Alanine dehydrogenase mutant and application thereof

Abstract

The application discloses an alanine dehydrogenase mutant, wherein 1-3 amino acids in positions 165, 177, 178, 180, 199, 200, 217, 218, 219, 225, 237 and 238 of the wild-type alanine dehydrogenase amino acid sequence shown in SEQ ID NO. 1 are respectively mutated into one of lysine, arginine, glutamic acid, proline, valine, glycine, alanine and serine. The alanine dehydrogenase mutant has significant preference for NAD(H) analogues, can produce L-alanine or pyruvic acid by using NAD analogues and NADH analogues, and does not cause interference problems. The application further discloses application of the alanine dehydrogenase mutant.

Description

Technical Field

[0001] This application relates to an enzyme, which belongs to the fields of metabolic engineering and synthetic biology. Background Technology

[0002] L-Alanine is widely used in pharmaceuticals, cosmetics, and veterinary medicine. For example, it can be used in parenteral formulations with other amino acids, as a pre- and post-operative food for clinical use, and as an animal feed supplement. Furthermore, due to its sweet taste, alanine is also used as a food additive. Early biosynthesis of L-alanine involved glutamate dehydrogenase converting α-ketoglutarate, ammonia, and NADH into glutamate, NAD, and water. In a second step, the amino group of the newly formed glutamate is transferred to pyruvate via aminotransferase, converting pyruvate back to alanine and regenerating α-ketoglutarate. However, this is a multi-step process requiring a continuous supply of enzymes and substrates, and the high production cost limits its application. Therefore, the microbial production of L-alanine using alanine dehydrogenase has attracted considerable attention.

[0003] Pyruvate dehydrogenase (AlaDH) catalyzes reversible reactions: oxidative deamination of L-alanine to pyruvate or reductive amination of pyruvate to L-alanine. Oxidative deamination requires NAD as a cofactor, while reductive amination requires NADH. Besides producing L-alanine, alanine dehydrogenase can also be used to produce pyruvate. Pyruvate is a crucial component of various endogenous metabolic pathways, playing a vital role in bioenergy metabolism and serving as a precursor for the synthesis of many compounds. In specific scenarios, alanine dehydrogenase can convert excess / waste L-alanine into pyruvate, further producing other pyruvate-derived compounds such as acetoin, butanol, and butyrate.

[0004] Redox cofactors NAD / NADH and their phosphorylated forms NADP / NADPH are closely related to product formation in microbial cell factories. They have important applications in in vitro chemical synthesis and in vivo metabolic engineering. Approximately 80% of known intracellular redox enzymes require nicotinamide adenine dinucleotide as a cofactor (Current opinion in biotechnology 2020, 66, 217-226). This means that electron transfer processes mediated by the redox cofactor NAD result in electrons being dispersed throughout the metabolic network, making it difficult to regulate a single NAD-dependent enzyme catalysis process without affecting other metabolic processes. Therefore, with the development of synthetic biology, the development of NAD analogues has gradually gained attention in order to reduce the cost of biocatalytic industrial production and minimize interference with intracellular metabolism. Examples include nicotinamide cytosine dinucleotide (NCD), nicotinamide thymine dinucleotide (NTD), nicotinamide uracil dinucleotide (NUD), and nicotinamide pseudouracil dinucleotide (NpUD) (J Am Chem Soc 2011, 133(51), 20857-62; Tetrahedron Letters 2022, 88). Using NAD analogues and enzymes that recognize them, more economical and efficient biocatalytic systems can be constructed.

[0005] Although there is considerable research on the production of L-alanine using alanine dehydrogenase, there is still very little research on the directed evolution modification of alanine dehydrogenase to utilize NAD analogs. No literature reports how to modify alanine dehydrogenase to utilize NAD analogs. Modifying alanine dehydrogenase to recognize NAD(H) analogs could achieve the production of L-alanine or pyruvate without disrupting intracellular redox balance. Because intracellular redox reactions are continuous, complex, and precise, existing modification techniques, while purposefully altering enzyme function, all interfere with intracellular metabolic pathways in practical applications, making it impossible to achieve a complete intracellular production process. Summary of the Invention

[0006] Based on the above technical problems, this application obtains an alanine dehydrogenase mutant with a preference for NAD(H) analogues through directed evolution, enabling it to achieve the production of L-alanine or pyruvate and uncoupling from NAD(H), while avoiding the problem of interference with intracellular reactions.

[0007] The present invention provides one aspect of an alanine dehydrogenase mutant, characterized in that the alanine dehydrogenase mutant mutates one to three amino acids from positions 165, 177, 178, 180, 199, 200, 217, 218, 219, 225, 237, and 238 of the wild-type alanine dehydrogenase amino acid sequence shown in SEQ ID NO.1 to one of lysine, arginine, glutamic acid, proline, valine, glycine, alanine, and serine, respectively.

[0008] The alanine dehydrogenase mutant described in this invention is selected from any one of the mutants obtained by performing the following mutations based on the wild-type alanine dehydrogenase shown in SEQ ID NO.1:

[0009] The 177th amino acid is mutated from threonine to lysine or arginine; or

[0010] The 180th amino acid was mutated from threonine to lysine; or

[0011] The 199th amino acid was mutated from asparagine to arginine; or

[0012] The 200th amino acid is mutated from alanine to arginine; or

[0013] The 217th amino acid was mutated from leucine to lysine; or

[0014] The 218th amino acid was mutated from methionine to arginine; or

[0015] The 219th amino acid is mutated from serine to glutamic acid or proline; or

[0016] The 225th amino acid was mutated from alanine to proline; or

[0017] The 237th amino acid was mutated from alanine to valine; or

[0018] The amino acid at position 238 is mutated from valine to arginine; or

[0019] The 200th amino acid is mutated from alanine to arginine, and the 178th amino acid is mutated from alanine to valine; or

[0020] The 200th amino acid is mutated from alanine to arginine, and the 180th amino acid is mutated from threonine to glycine; or

[0021] The 200th amino acid is mutated from alanine to arginine, and the 219th amino acid is mutated from serine to glutamic acid; or

[0022] The 225th amino acid is mutated from alanine to proline, and the 165th amino acid is mutated from valine to alanine; or

[0023] The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 177 is mutated from threonine to lysine; or

[0024] The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 178 is mutated from alanine to valine; or

[0025] The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 180 is mutated from threonine to lysine; or

[0026] The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 199 is mutated from asparagine to arginine; or

[0027] The 225th amino acid is mutated from alanine to proline, and the 200th amino acid is mutated from alanine to lysine; or

[0028] The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 217 is mutated from leucine to serine; or

[0029] The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 219 is mutated from serine to glutamic acid; or

[0030] The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 238 is mutated from valine to alanine; or

[0031] The 200th amino acid is mutated from alanine to arginine, the 178th amino acid from alanine to valine, and the 219th amino acid from serine to glutamic acid; or

[0032] The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 165 is mutated from valine to alanine, and the amino acid at position 219 is mutated from serine to glutamic acid; or

[0033] The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 177 is mutated from threonine to lysine, and the amino acid at position 219 is mutated from serine to glutamic acid; or

[0034] The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 178 is mutated from alanine to valine, and the amino acid at position 219 is mutated from serine to glutamic acid; or

[0035] The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 180 is mutated from threonine to lysine, and the amino acid at position 219 is mutated from serine to glutamic acid; or

[0036] The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 199 is mutated from asparagine to arginine, and the amino acid at position 219 is mutated from serine to glutamic acid; or

[0037] The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 200 is mutated from alanine to lysine, and the amino acid at position 219 is mutated from serine to glutamic acid; or

[0038] The amino acid at position 225 was mutated from alanine to proline, the amino acid at position 238 was mutated from valine to alanine, and the amino acid at position 219 was mutated from serine to glutamic acid.

[0039] The mutant alanine dehydrogenase described in this invention uses an NAD(H) analogue as a cofactor.

[0040] In this application, NAD(H) analogues include NAD analogues and NADH analogues.

[0041] The NAD analogue is at least one of NCD, NUD, NTD, or NpUD.

[0042] The NAD analogues are one or more of NCD, NUD, NTD, or NpUD, and their chemical structures are as follows:

[0043]

[0044] Reduced NAD analogues are one or more of NCDH, NUDH, NTDH, or NpUDH, and their chemical structures are as follows:

[0045]

[0046] The present invention provides yet another aspect, providing a DNA sequence encoding, characterized in that it encodes the DNA sequence corresponding to a mutant of the said alanine dehydrogenase.

[0047] The present invention provides yet another aspect, providing a protein expression vector for controlled expression, characterized in that the DNA coding sequence corresponding to the mutant of the alanine dehydrogenase is cloned in the protein expression vector.

[0048] In this invention, the NADH analogs NCDH, NUDH, NTDH, or NpUDH are obtained by enzymatic or chemical reduction of NCD, NUD, NTD, or NpUD. The regenerating enzymes for the enzymatic reduction of NCD, NUD, NTD, and NpUD are malate enzyme ME-L310R / Q401C, D-lactate dehydrogenase DLDH-V152R / I177K / N213I, and phosphorylation dehydrogenase. PDH-I151R / P176R / M207A, formate dehydrogenase FDH-V198I / C256I / P260S / E261P / S381N / S383F, methanol dehydrogenase MDH-Y171R / I196V / V237T / N240E / K241A, or formaldehyde dehydrogenase FalDH-A192R / L233V / L236V, or one or more of these enzymes;

[0049] The regeneration enzymes for the enzymatic reduction of NCD, NUD, NTD, and NpUD correspond to one or more of the following regeneration substrates: malic acid compounds, phosphite compounds, D-lactic acid compounds, formic acid compounds, methanol compounds, or formaldehyde compounds; wherein the malic acid compound is one or more of malic acid and malate; the phosphite compound is one or more of phosphorous acid and phosphite salts; the D-lactic acid compound is one or more of D-lactic acid and D-lactic acid salts; the formic acid compound is one or more of formic acid and formate salts; the methanol compound is one or more of methanol and deuterated methanol; and the formaldehyde compound is one or more of formaldehyde and deuterated formaldehyde. The reaction system for the enzymatic regeneration of NADH analogs is a buffer solution at pH 7.5, containing 1 mM-20 mM NAD analog, 10 U-500 U regeneration enzyme, and 2 mM-100 mM regeneration substrate. The reaction is carried out at 20℃-40℃ for 20 min-4 h. The buffer solution is one or more of phosphate, Tris-HCl, MES, or HEPES buffer.

[0050] The chemical reduction of NCD, NUD, NTD and NpUD is carried out in an aqueous solution, using one or more of the following as reducing agents: Na2S2O4, NaBH4, NaBH(Et)3, NaBH3CN or CH4N2O2S. The ratio of NAD analog to reducing agent is 1:2-4, and the reaction is carried out at 20℃-40℃ for 20 min-2 h.

[0051] The present invention provides another aspect, providing an application of the mutant of the alanine dehydrogenase, characterized in that the alanine dehydrogenase mutant, in a buffer system of pH 5-10, utilizes an NAD analogue as a cofactor to catalyze the oxidative deamination of L-alanine to pyruvate.

[0052] The buffer system includes, but is not limited to, at least one of phosphate, Tris HCl, MES, CAPS or HEPES buffer.

[0053] The alanine dehydrogenase mutant, in a buffer system at pH 5-9, is coupled with malate enzyme ME-L310R / Q401C, D-lactate dehydrogenase DLDH-V152R / I177K / N213I, phosphite dehydrogenase PDH-I151R / P176R / M207A, formate dehydrogenase FDH-V198I / C256I / P260S / E261P / S381N / S383F, methanol dehydrogenase MDH-Y171R / I196V / V237T / N240E / K241A, or formaldehyde dehydrogenase FalDH-A192R / L233V / L236V, and the corresponding regeneration substrate, to catalyze the reductive amination of pyruvate to synthesize L-alanine using NAD analogues as cofactors.

[0054] The NAD analogue-dependent alanine dehydrogenase mutant is co-expressed in microbial cells with NTT4 protein derived from Chlamydia or AtNDT2 protein derived from Arabidopsis, catalyzing the production of pyruvate from L-alanine intracellularly. The NAD analogue is transported into the cell via NTT4 protein derived from Chlamydia or AtNDT2 protein derived from Arabidopsis.

[0055] The NAD analogue-dependent alanine dehydrogenase mutant, along with NTT4 derived from Chlamydia or AtNDT2 derived from Arabidopsis, and the regenerating NADH analogues: malate enzyme ME-L310R / Q401C, D-lactate dehydrogenase DLDH-V152R / I177K / N213I, phosphorylation dehydrogenase PDH-I151R / P176R / M207A, and formate dehydrogenase FDH- One or more of the following enzymes are co-expressed in microbial cells: V198I / C256I / P260S / E261P / S381N / S383F, methanol dehydrogenase MDH-Y171R / I196V / V237T / N240E / K241A, or formaldehyde dehydrogenase FalDH-A192R / L233V / L236V. These enzymes catalyze the reductive amination of pyruvate to L-alanine within the cell.

[0056] NAD analogues are transported into the cell via NTT4, derived from Chlamydia, or AtNDT2, derived from Arabidopsis.

[0057] The microbial cells expressing the alanine dehydrogenase mutant and used for intracellular catalysis of the interconversion of alanine and L-alanine are at least one of the following: prokaryotic microorganisms: *Escherichia coli*, *Lactococcus lactis*; eukaryotic microorganisms: *Saccharomyces cerevisiae*, *Trichoderma reesei*.

[0058] The beneficial effects that this application can produce include:

[0059] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: The alanine dehydrogenase mutant obtained by the present invention has a significant preference for NAD(H) analogues, and can use NAD analogues and NADH analogues to produce L-alanine or pyruvate without causing interference problems. Attached Figure Description

[0060] none Detailed Implementation

[0061] To facilitate understanding of the present invention, the invention will be described more fully and in detail below with reference to preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0062] The alanine dehydrogenase used in this invention is derived from Geobacillus stearothermophilus XL-65-6, UniProt Primary accession: A8QVZ6.

[0063] The malicase ME used in this application is derived from Escherichia coli K12, UniProt Primary accession: P26616. The mutation sites of its mutant ME-L310R are: amino acid 310 changed from L to R; amino acid 310 changed from L to R and amino acid 401 changed from Q to V; amino acid 310 changed from L to R and amino acid 401 changed from Q to V; amino acid 310 changed from L to R and amino acid 401 changed from Q to C; amino acid 310 changed from L to R and amino acid 401 changed from Q to S; amino acid 310 changed from L to R and amino acid 401 changed from Q to S; and amino acid 310 changed from L to R and amino acid 401 changed from Q to G.

[0064] The D-lactate dehydrogenase DLDH used in this application is derived from Lactobacillus helveticus, UniProtPrimary accession: P30901. The mutant DLDH-V152R / I177K / N213I has the following mutation sites: amino acid 152 is changed from V to R, amino acid 177 is changed from I to K, and amino acid 213 is changed from N to I.

[0065] The phosphorylated dehydrogenase PDH used in this application is derived from Ralstonia sp. strain 4506, UniProt Primary accession: G4XDR8. The mutant PDH-I151R / P176E has the following mutation sites: amino acid 151 changes from I to R and amino acid 176 changes from P to E; the mutant PDH-I151R / P176E / M207A has the following mutation sites: amino acid 151 changes from I to R, amino acid 176 changes from P to E, and amino acid 207 changes from M to A; the mutant PDH-I151R / P176R / M207A has the following mutation sites: amino acid 151 changes from I to R, amino acid 176 changes from P to R, and amino acid 207 changes from M to A.

[0066] The formate dehydrogenase FDH used in this application is derived from Pseudomonas sp. 101, Uniprot Primary accession: P33160. The mutant FDH-V198I / C256I / P260S / E261P / S381N / S383F has the following mutation sites: amino acid 198 changes from V to I, amino acid 256 changes from C to I, amino acid 260 changes from P to S, amino acid 261 changes from E to P, amino acid 381 changes from S to N, and amino acid 383 changes from S to F.

[0067] The formaldehyde dehydrogenase FADH used in this application is derived from *Pseudomonas putida*, Uniprot Primary accession: P46154. The mutant FADH-A192R / L223V / L236V has the following mutation sites: amino acid 192 changes from A to R, amino acid 223 changes from L to V, and amino acid 236 changes from L to V.

[0068] The methanol dehydrogenase MDH used in this application is derived from Bacillus stearothermophilus, Uniprot Primary accession: P42327. The mutant MDH-Y171R / I196V / V237T / N240E / K241A has the following mutation sites: amino acid 171 changes from Y to R, amino acid 196 changes from I to V, amino acid 237 changes from V to T, amino acid 240 changes from N to E, and amino acid 241 changes from E to A.

[0069] The mutant dehydrogenase used in this application utilizes... The amino acid mutation was introduced using a single point mutation kit.

[0070] The purified enzymes used in this application 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.

[0071] The crude enzyme solution was obtained as follows when determining alanine dehydrogenase activity: Glycerol-containing bacteria were activated overnight by streak plating for protein expression. Activated bacteria were selected and cultured in 24-well plates for 48 hours (2.5 mL of medium per well; LB medium was supplemented with 50 μg / mL kanamycin and 0.5 mM IPTG). The plates were centrifuged at 4000g for 5 minutes, the supernatant was discarded, and 250 μL of cell lysis buffer was added. The plates were then frozen at -80℃ for at least 1 hour to aid lysis, followed by lysis at 37℃ and 200 rpm for 2 hours. Centrifugation at 4000g for 5 minutes yielded the whole-cell lysis supernatant. The cell lysis buffer consisted of: 10 mM Tris-HCl buffer (pH 8.0), 1 mM MgCl2, 1 mg / mL lysozyme, and 0.1 mg / mL DNase I.

[0072] Pyruvate Determination: The pyruvate content in the reaction solution was 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) with an IonPac AG11-HC anion exchange protectant (50 mm × 4 mm) was used. Analytical conditions: mobile phase 5 mM NaOH, flow rate 1 mL / min, column temperature 30 °C, injection volume 25 μL. Regeneration buffer 30 mM H₂SO₄, nitrogen pressure 40 psi.

[0073] 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 °C, injection volume 25 μL.

[0074] Example 1: Construction of a NAD(H) analog-dependent alanine dehydrogenase mutant

[0075] Alanine dehydrogenase (AlaDH) from the thermophilic bacterium *Geobacillus stearothermophilus* XL-65-6 was synthesized based on its amino acid sequence (SEQ ID NO.1) and optimized according to the species *Escherichia coli* (SEQ ID NO.2). Using the 8hye crystal structure as a structural guide and combining semi-rational design, the cofactor binding region of alanine dehydrogenase was modified. The resulting mutated recombinant plasmid was electroporated into *E. coli* cells, yielding the corresponding site-directed mutant and mutant library.

[0076] Example 2: Screening of NAD(H) analog-dependent alanine dehydrogenase mutants

[0077] The oxidative deamination reaction of L-alanine: H₂O + L-alanine + NAD = H₂O + +NADH+NH4 + The crude enzyme activity of alanine dehydrogenase mutants was detected by combining pyruvate with an MTT / PES colorimetric reaction. The reaction system consisted of 50 mM HEPES (pH 7.5), 10 mM L-alanine (L-Ala), 0.1 mM NXD (NXD represents the NAD analog mentioned in claim 4), 0.4 mM MTT, 1 mM PES, and ultrapure water to a final volume of 90 μL. 10 μL of crude enzyme solution was added to initiate the reaction. After mixing, the absorbance at 570 nm was measured at 30 °C. Mutants exhibiting increased NAD analog activity were selected by comparing the NAD analog activity of the alanine dehydrogenase mutants with their NAD analog activity. Specific crude enzyme activity data are shown in the table below.

[0078] Table 1. Crude enzyme activity of alanine dehydrogenase and its mutants against NAD analogues.

[0079]

[0080]

[0081] 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).

[0082] Example 3: Kinetic determination of pure enzymes of GsAlaDH and its mutants

[0083] Several mutants with high NCD activity from Example 2 were selected for protein expression and purification. The resulting pure enzymes were used to determine the kinetics of alanine oxidative deamination.

[0084] The kinetic assay system (100 μL system) consisted of 50 mM HEPES (pH 7.5), 10 mM L-alanine (L-Ala), 0.4 mM MTT, and 1 mM PES. Each enzyme was diluted at different factors for detection, with NAD or NCD concentrations varying in a gradient. Water was added to bring the volume to 100 μL. Different concentrations of cofactors were added to initiate the reaction. After mixing, the absorbance at 570 nm was measured at 30 °C. Three replicates were performed for each sample. All enzyme solutions were diluted 50x and added to the reaction system in 10 μL. The stock solutions of NAD were 0-50 mM and NCD were 0-100 mM. 10 μL of coenzyme was added to each reaction system to initiate the reaction. The actual cofactor concentration gradients in the systems were 0.31, 0.61, 1.22, 2.44, 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000, and 10000 μM. Dilutes were performed from highest to lowest concentration using a 2:1 dilution method, with the diluted solution being 1 M HEPES (pH 7.5).

[0085] The measured kinetic data are shown in the table below:

[0086] Table 2 Kinetics of alanine dehydrogenase and its mutants

[0087]

[0088]

[0089] Compared with the wild type, the six selected mutants all showed improved catalytic efficiency for NCD. Except for mutant A225P / V165A, the other mutants showed reduced catalytic efficiency for NAD, indicating a preference for NCD.

[0090] Example 4: Alanine dehydrogenase and NAD analogue catalyze the oxidative deamination of L-alanine to pyruvate.

[0091] Alanine dehydrogenase mutants can use NAD analogues as cofactors to convert the substrate L-alanine into the important intermediate pyruvate, which can be used to synthesize more high-value-added products.

[0092] A representative reaction system was as follows: 50 mM HEPES (pH 7.5), 2 mM L-alanine (L-Ala), 0.1 mM NCD, and 2 mg / mL alanine dehydrogenase, reacted at 37 °C for 120 min. Reaction termination: 30 μL of the reaction solution was added to 270 μL of termination solution (methanol:acetonitrile:water = 4:4:1), vortexed, and centrifuged at 14000 g for 10 min at 10 °C. The supernatant was transferred to a new tube, centrifuged at 14000 g for 20 min at 10 °C, and the supernatant was loaded for ion chromatography.

[0093] Table 3. Alanine dehydrogenase and its mutant pure enzymes synthesize pyruvate.

[0094] Amount of pyruvate (mM) Wild type (WT) 0.28mM A200R / S219E 1.54mM A225P / S219E 1.32mM A225P / V165A / S219E 1.89mM A225P / V238A / S219E 1.67mM

[0095] The mutated alanine mutant showed an increased yield of pyruvate produced using the NAD analog NCD, with a substrate molar conversion rate of up to 95%.

[0096] Example 5: Alanine dehydrogenase and NAD analogue catalyze the reductive amination of pyruvate to produce L-alanine.

[0097] Alanine dehydrogenase mutants can utilize reduced NAD analogues as cofactors to convert substrates pyruvate and ammonium ions into alanine. By coupling an enzyme that utilizes NAD analogues to provide the reducing driving force for alanine dehydrogenase, L-alanine can be generated in a one-pot process.

[0098] The formate dehydrogenase mutant FDH-V198I / C256I / P260S / E261P / S381N / S383F can utilize NCD as a cofactor to generate carbon dioxide from the substrate formate, while simultaneously converting NCD into NCDH. The generated NCDH can provide a reduction driving force for the alanine dehydrogenase mutant, enabling it to convert pyruvate and ammonium ions into alanine. Similarly, the malate dehydrogenase mutant ME-L310R / Q401C, D-lactate dehydrogenase DLDH-V152R / I177K / N213I, phosphite dehydrogenase PDH-I151R / P176R / M207A, methanol dehydrogenase MDH-Y171R / I196V / V237T / N240E / K241A, or formaldehyde dehydrogenase FalDH-A192R / L233V / L236V can all convert NCD to NCDH. These mutant enzymes can then provide reducing power for alanine dehydrogenase to produce L-alanine.

[0099] A representative reaction system consisted of: 50 mM HEPES (pH 7.5), 10 mM formic acid, 100 μM NCD, 2 mM pyruvate, 200 mM NH4Cl, 0.1 U / mL FDH 3A3, and 2 mg / mL alanine dehydrogenase. The reaction was carried out at 37 °C for 120 min. Reaction termination: 30 μL of the reaction solution was added to 270 μL of termination solution (methanol:acetonitrile:water = 4:4:1), and the mixture was shaken and centrifuged at 14000 g for 10 min at 10 °C. The supernatant was transferred to a new tube, centrifuged at 14000 g for 20 min at 10 °C, and the supernatant was loaded onto an ion chromatography sample.

[0100] Table 4. Synthesis of L-alanine by alanine dehydrogenase and its mutant purified enzymes.

[0101] L-alanine amount (mM) Wild type (WT) 0.46mM A200R / S219E 1.72mM A225P / S219E 1.63mM A225P / V165A / S219E 1.98mM A225P / V238A / S219E 1.89mM

[0102] The mutated alanine mutant showed an increased yield of L-alanine produced using the NAD analog NCD, with a substrate molar conversion rate of up to 99%.

[0103] When NCDH was generated using the malate dehydrogenase mutant ME-L310R / Q401C, the reaction system consisted of 50 mM HEPES (pH 7.5), 20 mM malate, 10 mM MgCl2, 100 μM NCD, 200 mM NH4Cl, 0.1 U / mL ME-L310R / Q401C, and 2 mg / mL alanine dehydrogenase. When NCDH was generated using D-lactate dehydrogenase DLDH-V152R / I177K / N213I, the reaction system consisted of 50 mM HEPES (pH 7.5), 20 mM sodium lactate, 100 μM NCD, 200 mM NH4Cl, and 0.1 U / mL alanine dehydrogenase. DLDH-V152R / I177K / N213I, 2 mg / mL alanine dehydrogenase; when using phosphite dehydrogenase PDH-I151R / P176R / M207A to produce NCDH, the reaction system is 50 mM HEPES (pH 7.5), 10 mM phosphorous acid, 100 μM NCD, 200 mM NH4Cl, 0.1 U / mL PDH-I151R / P176R / M207A, 2 mg / mL alanine dehydrogenase; when using methanol dehydrogenase MDH-Y171R / I196V / V237T / N240E / K241A to produce NCDH, the reaction system is 50 mM HEPES (pH 7.5), 800 mM methanol, 100 μM NCD, 200 mM... NH4Cl, MDH-Y171R / I196V / V237T / N240E / K241A, 2 mg / mL alanine dehydrogenase; when using formaldehyde dehydrogenase FalDH-A192R / L233V / L236V to produce NCDH, the reaction system is 50 mM HEPES (pH 7.5), 10 mM formaldehyde, 100 μM NCD, 200 mM NH4Cl, 0.1 U / mL PDH-I151R / P176R / M207A, 2 mg / mL alanine dehydrogenase.

[0104] Example 6: Pyruvate production mediated by NAD analog-dependent alanine dehydrogenase in microbial cells.

[0105] By simultaneously expressing NAD(H)-dependent alanine dehydrogenase and NAD analog transporter in the host cell, a biocatalytic system dependent on NAD analogs is formed. This biocatalytic system is activated when the substrate and NAD analog in the culture medium enter the host cell. Therefore, NAD(H)-dependent alanine dehydrogenase can convert extracellularly transported NAD analogs into pyruvate via alanine dehydrogenase, independent of intracellular NAD(H) levels. The following describes the construction of a high-pyruvate-producing strain dependent on NAD(H) analogs using *Escherichia coli* BL21(DE3) as the host bacterium. The NAD transporter AtNDT2 (Accession NO.NC_003070) has a broad substrate spectrum (Palmieri F, et al. *J Biol Chem*, 2009, 284, 31249-31259) and can transport NCD. The expression of the AtNDT2 gene, which expresses the transporter protein, was controlled by the gapAP1 promoter (Charpentier B, et al. J Bacteriol, 1994, 176, 830-839).

[0106] The gene encoding the alanine dehydrogenase mutant A225P / V165A / S219E is controlled by an isopropyl galactothiosulfate (IPTG)-induced lac promoter. The two expression cassettes were cloned into the same plasmid by replacing the LacZ gene in pUC18 to obtain an engineered plasmid. This engineered plasmid was introduced into E. coli BL21(DE3) to obtain the engineered strain E. coli HYH 001. The engineered strain E. coli HYH 001 was induced to express the three functional proteins mentioned above in LB medium with 100 μg / mL ampicillin and 1 mM IPTG added. The culture was carried out at 30℃ and 200 rpm for 48 h, followed by centrifugation at 2000 × g for 6 min to collect the cells. The cells were washed and resuspended in MOPS medium at pH 7.5, and the cell density (OD600 nm) was adjusted to 10. 10 mM L-alanine and 5 mM NCD were added to the above-mentioned engineered bacterial suspension, and the mixture was reacted for 4 h at 37 °C and 200 rpm in a shaker. 100 μL of the reaction solution was then added to 900 μL of an acetonitrile-water mixture (acetonitrile:water = 4:1) to terminate the reaction. Ion chromatography detected the formation of 8.6 mM L-alanine. In the control groups without NCD or L-alanine, the L-alanine concentrations were 1.8 mM, 0.7 mM, and 2.1 mM, respectively. The experimental results indicate that during whole-cell catalysis…

[0107] The formate dehydrogenase mutant FDH-V198I / C256I / P260S / E261P / S381N / S383F provides reducing power to the alanine dehydrogenase mutant A225P / V165A / S219E by oxidizing formate, enabling it to utilize the non-natural reducing power NCDH to reduce and amination pyruvate, producing more L-alanine.

[0108] Example 7: L-alanine production mediated by NAD analog-dependent alanine dehydrogenase in microbial cells.

[0109] A biocatalytic system dependent on NAD(H) is formed by simultaneously expressing NAD(H)-dependent alanine dehydrogenase, a regenerating enzyme for NADH analogs, and an NAD analog transporter in the host cell. This system is activated when the regenerating substrate and NAD analogs in the culture medium enter the host cell. Therefore, NAD(H)-dependent alanine dehydrogenase is used to convert extracellularly transported NAD analogs into NADH analogs intracellularly via the action of NADH regenerating enzymes, independent of intracellular NAD(H) levels. The reducing power derived from NADH analogs drives alanine dehydrogenase to produce L-alanine. This achieves the uncoupling of L-alanine production from endogenous NADH. The following example illustrates this process using *Escherichia coli* BL21(DE3) as the host bacterium to construct an engineered strain for formic acid-driven L-alanine production. The NAD transporter AtNDT2 (Accession NO.NC_003070) has a broad substrate spectrum (Palmieri F, et al. J Biol Chem, 2009, 284, 31249-31259) and can transport NCD. The expression of the AtNDT2 transporter gene is controlled by the gapAP1 promoter (Charpentier B, et al. J Bacteriol, 1994, 176, 830-839).

[0110] Genes encoding the alanine dehydrogenase mutant A225P / V165A / S219E and the formate dehydrogenase mutant FDH-V198I / C256I / P260S / E261P / S381N / S383F were cloned into the same plasmid by replacing the LacZ gene in pUC18, using an isopropyl galactothioglycolate (IPTG)-induced lac promoter to obtain an engineered plasmid. This engineered plasmid was then introduced into E. coli BL21(DE3) to obtain the engineered strain E. coli HYH 002. The engineered strain E. coli HYH 002 was induced to express the three functional proteins in LB medium supplemented with 100 μg / mL ampicillin and 1 mM IPTG. The culture was incubated at 25°C and 200 rpm for 48 h, and the cells were collected by centrifugation at 2000 × g for 6 min. The bacterial cells were washed and resuspended in MOPS medium at pH 7.5, and the cell density (OD600nm) was adjusted to 10. 50 mM sodium formate, 20 mM pyruvate, 200 mM ammonium chloride, and 5 mM NCD were added to the engineered bacterial suspension. The mixture was reacted at 37°C and 200 rpm for 4 h in a shaker. 100 μL of the reaction solution was then added to 900 μL of an acetonitrile-water mixture (acetonitrile:water = 4:1) to terminate the reaction. Ion chromatography detected the formation of 12.1 mM L-alanine. In the control groups without formic acid, NCD, or pyruvate, the L-alanine concentrations were 1.8 mM, 0.7 mM, and 2.1 mM, respectively. Experimental results show that, during whole-cell catalysis, the formate dehydrogenase mutant FDH-V198I / C256I / P260S / E261P / S381N / S383F provides reducing power to the alanine dehydrogenase mutant A225P / V165A / S219E by oxidizing formate, enabling it to utilize the non-natural reducing power NCDH to reduce and amination pyruvate, producing more L-alanine.

[0111] Amino acid sequence of wild-type alanine dehydrogenase:

[0112] SEQ ID NO.1

[0113] MKIGIPKEIKNNENRVAITPAGVMTLVKAGHDVYVETEAGAGSGFSDSEYEKAGAVIVTKAEDAWAAEMVLKVKEPLAEEFRYFRPGLILFTYLHLAAAEALTKALVEQKVVGIAYETVQLANGSLPLLTPMSEVAGRMSVQVGAQFLEKPHGGKGILLGGVPGVRRGKVTIIGGGTAGTNAAKIAVGLGADVTILDINAERLRELDDLFGDQVTTLMSNSYHIAECVRESDLVVGAVLIPGAKAPKLVTEEMVRSMTPGSVLVDVAIDQGGIFETTDRVTTHDDPTYVKHGVVHYAVANMPGAVPRTSTFALTNVTIPYALQIANKGYRAACLDNPALLKGINTLDGHIVYEAVAAAHNMPYTDVHSLLQG

[0114] Nucleotide sequence of wild-type alanine dehydrogenase:

[0115] SEQ ID NO.2

[0116]

[0117] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. An alanine dehydrogenase mutant, characterized in that, The alanine dehydrogenase mutant is created by mutating one to three amino acids from positions 165, 177, 178, 180, 199, 200, 217, 218, 219, 225, 237, and 238 of the wild-type alanine dehydrogenase amino acid sequence shown in SEQ ID NO.1 to one of the following: lysine, arginine, glutamic acid, proline, valine, glycine, alanine, and serine.

2. The mutant according to claim 1, characterized in that, The alanine dehydrogenase mutant is selected from any one of the mutants obtained by performing the following mutations based on the wild-type alanine dehydrogenase shown in SEQ ID NO.1: The 177th amino acid is mutated from threonine to lysine or arginine; or The 180th amino acid was mutated from threonine to lysine; or The 199th amino acid was mutated from asparagine to arginine; or The 200th amino acid is mutated from alanine to arginine; or The 217th amino acid was mutated from leucine to lysine; or The 218th amino acid was mutated from methionine to arginine; or The 219th amino acid is mutated from serine to glutamic acid or proline; or The 225th amino acid was mutated from alanine to proline; or The 237th amino acid was mutated from alanine to valine; or The amino acid at position 238 is mutated from valine to arginine; or The 200th amino acid is mutated from alanine to arginine, and the 178th amino acid is mutated from alanine to valine; or The 200th amino acid is mutated from alanine to arginine, and the 180th amino acid is mutated from threonine to glycine; or The 200th amino acid is mutated from alanine to arginine, and the 219th amino acid is mutated from serine to glutamic acid; or The 225th amino acid is mutated from alanine to proline, and the 165th amino acid is mutated from valine to alanine; or The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 177 is mutated from threonine to lysine; or The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 178 is mutated from alanine to valine; or The 225th amino acid was mutated from alanine to proline, and the 180th amino acid was mutated from threonine to lysine; or The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 199 is mutated from asparagine to arginine; or The 225th amino acid is mutated from alanine to proline, and the 200th amino acid is mutated from alanine to lysine; or The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 217 is mutated from leucine to serine; or The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 219 is mutated from serine to glutamic acid; or The amino acid at position 225 is mutated from alanine to proline, and the amino acid at position 238 is mutated from valine to alanine; or The 200th amino acid is mutated from alanine to arginine, the 178th amino acid from alanine to valine, and the 219th amino acid from serine to glutamic acid; or The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 165 is mutated from valine to alanine, and the amino acid at position 219 is mutated from serine to glutamic acid; or The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 177 is mutated from threonine to lysine, and the amino acid at position 219 is mutated from serine to glutamic acid; or The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 178 is mutated from alanine to valine, and the amino acid at position 219 is mutated from serine to glutamic acid; or The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 180 is mutated from threonine to lysine, and the amino acid at position 219 is mutated from serine to glutamic acid; or The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 199 is mutated from asparagine to arginine, and the amino acid at position 219 is mutated from serine to glutamic acid; or The amino acid at position 225 is mutated from alanine to proline, the amino acid at position 200 is mutated from alanine to lysine, and the amino acid at position 219 is mutated from serine to glutamic acid; or The amino acid at position 225 was mutated from alanine to proline, the amino acid at position 238 was mutated from valine to alanine, and the amino acid at position 219 was mutated from serine to glutamic acid.

3. The mutant of alanine dehydrogenase according to claim 1, characterized in that, The alanine dehydrogenase mutant uses NAD analogues and / or NADH analogues as cofactors. The NAD analogue is at least one of NCD, NUD, NTD, or NpUD, and the reduced NADH analogue is at least one of NCDH, NUDH, NTDH, or NpUDH.

4. A DNA sequence encoding, characterized in that, The DNA sequence corresponding to the mutant of the alanine dehydrogenase described in claim 1 is encoded.

5. A protein expression vector for controlled expression, characterized in that, The DNA coding sequence corresponding to the mutant of the alanine dehydrogenase of claim 1 was cloned into the protein expression vector.

6. The application of the mutant of alanine dehydrogenase according to claim 1, characterized in that, The alanine dehydrogenase mutant, in a buffer system with pH 5-10, utilizes an NAD analogue as a cofactor to catalyze the oxidative deamination of L-alanine to pyruvate.

7. The application according to claim 6, characterized in that, The buffer system includes, but is not limited to, at least one of phosphate, TrisHCl, MES, CAPS or HEPES buffer. The alanine dehydrogenase mutant, in a buffer system at pH 5-9, is coupled with malate enzyme ME-L310R / Q401C, D-lactate dehydrogenase DLDH-V152R / I177K / N213I, phosphite dehydrogenase PDH-I151R / P176R / M207A, formate dehydrogenase FDH-V198I / C256I / P260S / E261P / S381N / S383F, methanol dehydrogenase MDH-Y171R / I196V / V237T / N240E / K241A, or formaldehyde dehydrogenase FalDH-A192R / L233V / L236V, and the corresponding regeneration substrate, to catalyze the reductive amination of pyruvate to synthesize L-alanine using NAD analogues as cofactors.

8. The application according to claim 6, characterized in that, The NAD analogue-dependent alanine dehydrogenase mutant is co-expressed in microbial cells with NTT4 protein derived from Chlamydia or AtNDT2 protein derived from Arabidopsis, catalyzing the production of pyruvate from L-alanine intracellularly. The NAD analogue is transported into the cell via NTT4 protein derived from Chlamydia or AtNDT2 protein derived from Arabidopsis.

9. The application according to claim 7, characterized in that: The NAD analogue-dependent alanine dehydrogenase mutant, along with NTT4 derived from Chlamydia or AtNDT2 derived from Arabidopsis, and the regenerating NADH analogues: malate enzyme ME-L310R / Q401C, D-lactate dehydrogenase DLDH-V152R / I177K / N213I, phosphorylation dehydrogenase PDH-I151R / P176R / M207A, and formate dehydrogenase FDH- One or more of the following enzymes—V198I / C256I / P260S / E261P / S381N / S383F, methanol dehydrogenase MDH-Y171R / I196V / V237T / N240E / K241A, or formaldehyde dehydrogenase FalDH-A192R / L233V / L236V—are co-expressed in microbial cells, catalyzing the reductive amination of pyruvate to L-alanine. The NAD analogue is transported into the cell via NTT4, derived from Chlamydia, or AtNDT2, derived from Arabidopsis.

10. The application according to claim 8 or 9, further characterized in that: The microbial cells expressing the alanine dehydrogenase mutant and used for intracellular catalysis of the interconversion of alanine and L-alanine are at least one of the following: prokaryotic microorganisms: *Escherichia coli*, *Lactococcus lactis*; eukaryotic microorganisms: *Saccharomyces cerevisiae*, *Trichoderma reesei*.

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