ ID NO. 2). Using the 2EUH crystal structure as a structural guide and combining semi-rational design, the cofactor binding region (amino acids 145-252) of the non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase was modified. The mutated recombinant plasmid was then electroporated into *E. coli* cells, yielding the corresponding site-directed mutant and mutant library.

[0063] Rational and semi-rational library construction utilizes restriction-free cloning (RF cloning) methods. One-step or two-step PCR systems are used to perform site-directed, combinatorial, or saturation mutations at selected sites, choosing appropriate degenerate codons and designing primers. For single-site mutations, a pair of inversely complementary upstream and downstream primers are designed, using wild-type or mutant vectors as templates for RF cloning. For two-site or multi-site libraries, upstream or downstream primers are designed at different sites to amplify the fragment between the two mutation sites. The fragment is purified using a gel extraction kit to obtain a PCR product containing degenerate codons, which is then used for RF cloning with a template vector.

[0064] The one-step PCR system: The RF cloning PCR system contains (50 μL): 20 ng template vector, 10 mM each of forward and reverse primers, and 5×PrimeSTAR. TM Buffer (Mg) 2+ 10 μL of dNTP Mixture (2.5 mM each), 4 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and ultrapure water were added to a final volume of 50 μL. The PCR reaction conditions were: 98℃ pre-denaturation for 5 min, followed by 98℃ denaturation for 10 s, 65℃ annealing for 5 s, and 72℃ extension for 6 min 50 s. The annealing temperature was decreased by 1℃ for the first 10 cycles, and then annealed at 55℃ for the last 5 cycles, for a total of 15 cycles. To ensure sufficient transformants, the volume was increased to 200 μL. The PCR products were then purified.

[0065] The two-step PCR system: The RF cloning PCR system contains (50 μL): 50 ng template vector, 200 ng fragment containing the mutation site, 5×PrimeSTAR... TMBuffer (Mg) 2+ 10 μL of dNTP Mixture (2.5 mM each), 4 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and ultrapure water were added to a final volume of 50 μL. The PCR reaction conditions were: 98℃ pre-denaturation for 5 min, followed by 98℃ denaturation for 10 s, 65℃ annealing for 5 s, and 72℃ extension for 6 min 50 s. The annealing temperature was decreased by 1℃ for the first 10 cycles, and then annealed at 55℃ for the last 5 cycles, for a total of 15 cycles. To ensure sufficient transformants, the volume was increased to 200 μL. The PCR products were then purified.

[0066] The plasmid template in the PCR product was digested with the restriction enzyme Dpn I. The digestion reaction system was: 8 μL of RF cloning product, 1 μL of Dpn I, and 1 μL of 10×T Buffer, digested at 37℃ for 4 h. Then, 10 μL of the digested sample was transformed into E. coli BL21(DE3) electroporation competent cells. 100 μL of the rejuvenated bacterial culture was plated on LB agar plates containing 50 μg / mL kanamycin. The remaining bacterial culture was mixed with 60% glycerol to a final glycerol concentration of 15% (v / v) and frozen at -80℃. After the transformants grew, an appropriate amount of the remaining bacterial culture was plated on the same plate according to the number of transformants and the number of transformants required for the library. Four transformants from each library were selected for sequencing to verify the library quality. The number of screened transformants should reach 95% of the theoretical value of the corresponding library size.

[0067] Random mutant libraries were constructed using GenStar's StarMut Random Mutagenesis Kit to obtain random mutant fragments. Primer design principles: approximately 20-45 bp in length, GC content 40-60%. Fragments containing random mutations were first obtained using the kit, and then these fragments were used as large primers to construct random mutant libraries via RF cloning.

[0068] The random mutation PCR reaction system contained (50 μL*2): 10 ng template plasmid, 0.2 μM each of forward and reverse primers, 25 μL of 2xStarMut Random PCR Mix (extension rate 1 min / kb), 8 μL of StarMut Enhancer, and then added ultrapure water to 50 μL.

[0069] The PCR reaction conditions were: 95 °C pre-denaturation for 2 min, followed by 94 °C denaturation for 30 s, 55 °C annealing for 1 min, and 72 °C extension for 1 min, for a total of 25 cycles, with a final extension at 72 °C for 7 min. The amplified samples were then subjected to agarose gel electrophoresis to recover the target fragment. Fragment 1 was extended for 30 s (412 bp), fragment 2 for 1 min 0.1 s (1011 bp), and fragment 3 for 1 min 25 s (1393 bp). The recovered fragments were used as large primers for RF cloning.

[0070] The RF cloning PCR system contains (50 μL * 4): 20 ng template vector, 200 ng fragment containing the mutation, and 5×PrimeSTAR Buffer (Mg). 2+ 10 μL of dNTP Mixture (2.5 mM each), 4 μL of PrimeSTAR HS DNA Polymerase (2.5 U / μL), and ultrapure water were added to a final volume of 50 μL. The PCR reaction conditions were: 98 °C pre-denaturation for 5 min, followed by 98 °C denaturation for 10 s, 65 °C annealing for 5 s, and 72 °C extension for 6 min 50 s. The annealing temperature was decreased by 1 °C for the first 10 cycles, and then annealed at 55 °C for the last 6 cycles, for a total of 16 cycles. The PCR product was then purified.

[0071] The plasmid template in the PCR product was digested with the restriction enzyme Dpn I. The digestion reaction system was as follows: 50-100 μL of RF cloning product, 1 μL of Quick Cut Dpn I, and 1 μL of 10×Quick Cut Buffer. Digestion was carried out at 37℃ for 30 min-1 h. Then, 10 μL of the digested sample was transformed into E. coli BL21(DE3) electroporation competent cells. Four transformants were selected from each condition and sent to Sangon Biotech for sequencing to verify library quality (mutation coverage and frequency).

[0072] Example 2: Screening for NAD analog-dependent nonphosphorylated glyceraldehyde triphosphate dehydrogenase mutants

[0073] Library screening consisted of primary screening and secondary screening. Since wild-type GAPN uses NADP as a cofactor, the initial screening used wild-type as a control to screen for mutants with reduced NADP preference and increased NAD analog activity. Subsequent screenings used mutants with better performance as controls for further selection. The screening process included cell culture, primary screening colorimetric reaction, candidate mutant culture, secondary screening, and crude enzyme activity assay, ultimately yielding mutants with increased NAD analog preference. The colorimetric reaction and crude enzyme activity assay employed an enzyme-coupled method.

[0074] (1) Initial screening method

[0075] To amplify the reaction signal, a coupled colorimetric reaction method was chosen for the initial screening to determine the crude enzyme activity.

[0076] Single colonies were picked up with sterile toothpicks and placed into 96-well plates. The culture medium was LB with 50 μg / mL kanamycin and 0.5 mM IPTG (400 μL per well). Expression was induced at 30°C and 600 rpm for 48 h. Upon harvesting, the cells were centrifuged at 4000 g for 3 min, the supernatant was discarded, and 180 μL of cell lysis buffer was added to each well. The cells were then incubated at -80°C for at least 1 h to aid lysis, followed by lysis at 37°C and 600 rpm for 2 h. Using a high-throughput cell culture and activity analysis system, 20 μL of the crude enzyme supernatant was transferred to a microplate, followed by 80 μL of chromogenic reagent. The absorbance at 570 nm was measured at 30°C. After all samples were analyzed, the endpoint absorbance at 570 nm was measured. (H11 of each microplate contains the template for the library, and H12 contains the blank culture medium.)

[0077] Cell lysis buffer composition (with DTT): 10 mM Tris-HCl buffer (pH 8.0), 1 mM MgCl2, 1 mg / mL lysozyme, 0.1 mg / mL DNase I, 1.5 mM DTT.

[0078] Colorimetric solution (80 μL / well): 50 mM HEPES (pH 7.5), 10 mM fructose 1,6-bisphosphate (FBP), 0.05 mg / mL FbaA purified enzyme, 300 μM cofactors (NADP / NAD / NCD / NUD / NpUD), 400 μM MTT, 1 mM PES, and bring the volume to 80 μL with ultrapure water.

[0079] (2) Secondary screening method

[0080] Transformants with decreased NADP activity and increased NAD analog activity obtained from the initial screening were used for secondary screening. Single colonies found on the initial screening plates were reactivated on antibiotic-resistant plates. Bacterial colonies were picked and induced in 24-well plates for 48 h (2.5 mL of bacterial culture per well, LB medium supplemented with 50 μg / mL kanamycin and 0.5 mM IPTG). The plates were centrifuged at 4000 g for 5 min, the supernatant was discarded, and 250 μL of cell lysis buffer was added. The plates were then frozen at -80℃ for at least 1 h to aid lysis, followed by lysis at 37℃ and 200 rpm for 2 h. After centrifugation at 4000 g for 5 min, 15 μL of the supernatant crude enzyme solution was transferred to a 96-well UV-Vis microplate containing the reaction solution. The reaction was initiated, and absorbance changes were measured at 30℃ and 340 nm.

[0081] Cell lysis buffer composition (with DTT): 10 mM Tris-HCl buffer (pH 8.0), 1 mM MgCl2, 1 mg / mL lysozyme, 0.1 mg / mL DNase I, 1.5 mM DTT.

[0082] Crude enzyme activity assay system (100 μL / well): 50 mM HEPES (pH 7.5), fructose 1,6-bisphosphate (FBP), 0.05 mg / mL FbaA pure enzyme, 200-500 μM cofactors (NADP / NAD / NCD / NUD / NpUD), 15 μL of GAPN and mutant crude enzyme solution, and ultrapure water to 100 μL.

[0083] By comparing the activity of non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase utilizing NAD analogs with its activity utilizing NADP, mutants exhibiting increased activity towards NAD analogs were selected. Specific crude enzyme activity data are shown in the table below:

[0084] Table 1. Crude enzyme activities of nonphosphorylated glyceraldehyde triphosphate dehydrogenase and its mutants against NAD analogues. , , , .

[0085] The selected mutants showed increased crude enzyme activity for NAD analogs compared to the wild type, indicating a preference for NAD analogs. Among the various analogs, the mutants exhibited the highest activity and the strongest preference for nicotinamide cytosine dinucleotide (NCD).

[0086] Table 2 Description of site mutations in mutants , , , , .

[0087] Example 3: Cofactor kinetics determination of NAD analog-dependent nonphosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant

[0088] The kinetics of the mutant on NADP, NAD and NCD (cofactors) were determined.

[0089] Cofactor kinetic assay system (100 μL system): 50 mM HEPES (pH 7.5), 10 μL of enzyme solution with a final concentration of 0.01-0.03 mg / mL (fixed at 0.2 mM DL-G3P), 0.05-10 mM NADP, NAD, or NCD, and water to a final volume of 100 μL. Different concentrations of DL-G3P were added to initiate the reaction. After mixing, the absorbance at 340 nm was measured at 30 ℃. Three replicates were performed for each sample. The cofactor stock solution concentration was 0.5-100 mM (prepared with HEPES, pH 7.5).

[0090] The data measured by the ELISA reader were converted into the formazan formation rate v (μM / s) according to Beer-Lambert's law. The conversion formula is as follows:

[0091]

[0092] Where ΔA / min represents the change in absorbance at 340 nm, in mOD / min; k is the N×DH molar extinction coefficient, 6.22 mM. -1 ·cm -1 b is the optical path measured by the microplate reader; the optical path of the Nest microplate is 0.3125 cm; 60 is the conversion between minutes and seconds.

[0093] Substrate concentration and reaction rate were input into Origin for nonlinear fitting (Data selection was set to No Weighting during fitting).

[0094] The enzyme kinetics to cofactors were fitted using the Michaelis-Menten equation, which is as follows:

[0095]

[0096] Where x is the cofactor concentration, in μM in this experiment; y is the reaction rate, in μM / s in this experiment; Vmax is the maximum enzyme reaction rate, in the same unit as y; Km is the Michaelis constant, representing the substrate concentration at which the reaction rate reaches half of the maximum reaction rate, in μM in this experiment.

[0097] After fitting, Km and Vmax data are obtained. kcat data are calculated using the Vmax value.

[0098]

[0099] kcat is the turnover number, which represents the number of times the enzyme converts the substrate into the product per unit time at each enzyme active site. In this experiment, the unit is seconds (s). -1Vmax represents the maximum reaction rate of the enzyme, with the same unit as Y; Et represents the concentration of the enzyme catalytic site, in this experiment, the unit is μM. Et = enzyme concentration in the system (mg / mL) / relative molecular mass of the enzyme monomer. The relative molecular mass of the enzyme monomer can be calculated by inputting the amino acid sequence on the website: https: / / web.expasy.org / compute_pi / The principle of mutant cofactor kinetics determination is as follows: Figure 1 As shown, the kinetic results are as follows:

[0100] Table 3. Cofactor kinetics data of nonphosphorylated glyceraldehyde triphosphate dehydrogenase mutant. , .

[0101] Among them, mutant 1-F-AQVPR represents mutant I20F / P179A / T180Q / R209V / G210P / I234R, 2-F-AQWPR represents mutant I20F / P179A / T180Q / R209W / G210P / I234R, 3-F-SERR represents mutant I20F / P179S / T180E / G210R / I234R, 4-F-SEPRR represents mutant I20F / P179S / T180E / R209P / G210R / I234R; 5-FS-SEPRR represents mutant I20F / A36S / P179S / T1 80E / R209P / G210R / I234R, 6-MV-SERR represents the mutant I20M / A36V / P179S / T180E / G210R / I234R, 7-MV-SEKRR represents the mutant I20M / A36V / P179S / T180E / R209K / G210R / I234R, 8-MI-SERR represents the mutant I20M / A36I / P179S / T180E / G210R / I234R, and 9-LT-SELRR represents the mutant I20L / A36T / P179S / T180E / R209L / G210R / I234R.

[0102] NCD / NADP means (k cat / K m ) NCD / (k cat / K m ) NADP NCD / NAD means (k cat / K m ) NCD / (k cat / K m ) NADThe site mutation details of the above nine mutants are shown in Table 2.

[0103] Cells contain two natural cofactors, NAD and NADP. Wild-type non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase specifically utilizes NADP as a cofactor. After a series of modifications, the mutants showed a significant decrease in the catalytic efficiency for NADP and a slight increase in the catalytic efficiency for NAD. The highest efficiency was achieved by the mutant 7-MV-SEKRR, which showed a catalytic efficiency of 0.39 mM for NAD. -1 s -1 Compared to the wild type, the catalytic efficiency for NAD was increased by one order of magnitude; the catalytic efficiency of the above mutants for the NAD analog NCD was significantly improved, with the mutant 3-F-SERR showing the highest catalytic efficiency for NCD at 1.89 mM. -1 s -1 Compared to the wild type, the catalytic efficiency of these mutants for NCD was increased by three orders of magnitude. These mutants exhibited higher catalytic efficiency for NCD than for the natural cofactors NADP and NAD, demonstrating an NCD preference. (Preference is expressed as the ratio of the mutant's catalytic efficiency for NCD to its catalytic efficiency for NADP or NAD, respectively: (k...) cat / K m ) NCD / (k cat / K m ) NAD(P) )

[0104] Example 4: Production of reduced NAD analogues

[0105] The mutants 3-F-SERR (representing mutant I20F / P179S / T180E / G210R / I234R), 6-MV-SERR (representing mutant I20M / A36V / P179S / T180E / G210R / I234R), 7-MV-SEKRR (representing mutant I20M / A36V / P179S / T180E / R209K / G210R / I234R), and 8-MI-SERR (representing mutant I20M / A36I / P179S / T180E / G210R / I234R) from Example 3 were selected for the production of the reduced NAD analog NCDH.

[0106] The reaction conditions were as follows: 50 mM fructose-1,6-bisphosphate (FBP), 0.1 mg / mL FbaA purified enzyme, 20 mM cofactor, pH 7.5, and reaction at 37℃. The formation of the reduced coenzyme NAD analog NCDH was detected by monitoring the absorbance at 340 nm (reduced NAD analogs have a characteristic absorption peak at 340 nm).

[0107] After 2 hours of reaction, samples were taken to determine the formation of reduced NAD analogues. Since the molar extinction coefficient of the reduced coenzyme NAD analogue was unknown, it was determined based on the molar extinction coefficient of NADH (6.22 mM). 1 cm -1 The amount of reduced coenzyme NAD analog NCDH generated was estimated based on Lambert-Beer's law, and the results are shown in Table 4. The highest conversion rate was 90%.

[0108] Compared with traditional chemical reduction methods, this method has milder reaction conditions, stronger reaction specificity, and no over-reduction. Compared with other enzymatic reduction methods, the reaction catalyzed by this enzyme is irreversible, which is conducive to the formation of reduced products.

[0109] Table 4. NCDH generation

[0110] Example 5: Preparation of alanine by reductive amination of pyruvate catalyzed by a system of non-phosphorylated glyceraldehyde triphosphate dehydrogenase, alanine dehydrogenase A225P / S219E, and NAD analog.

[0111] Alanine dehydrogenase A225P / S219E prefers the NAD analog NCD, and the reductive amination reaction to produce alanine requires a reduced analog as a cofactor. The reduced cofactor produced by non-phosphorylated glyceraldehyde triphosphate dehydrogenase is utilized by alanine dehydrogenase, achieving a cofactor cycle while simultaneously generating the bulk chemical alanine, which shows promising application potential.

[0112] A representative experimental procedure: 10 mM fructose-1,6-bisphosphate (FBP), 0.1 mg / mL FbaA purified enzyme, 5 mM pyruvate, 50 mM NH4Cl, 0.1 mM NCD, 0.1 mg / mL nonphosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant 3-F-SERR (representing mutant I20F / P179S / T180E / G210R / I234R), and 0.1 mg / mL alanine dehydrogenase A225P / S219E. The reaction was carried out at 37℃ for 1 h.

[0113] When the reaction is terminated: 100 μL of sample was taken at each time point and added to 900 μL of termination solution (methanol:acetonitrile:water = 4:4:1). After shaking and mixing, the solution was centrifuged at 14000 g for 20 min at 10℃. The supernatant was then used for ion chromatography to detect pyruvate and L-alanine.

[0114] Pyruvate detection: Ion chromatography conductivity detector (organic acid system), IonPac AS11-HC Analytical Column (250×2 mm), IonPac AG11-HC Guard Column (50×4 mm). Flow rate: 1 mL / min. Isocratic analysis with 5 mM NaOH for 20 min. Regeneration solution: 30 mM H2SO4, nitrogen pressure: 30 kPa.

[0115] Determination of L-alanine: The content of L-alanine in solution was analyzed using a Dionex ICS-2500 ion chromatography system in integrated amperometric detection mode. A Dionex AminoPac PA10 analytical column (250 mm × 2 mm) and a Dionex AminoPac PA10 guard column (50 mm × 2 mm) were used. Analytical conditions: gradient elution of 50 mM NaOH to 60 mM NaOH and 760 mM ammonium acetate, flow rate 0.2 mL / min, column temperature 30℃, injection volume 25 μL.

[0116] The results showed that the reaction solution contained 0.5 mM pyruvate and 4.5 mM alanine, indicating that the cyclic cofactor NCD between nonphosphorylated glyceraldehyde triphosphate dehydrogenase and alanine dehydrogenase A225P / S219E provided reducing power for the reductive amination of pyruvate, thus achieving NCD-mediated alanine synthesis.

[0117] Example 6: Preparation of lactate from pyruvate by a system of non-phosphorylated glyceraldehyde triphosphate dehydrogenase, D-lactate dehydrogenase DLDH-V152R, and NAD analogs.

[0118] D-lactate dehydrogenase DLDH-V152R prefers the NAD analog NUD and requires a reduced analog as a cofactor. The reduced cofactor produced by non-phosphorylated glyceraldehyde triphosphate dehydrogenase is utilized by DLDH-V152R, achieving cofactor cycling while simultaneously generating the bulk chemical D-lactate, which shows promising application potential.

[0119] A representative experimental procedure: 10 mM fructose-1,6-bisphosphate (FBP), 0.1 mg / mL FbaA purified enzyme, 5 mM pyruvate, 0.1 mM NUD, 0.1 mg / mL nonphosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant 17-SELRR (representing mutant P179S / T180E / R209L / G210R / I234R), and 0.1 mg / mL DLDH-V152R. The reaction was carried out at 37℃ for 2 h.

[0120] When the reaction is terminated: 100 μL of sample was taken at each time point and added to 900 μL of termination solution (methanol:acetonitrile:water = 4:4:1). After shaking and mixing, the solution was centrifuged at 14000 g for 20 min at 10℃. The supernatant was then used for ion chromatography to detect pyruvate and lactic acid.

[0121] The contents of pyruvate and lactic acid in the reaction solution were analyzed and determined using a Dionex ICS-2500 ion chromatography system in ED50 pulsed electrochemical detection mode. An IonPac AS11-HC anion exchange column (200 mm × 4 mm) and an IonPac AG11-HC anion exchange protectant (50 mm × 4 mm) were used. Analytical conditions: mobile phase 24 mM NaOH, flow rate 1 mL / min, column temperature 30 °C, injection volume 25 μL.

[0122] The test results showed that the reaction solution contained 1.2 mM pyruvate and 3.7 mM D-lactic acid. This indicates that NUD-mediated D-lactic acid synthesis is achieved by circulating the cofactor NUD between nonphosphorylated glyceraldehyde triphosphate dehydrogenase and DLDH-V152R, providing reducing power for the reduction of pyruvate to lactate.

[0123] Example 7: Preparation of malic acid by reductive carboxylation of pyruvate catalyzed by a system of non-phosphorylated glyceraldehyde triphosphate dehydrogenase, malate enzyme ME-L310R / Q401V and NAD analog.

[0124] Malicase ME-L310R / Q401V utilizes the NAD analog NpUD. The reaction catalyzed by this enzyme requires a reduced analog as a cofactor. The reduced cofactor produced by non-phosphorylated glyceraldehyde triphosphate dehydrogenase is utilized by malicase ME-L310R / Q401V, achieving cofactor cycling while simultaneously generating the bulk chemical L-malic acid, which has certain application prospects.

[0125] A representative experimental procedure was as follows: 10 mM fructose-1,6-bisphosphate (FBP), 0.1 mg / mL FbaA purified enzyme, 5 mM pyruvate, 10 mM NaHCO3, 2 mM MgCl2, 0.1 mM NpUD, 0.1 mg / mL non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant 3-F-SERR (representing mutant I20F / P179S / T180E / G210R / I234R), and 0.1 mg / mL malate enzyme ME-L310R / Q401V. The reaction was carried out at 37℃ for 2 h.

[0126] At reaction termination: 100 μL of sample was taken at each time point and added to 900 μL of termination solution (methanol:acetonitrile:water = 4:4:1). After shaking and mixing, the mixture was centrifuged at 14000 g for 20 min at 10°C. The supernatant was then used for ion chromatography to detect pyruvate and malic acid. The detection method was the same as in Example 6.

[0127] The test results showed that the reaction solution contained 1.8 mM pyruvate and 3.0 mM L-malate. This indicates that the NpUD-mediated synthesis of L-malate is achieved by circulating the cofactor NpUD between non-phosphorylated glyceraldehyde triphosphate dehydrogenase and malate enzyme ME-L310R / Q401V to provide reducing power for the reductive carboxylation reaction of pyruvate.

[0128] Example 8: Screening for growth coupling using non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase

[0129] Principle: In *E. coli* with the endogenous genes gapA (encoding glyceraldehyde-3-phosphate dehydrogenase) and mgsA (encoding methylglyoxal synthase) knocked out, the glycolysis pathway is blocked, and the strain cannot grow in a medium with glucose as the sole carbon source. By introducing a non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant into this strain, and simultaneously providing the NAD analog NCD, an NCD-mediated growth-dependent platform strain is constructed. The glycolysis pathway then becomes an NCD-dependent glycolysis pathway, changing from producing two molecules of NADH to producing two molecules of NCDH. Furthermore, without the additional introduction of an enzyme capable of utilizing NCDH, the platform strain will also be unable to grow in a medium with glucose as the sole carbon source due to redox imbalance. This characteristic can be used as a growth-dependent screening platform to screen for enzyme elements that utilize NCDH. (See diagram below.) Figure 2 As shown.

[0130] We introduced malicases with different cofactor preferences (ME: NAD-biased, ME-L310R / Q401C: NCD-biased) into the platform strain to verify whether the strain could be restored to grow in glucose medium.

[0131] The conventional culture medium formulations used for each stage of the platform strain are as follows: SGC medium is the conventional culture medium for the platform strain, and glucose medium is the culture medium used to verify the function of the platform strain.

[0132] SGC medium: 50 mM sodium succinate, 50 mM glycerol, 0.1% (w / v) casein amino acid hydrolysate, 0.5 g / L NaCl, 1 g / L NH4Cl, 6.8 g / L Na2HPO4 (48 mM), 3 g / L KH2PO4 (22 mM), 2 mM MgSO4, 0.1 mM CaCl2, pH 7.0.

[0133] Glucose medium: 0.4% glucose, 0.5 g / L NaCl, 1 g / L NH4Cl, 6.8 g / L Na2HPO4 (48 mM), 3 g / L KH2PO4 (22 mM), 2 mM MgSO4, 0.1 mM CaCl2, pH 7.0.

[0134] Add 1.5-2% agar powder when preparing plates. Antibiotic concentrations used: kanamycin 50 μg / mL, chloramphenicol 34 μg / mL, carbenicillin 50 μg / mL. Add the appropriate concentration of antibiotic to the corresponding liquid or solid culture medium as needed.

[0135] Construction of engineered strains: The expression vectors of malicases (ME and ME-L310R / Q401C) with different cofactor preferences and the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant 6-MV-SERR (representing mutant I20M / A36V / P179S / T180E / G210R / I234R) were transformed into engineered strains with gapA and mgsA gene knockout (see Proceedings of the National Academy of Sciences 2018, 115 (14), 3538-3546.) and NCD synthesis module integrated into the genome (see Nature communications 2021, 12 (1), 2116.) using conventional E. coli transformation methods to obtain the target engineered strain.

[0136] Growth curve determination procedure: Mycelial growth was picked from plates and cultured in 2 mL of SGC+ resistant medium, activated at 37℃ and 200 rpm for 24 h. The OD of the seed culture was then measured. 600 Transfer to 2 mL of SGC+ corresponding resistance induction medium to induce initial OD 600 The initial OD was 0.1, and protein expression was induced at 30℃ and 200 rpm for 24 h. The bacterial cells were washed twice with M9 salt solution, and then a certain volume of the washed seed culture was inoculated into glucose medium with the corresponding resistance to achieve the initial OD.600 The value was 0.2. The growth of the strain at 30°C (600 nm absorbance) was measured using a Bioscreen C growth curve analyzer.

[0137] Growth curve measurement results are as follows Figure 3 As shown, all strains expressed the non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant 6-MV-SERR. When intracellular NCD synthesis occurred in the NCD synthesis module, samples expressing the NCD-preferred malicase ME-L310R / Q401C grew faster than those expressing the NAD-preferred malicase ME. Furthermore, the group with NCD synthesis grew faster than the group without induced NCD synthesis, indicating the effectiveness of the NCD-mediated growth selection platform. Non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase, as a key component, played a role in regenerating reduced NCD and is a necessary condition for the platform strains to perform their selection function.

[0138] Example 9: Non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase for growth-coupled production

[0139] Non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase mutants can also be used in growth-coupled production, such as... Figure 4 As shown. The principle is similar to that of Example 8. In *E. coli* with the endogenous genes gapA (encoding glyceraldehyde-3-phosphate dehydrogenase) and mgsA (encoding methylglyoxal synthase) knocked out, a non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant was introduced, along with the NAD analog NCD, to construct an NCD-mediated growth-dependent platform strain. This platform strain can be used as a growth-coupled production platform. Malases with different cofactor preferences (ME: NAD-biased, ME-L310R / Q401C: NCD-biased) were introduced into the platform strain to verify whether the strain's growth in glucose medium could be restored.

[0140] Production culture medium: 0.4% glucose, 20 mM sodium pyruvate, 0.5 g / L NaCl, 1 g / L NH4Cl, 6.8 g / L Na2HPO4 (48 mM), 3 g / L KH2PO4 (22 mM), 2 mM MgSO4, 0.1 mM CaCl2, pH 7.0.

[0141] Construction of engineered strains: The expression vectors of alanine dehydrogenase A225P / S219E / V165A and the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant 6-MV-SERR (representing mutant I20M / A36V / P179S / T180E / G210R / I234R) were transformed into E. coli using conventional transformation methods. gapA, mgsAThe target engineered strain is obtained from an engineered strain with gene knockout (see Proceedings of the National Academy of Sciences 2018, 115 (14), 3538-3546.) and simultaneous integration of the NCD synthesis module into the genome (see Nature communications 2021, 12 (1), 2116.).

[0142] A representative experimental procedure: Mycelial growth was picked from a plate and cultured in 2 mL of SGC+ resistant medium (same formulation as in Example 8), activated at 37°C and 200 rpm for 24 h, and the OD of the seed culture was measured. 600 Transfer to 2 mL of SGC+ corresponding resistance induction medium to induce initial OD 600 The initial OD was 0.1, and protein expression was induced at 30℃ and 200 rpm for 24 h. The bacterial cells were washed twice with M9 salt solution, and then a certain volume of the washed seed culture was inoculated into the corresponding antibiotic production medium to achieve the initial OD. 600 The value was 0.2, and the mixture was cultured at 30°C to produce alanine. The alanine detection method was the same as in Example 5.

[0143] After 48 h of cultivation, 12 mM alanine was detected, while the control strain, which did not express glyceraldehyde-3-phosphate dehydrogenase, did not grow, thus achieving a production model where growth is equivalent to production. This method couples alanine production with the growth of engineered strains, realizing the production of alanine mediated by the non-natural coenzyme NCD. Compared with existing methods for microbial synthesis of alanine, this method reduces the crosstalk problem of natural coenzymes within the cell.

[0144] Example 10: Crystal structure analysis of the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant

[0145] The crystal structure of the non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant 6-MV-SERR was determined. The purified protein concentration was 20 mg / mL, stored in a buffer solution consisting of 20 mM HEPES (pH 7.5), 200 mM NaCl, and 1 mM TCEP. Crystallization was performed using the hanging drop method and incubated at 18°C. Crystallization droplets were prepared by mixing 1 μL of protein solution with 1 μL of pooling solution. Initial crystallization conditions were obtained through systematic screening, and subsequently optimized to obtain crystals suitable for X-ray diffraction. Crystals used for data collection were grown in an optimized system with a pooling solution consisting of 0.1 M KCl, 0.1 M Tris (pH 8.0), and 15% (w / v) methoxy polyethylene glycol 2000. Before collecting diffraction data, the crystals were briefly immersed in a cryoprotectant solution containing 20% ​​glycerol (v / v) and then rapidly frozen in liquid nitrogen. X-ray diffraction data acquisition was conducted at the BL02U1 beamline of the Shanghai Synchrotron Radiation Facility, with a maximum resolution of 2.0 angstroms. XDS and aimless software were used to process the diffraction data.

[0146] The 6-MV-SERR crystal structure was obtained with a resolution of 2.0 Å, and one unit cell consisted of four protein molecules. The obtained crystal structure is shown below. Figure 5 and Figure 6 As shown.

[0147] Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A nonphosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant with a preference for NAD analogues, characterized in that, The non-phosphorylated glyceraldehyde-3-phosphate dehydrogenase mutant was obtained by mutating the amino acid sequence as shown in SEQ ID NO.

1. The site mutation and the corresponding amino acid sequence of the mutant are as follows: (1) Mutant 1-F-AQVPR: is obtained by changing amino acid I to R, amino acid I to F, amino acid P to A, amino acid T to Q, amino acid R to V, and amino acid G to P in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of mutant 1-F-AQVPR is shown in SEQ ID NO.

3. (2) Mutant 2-F-AQWPR: is obtained by changing amino acid I to R, amino acid I to F, amino acid P to A, amino acid T to Q, amino acid W to W, and amino acid G to P in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of mutant 2-F-AQWPR is shown in SEQ ID NO.

4. (3) Mutant 3-F-SERR: is obtained by changing amino acid I to R, amino acid I to F, amino acid P to S, amino acid T to E, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of mutant 3-F-SERR is shown in SEQ ID NO.

5. (4) Mutant 4-F-SEPRR: is obtained by changing amino acid I to R, amino acid I to F, amino acid P to S, amino acid T to E, amino acid R to P, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of mutant 4-F-SEPRR is shown in SEQ ID NO.

6. (5) Mutant 5-FS-SEPRR: is obtained by changing amino acid I to R, amino acid I to F, amino acid A to S, amino acid P to S, amino acid T to E, amino acid R to P, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of mutant 5-FS-SEPRR is shown in SEQ ID NO.

7. (6) Mutant 6-MV-SERR: is obtained by changing amino acid I to R, amino acid I to M, amino acid A to V, amino acid P to S, amino acid T to E, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of mutant 6-MV-SERR is shown in SEQ ID NO.

8. (7) Mutant 7-MV-SEKRR: is obtained by changing amino acid I to R, amino acid I to M, amino acid A to V, amino acid P to S, amino acid T to E, amino acid K to K, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of mutant 7-MV-SEKRR is shown in SEQ ID NO.

9. (8) Mutant 8-MI-SERR: is obtained by changing amino acid I to R, amino acid I to M, amino acid A to I, amino acid P to S, amino acid T to E, and amino acid G to R in the amino acid sequence shown in SEQ ID NO.

1. The amino acid sequence of mutant 8-MI-SERR is shown in SEQ ID NO.

10. (9) Mutant 9-LT-SELRR: is obtained by changing amino acid 234 from I to R, amino acid 20 from I to L, amino acid 36 from A to T, amino acid 179 from P to S, amino acid 180 from T to E, amino acid 209 from R to L, and amino acid 210 from G to R in the amino acid sequence shown in SEQ ID NO.

11. (10) Mutant 10-AQR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to A, the 180th amino acid to Q, and the 234th amino acid to R. The amino acid sequence of mutant 10-AQR is shown in SEQ ID NO.

12. (11) Mutant 11-SQR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to S, the 180th amino acid to Q, and the 234th amino acid to R. The amino acid sequence of mutant 11-SQR is shown in SEQ ID NO.

13. (12) Mutant 12-SER: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 from P to S, the 180th amino acid from T to E, and the 234th amino acid from I to R. The amino acid sequence of mutant 12-SER is shown in SEQ ID NO.

14. (13) Mutant 13-AQVR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to A, the 180th amino acid to Q, the 210th amino acid to V, and the 234th amino acid to R. The amino acid sequence of mutant 13-AQVR is shown in SEQ ID NO.

15. (14) Mutant 14-AQTR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to A, the 180th amino acid to Q, the 210th amino acid to T, and the 234th amino acid to R. The amino acid sequence of mutant 14-AQTR is shown in SEQ ID NO.

16. (15) Mutant 15-SQVR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to S, the 180th amino acid to Q, the 210th amino acid to V, and the 234th amino acid to R. The amino acid sequence of mutant 15-SQVR is shown in SEQ ID NO.

17. (16) Mutant 16-SERR: is obtained by changing the 179th amino acid of the amino acid sequence shown in SEQ ID NO.1 to S, the 180th amino acid to E, the 210th amino acid to R, and the 234th amino acid to R. The amino acid sequence of mutant 16-SERR is shown in SEQ ID NO.

18. (17) Mutant 17-SELRR: is obtained by changing the amino acid position 179 from P to S, the amino acid position 180 from T to E, the amino acid position 209 from R to L, the amino acid position 210 from G to R, and the amino acid position 234 from I to R in the amino acid sequence shown in SEQ ID NO.

19. (18) Mutant 18-M-SERR: is obtained by changing the 20th amino acid of the amino acid sequence shown in SEQ ID NO.1 from I to M, the 179th amino acid from P to S, the 180th amino acid from T to E, the 210th amino acid from G to R, and the 234th amino acid from I to R. The amino acid sequence of mutant 18-M-SERR is shown in SEQ ID NO.

20.

2. The use of the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant according to claim 1 in the preparation of formulations coupled with multi-enzyme-driven reduction of NAD analogs.

3. The application according to claim 2, characterized in that, The non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant can catalyze the conversion of glyceraldehyde triphosphate to 3-phosphoglycerate and can convert NAD analogs to their reduced form.

4. The application according to claim 2, characterized in that, The non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant can catalyze the reductive amination of pyruvate to prepare alanine, the reductive reduction of pyruvate to prepare lactic acid, and the reductive carboxylation of pyruvate to prepare malic acid.

5. The application according to claim 2, characterized in that, The multienzyme includes at least one of malic acid oxidase, lactate dehydrogenase, alanine dehydrogenase, and fructose-1,5-bisphosphate aldolase.

6. The application according to claim 2, characterized in that, The reaction system for the coupled multi-enzyme-driven reduction of NAD analogues includes: a buffer system with pH 3-9, substrate concentration of 1-500 mM, NAD analogue concentration of 0.05-20 mM, enzyme concentration of 0.01-5 mg / mL, and reaction at 10-40℃ for 5 min-24 h.

7. The application of the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant of claim 1 in screening NAD analogue-mediated growth-dependent strains.

8. The use of the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant of claim 1 in promoting NAD analogue-mediated strain growth.

9. The application according to claim 7 or 8, characterized in that, The specific methods of application include: (1) Provide a screening platform for enzymes that utilize reduced NAD analogues: The non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant is expressed in an engineered strain with the gapA and / or mgsA gene knocked out as a screening platform strain. The mutant library of the enzyme to be screened is induced to be expressed in the platform strain. The mutant is cultured at 18-37℃ for 2-15 days in a medium with glucose as the sole carbon source. Only mutants of enzymes that can utilize NAD analogues can grow, so as to screen for enzymes that can utilize reduced NAD analogues. (2) Provide a growth-dependent production platform: By expressing the non-phosphorylated glyceraldehyde triphosphate dehydrogenase mutant in the engineered strain as a production platform, the enzyme utilizing the reduced NAD analog is further expressed. In a medium with glucose as the sole carbon source, the strain is cultured at 18-37℃ for 2-15 days. The strain accumulates products while growing, so as to achieve the coupling of the production of the target product and the growth of the strain.

10. The application according to any one of claims 2, 7, or 8, characterized in that, The NAD analogues include at least one of NCD, NUD, and NpUD.

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