Construction and application of polyphosphate kinase bsppk mutant and its producing strain

Abstract

The application discloses a polyphosphate kinase BsPPK mutant and construction and application of a producing strain thereof, and belongs to the technical field of genetic engineering. The application carries out site-directed mutation on polyphosphate kinase BsPPK from the genus Bredia through homologous modeling, molecular docking and multiple sequence alignment, mutates lysine at the 92th position of the wild type BsPPK into alanine, threonine at the 95th position into alanine, and serine at the 205th position into arginine, and obtains a combined mutant BsPPK-K92A / T95A / S205R. Enzyme activity determination results show that the specific enzyme activity of the mutant is increased by 324.4% compared with the wild type, and the catalytic efficiency on AMP is significantly improved. When the mutant is applied to synthesis of UDP-Gal and derivative products thereof, only 15 mM AMP is needed to achieve a similar yield obtained by using 30 mM AMP for the wild type, and the nucleotide consumption is reduced by 50%.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the construction and application of a polyphosphate kinase BsPPK mutant and its producing bacteria. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Adenosine-5'-triphosphate (ATP) is a high-energy phosphate compound that plays a crucial role in metabolic processes, enzyme catalysis, biosynthesis, cofactor synthesis, and cell-free protein synthesis. In industrial production, the application of in vitro ATP-dependent enzyme cascades is limited by the addition of the expensive substrate ATP and the accumulation of the byproduct adenosine diphosphate (ADP). Therefore, developing ATP regeneration systems is of great significance for designing efficient and economical cascade reactions.

[0004] Currently reported PPK enzymes are mainly divided into two families: PPK1 and PPK2. PPK1 preferentially catalyzes the conversion of phosphate groups from ATP to PolyP. n Transfer, synthesis of PolyP n+1 PPK2 preferentially utilizes AMP or ADP to synthesize ATP. Achbergerová et al. analyzed and compared the sources of Escherichia coli (E. coli) Escherichia coli PPK1 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa The sequence homolog of PPK2 confirmed the low amino acid sequence similarity between PPK1 and PPK2. Polyphosphate (polyP) / polyphosphate kinase (PPK)-based ATP regeneration systems are a widely studied ATP regeneration system. Many existing technologies have been reported, for example, those derived from *Rugellella* (…). Ruegeria pomeroyi RpPPK is used in the production of LacNAc, derived from *Radiata spp.* (a type of coccus). Deinococcus radiodurans DrPPK is used in the production of D-enulose. Among them, three classes of PPK2 family enzymes (PPK2-3) can use inexpensive and stable sodium hexametaphosphate (PolyP6) as a phosphate donor to catalyze the phosphorylation of adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) to synthesize expensive ATP, and are considered to have more practical application prospects.

[0005] A species derived from the genus *Bredella* has been disclosed in the prior art. Bulleidia sp.The discovery of the polyphosphokinase BsPPK (BsPPK) provides a new option for constructing ATP regeneration systems. This enzyme can use sodium hexametaphosphate as a phosphate donor and catalyze the phosphorylation of AMP and ADP into ATP.

[0006] In practical applications, when AMP is used as a phosphate acceptor, AMP first synthesizes ADP, which is then further phosphorylated to ATP. In this two-step reaction, the second step has relatively low catalytic efficiency, becoming the rate-limiting step in AMP-to-ATP synthesis, as shown by… k cat / K m AMP and k cat / K m ADP There is a certain gap, which to some extent affects the application efficiency of BsPPK in actual reactions using AMP as the initial nucleotide.

[0007] Therefore, developing a polyphosphate kinase mutant with further enhanced AMP catalytic activity to meet the needs of efficient ATP regeneration is of great practical significance. Summary of the Invention

[0008] In view of the above-mentioned prior art, the purpose of this invention is to provide a polyphosphate kinase mutant and its production strain, as well as its construction and application. The inventors, through database mining and comparison, screened out a strain derived from the genus *Bredella* (…). Bulleidia sp. ) polyphosphokinase, cloned into Escherichia coli High-efficiency expression of the recombinase was achieved in BL21(DE3), and its function was verified. To further improve the enzyme's catalytic activity for AMP, amino acid sites of the polyphosphokinase were mutated using homology modeling, molecular docking, and multiple sequence alignment methods, thus solving the problem. k cat / K m AMP Compare k cat / K m ADP The problem was reduced by one order of magnitude, resulting in mutants with significantly improved AMP catalytic efficiency.

[0009] The technical solution adopted in this invention is as follows:

[0010] In a first aspect, the present invention provides a polyphosphate kinase mutant, said mutant being a mutant obtained by substituting amino acids into the polyphosphate kinase having the amino acid sequence shown in SEQ ID NO.1, said mutant comprising at least one amino acid substitution selected from the following sites:

[0011] (a) Alanine at position 68 is mutated to glycine; the amino acid sequence is as shown in SEQ ID NO.2, A68G mutant;

[0012] (b) The lysine at position 92 is mutated to alanine; the amino acid sequence is as shown in SEQ ID NO.3 for the K92A mutant.

[0013] (c) Threonine at position 95 is mutated to alanine; the amino acid sequence is as shown in SEQ ID NO.4, T95A mutant;

[0014] (d) The serine at position 191 is mutated to glutamic acid; the amino acid sequence is as shown in SEQ ID NO.5 for the S191E mutant.

[0015] (e) Serine at position 205 is mutated to arginine; the amino acid sequence is as shown in SEQ ID NO.6 for the S205R mutant.

[0016] Preferably, the substitution is a mutation of serine at position 205 to arginine.

[0017] Preferably, the mutant contains at least two of the amino acid substitutions.

[0018] More preferably, the mutant is a K92A / T95A / S205R combined mutation, that is, it simultaneously contains a mutation of lysine at position 92 to alanine, a mutation of threonine at position 95 to alanine, and a mutation of serine at position 205 to arginine, and the amino acid sequence is shown in SEQ ID NO.7.

[0019] In a specific embodiment of the present invention, the mutant may contain a combination mutation of any two of the stated sites, preferably: alanine at position 68 mutated to glycine and lysine at position 92 mutated to alanine (A68G / K92A); alanine at position 68 mutated to glycine and serine at position 191 mutated to glutamic acid (A68G / S191E); alanine at position 68 mutated to glycine and threonine at position 95 mutated to alanine (A68G / T95A); lysine at position 92 mutated to alanine and threonine at position 95 mutated. The mutations are as follows: alanine (K92A / T95A); lysine at position 92 is mutated to alanine and serine at position 191 is mutated to glutamic acid (K92A / S191E); threonine at position 95 is mutated to alanine and serine at position 191 is mutated to glutamic acid (T95A / S191E); threonine at position 95 is mutated to alanine and serine at position 205 is mutated to arginine (T95A / S205R); serine at position 191 is mutated to glutamic acid and serine at position 205 is mutated to arginine (S191E / S205R).

[0020] In a specific embodiment of the present invention, the mutant may further comprise a combination of mutations at any three of the stated sites, preferably: alanine at position 68 mutated to glycine, lysine at position 92 mutated to alanine, and threonine at position 95 mutated to alanine (A68G / K92A / T95A); alanine at position 68 mutated to glycine, lysine at position 92 mutated to alanine, and serine at position 191 mutated to glutamic acid (A68G / K92A / S191E); alanine at position 68 mutated to glycine, lysine at position 92 mutated to alanine, and serine at position 205 mutated to arginine (A68G / K92A / S205R).

[0021] The mutations are as follows: Alanine at position 68 is replaced by glycine, threonine at position 95 is replaced by alanine, and serine at position 191 is replaced by glutamic acid (A68G / T95A / S191E); Alanine at position 68 is replaced by glycine, threonine at position 95 is replaced by alanine, and serine at position 205 is replaced by arginine (A68G / T95A / S205R); Alanine at position 68 is replaced by glycine, threonine at position 191 is replaced by glutamic acid, and serine at position 205 is replaced by arginine (A68G / S191E / S205R); Lysine at position 92 is replaced by alanine, threonine at position 95 is replaced by alanine, and serine at position 191 is replaced by arginine (A68G / T95A / S205R); The following mutations were made: 91st serine was mutated to glutamic acid (K92A / T95A / S191E); 92nd lysine was mutated to alanine; 95th threonine was mutated to alanine; and 205th serine was mutated to arginine (K92A / T95A / S205R); 92nd lysine was mutated to alanine; 191st serine was mutated to glutamic acid; and 205th serine was mutated to arginine (K92A / S191E / S205R); 95th threonine was mutated to alanine; 191st serine was mutated to glutamic acid; and 205th serine was mutated to arginine (T95A / S191E / S205R).

[0022] In a specific embodiment of the present invention, the mutant may further include any combination of mutations at four or five of the sites.

[0023] In a second aspect, the present invention provides a gene encoding the above-mentioned polyphosphate kinase mutant.

[0024] Thirdly, the present invention provides a recombinant vector carrying the above-mentioned genes.

[0025] Preferably, the recombinant vector is a pET series vector, a pRSF series vector, or a pCDF series vector as the expression vector.

[0026] Fourthly, the present invention provides microbial cells that express the above-mentioned polyphosphate kinase mutant, or carry the above-mentioned gene, or carry the above-mentioned recombinant vector.

[0027] Preferably, the microbial cells are expressed using bacteria or fungi as the host.

[0028] Fifthly, the present invention provides a method for improving the activity of polyphosphate kinase, the method comprising substituting amino acids of polyphosphate kinase as shown in SEQ ID NO.1, wherein the substitution is selected from at least one of the following sites: alanine at position 68 is mutated to glycine; or lysine at position 92 is mutated to alanine; or threonine at position 95 is mutated to alanine; or serine at position 191 is mutated to glutamic acid; or serine at position 205 is mutated to arginine.

[0029] In a sixth aspect, the present invention provides the application of the above-mentioned polyphosphate kinase mutant in the preparation of adenosine triphosphate (ATP).

[0030] In a seventh aspect, the present invention provides the application of the above-mentioned polyphosphate kinase mutant in the preparation of uridine diphosphate-galactose (UDP-Gal).

[0031] Eighthly, the present invention provides the use of the above-mentioned polyphosphate kinase mutant in the preparation of lactosyl-N-tetrasaccharide (LNT) and / or lactosyl-N-neotetrasaccharide (LNnT).

[0032] In a specific embodiment of the present invention, when the polyphosphate kinase mutant is applied to the synthesis of UDP-Gal and its derivatives (such as LNT and LNnT), the amount of nucleotides used can be reduced by 50%.

[0033] The present invention also provides a method for preparing lactosyl-N-tetrasaccharide (LNT). In the reaction system, ATP is regenerated using polyphosphate kinase mutant K92A / T95A / S205R to synthesize UDP-galactose, and then UDP-galactose is reacted with lactose-N-trisaccharide under the action of galactosyltransferase to generate LNT.

[0034] Preferably, the reaction system contains 0.1-4 mg each of the UDP-Gal de novo synthesis-related enzyme, galactosyltransferase, and the polyphosphate kinase mutant, as well as 1-50 mM each of galactose, lactose-N-trisaccharide, polyP6, AMP, and UTP; the reaction pH is 6-10, the reaction temperature is 20-40℃, and the reaction time is 1-30 h. The UDP-Gal de novo synthesis-related enzyme includes enzymes derived from... Streptococcus pneumoniae galactokinase (SpGalK), derived from Bifidobacterium longum UDP-pyrophosphorylase (BlUSP) and derived from Pasteurella multocida Inorganic pyrophosphatase (PmPpA); the galactosyltransferase is derived from... Chromobacterium violaceum The Cvβ3GalT enzymes are all known enzymes.

[0035] More preferably, the enzyme dosage is 0.5 mg / mL, the AMP concentration is 15 mM, the reaction temperature is 37℃, the pH is 8.0, and the reaction time is 20 h. Under these conditions, the LNT yield can reach 13.79 g / L.

[0036] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:

[0037] This invention first involves mutating the polyphosphokinase BsPPK, specifically by mutating the K at position 92 to A, the T at position 95 to A, and the S at position 205 to R, thus obtaining a BsPPK mutant. The BsPPK mutant of this invention has the following significant advantages:

[0038] The enzyme activity (U) of the polyphosphate kinase BsPPK mutant of the present invention AMP The enzyme activity reached 70-80 U / mg, which was 300-350% higher than that of wild-type BsPPK.

[0039] The BsPPK mutant polyphosphate kinase of the present invention, which k cat / K m ADP and k cat / K m AMP They reached 51.4 and 40.8 s⁻¹mM respectively. -1 Compared to wild-type BsPPK, its k cat / K m AMP It is approximately 20 times better than BsPPK.

[0040] The BsPPK mutant of the present invention exhibits enhanced pH stability, maintaining a residual enzyme activity of over 80% after incubation for 24 h in a pH range from 4.0 to 8.0. Particularly under acidic conditions (pH 4.0), the mutant's residual enzyme activity reaches 88%, an 83% increase compared to the wild type (48%), demonstrating excellent acid resistance. Attached Figure Description

[0041] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0042] Figure 1 A comparison diagram of the enzyme activities of wild-type polyphosphokinase BsPPK and its mutant.

[0043] Figure 2 The optimal temperature for the most beneficial mutant Mut-K92A / T95A / S205R.

[0044] Figure 3 The optimal pH for the most beneficial mutant Mut-K92A / T95A / S205R.

[0045] Figure 4 pH stability of the optimal mutant Mut-K92A / T95A / S205R after incubation in different pH buffers for 24 h.

[0046] Figure 5 These are the kinetic parameters of wild-type polyphosphate kinase BsPPK.

[0047] Figure 6 The results show the kinetic parameters for the most beneficial mutant Mut-K92A / T95A / S205R.

[0048] Figure 7 SDS-PAGE analysis of the most beneficial mutant Mut-K92A / T95A / S205R.

[0049] Figure 8 This is an SDS-PAGE analysis chart of BsPPK.

[0050] Figure 9 Application of BsPPK and the optimal mutant Mut-K92A / T95A / S205R for the one-pot in vitro synthesis of LNT.

[0051] Figure 10 This is a high-performance liquid chromatography (HPLC) chromatogram of the LNT synthesis results.

[0052] Figure 11 This is a mass spectrometry chromatogram of the LNT standard.

[0053] Figure 12 Mass spectrometry analysis of the product of BsPPK-driven regenerated ATP for one-pot in vitro synthesis of LNT.

[0054] Figure 13 Mass spectrometry analysis of the product of LNT synthesis in vitro using regenerated ATP driven by the mutant Mut-K92A / T95A / S205R.

[0055] Figure 14 High-performance liquid chromatography (HPLC) chromatogram of ATP synthesis by the most beneficial mutant Mut-K92A / T95A / S205R.

[0056] Figure 15 A comparison of enzyme activities between wild-type polyphosphate kinase BsPPK and its multi-point mutant. Detailed Implementation

[0057] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0059] the term:

[0060] In this invention, the term "WT" refers to the wild-type polyphosphate kinase.

[0061] The term "mutation site" as used in this invention refers to the specific location in the amino acid sequence of a protein where a substitution occurs.

[0062] The term "enzyme activity" used in this invention refers to the ability of an enzyme to catalyze a certain chemical reaction. In this invention, enzyme activity is expressed as specific activity, which refers to the enzyme activity per gram (g) of enzyme protein, with the unit being U / mg.

[0063] The term "wild-type BsPPK gene" as used in this invention refers to the nucleotide sequence encoding a polyphosphokinase with an amino acid sequence as shown in SEQ ID NO.1.

[0064] The terms "protein" and "protein protein" used in this invention can be used interchangeably.

[0065] The culture media involved in the following examples:

[0066] LB liquid medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0. Add kanamycin (50 μg / mL) before use. For solid medium, add 1.5% agar powder.

[0067] The experimental methods for enzyme activity assays involved in the following examples are as follows:

[0068] The reaction system was as follows: 5 mM MgSO4, 1 mM polyP6, 1 mM ADP or 1 mM AMP, 50 mM Tris-HCl (pH 8.0), 1 µg / mL BsPPK. The reaction was carried out at 37℃ for 30 min, then terminated with 0.1% trichloroacetic acid. HPLC analysis conditions were: 20 mmol / L potassium dihydrogen phosphate-dipoxatol phosphate (pH 6.0) as the mobile phase, flow rate 1.0 mL / min, column temperature 30℃, UV detection wavelength 254 nm, and column: AQ-C18 (250 mm × 4.6 mm, 5 µm). ATP standards of different concentrations were prepared, and a standard curve was plotted. Enzyme activity was defined as the amount of enzyme required to generate 1 µmol of ATP per minute, defined as one polyphosphokinase activity unit (U). Specific enzyme activity was calculated as the ratio of enzyme activity (U) to the amount of enzyme used (mg).

[0069] Experimental materials:

[0070] 1. Source of the strain

[0071] The polyphosphate kinase BsPPK gene involved in this invention is derived from the genus *Brendella* (…). Bulleidia sp. ) strains, specifically Bulleidia extructa The genomic sequence information of this strain can be found in the NCBI database (accession number: MEE1410690.1). The target gene was obtained through artificial synthesis, and its encoded amino acid sequence is shown in SEQ ID NO.1.

[0072] 2. Non-patent literature

[0073] The present invention relates to Bulleidia extructa The strain is based on existing technology; relevant information can be found in the following literature:

[0074] Downes J , Olsvik B , Hiom SJ ,et al.Bulleidia extructa gen. nov.sp. nov. isolated from the oral cavity.[J].International Journal of Systematic&Evolutionary Microbiology, 2000, 50 Pt 3(3):979.DOI:10.1099 / 00207713-50-3-979.

[0075] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0076] Example 1: Obtaining the BsPPK mutation site and constructing the strain

[0077] This invention retrieved the structural formulas of all ligand molecules (Sodium hexametaphosphate, ADP, and AMP) from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). Molecular docking and analysis were performed using Schrödinger 2023-1 software, where BsPPK was defined as the receptor protein. Protein preparation was performed using the default parameters of the Protein Preparation module before docking, including removing excess water molecules and adding hydrogen atoms. The ligand small molecules were pretreated using the LigPrep module under an OPLS4 force field.

[0078] This invention utilizes a 10 ns molecular dynamics simulation of the BsPPK structure obtained from AlphaFold2 modeling, selecting the last frame of the simulation as the initial structure for subsequent molecular docking. The docking process employs the Induced Fit Docking (IFD) module, selecting docking pockets based on previously reported protein data. The complex with the lowest energy is chosen as the optimal model based on the docking score.

[0079] Based on the above calculations and analysis, potential key amino acid sites that may be related to substrate binding and catalytic activity were preliminarily identified. Further screening and validation of mutation sites were conducted using the following methods:

[0080] (1) BsPPK belongs to the PPK2-III family. The amino acids in the core catalytic domain are highly conserved, so a rational design strategy based on sequence alignment can be adopted to screen for mutation hotspots. Previous studies have found that the amino acid residue pairs of A79, S106, I108, S111 and L285 are derived from Cytophaga hutchinsonii The catalytic activity of polyphosphokinase ChPPK is significantly affected. The inventors compared and analyzed the amino acid sequences of ChPPK and BsPPK, identifying five amino acid sites: A68, T95, T97, D127, and E269, which will be used for subsequent mutation studies of BsPPK.

[0081] Subsequent experiments verified that mutations at the A68 and T95 sites had a significant effect on enhancing enzyme activity and were included in the final mutant combination.

[0082] (2) Predicting mutation sites based on the HotSpot Wizard 3.0 web computation method

[0083] HotSpot Wizard 3.0 (https: / / loschmidt.chemi.muni.cz / hotspotwizard / ) is a web server for automatically designing mutations and smart libraries. Its identification of mutagenic hotspots is based on the integration of structural, functional, and evolutionary information obtained from multiple bioinformatics databases and computational tools. This study uses the HotSpot Wizard 3.0 web server to automatically identify mutational hotspots in BsPPK to improve its ADP and AMP phosphorylation activity. The study inputs a BsPPK PDB format structure file and selects a protein engineering strategy that identifies hotspots as "functional hotspots represented by highly variable residues located in catalytic pockets and / or channels." Based on the mutation frequency of individual amino acids, the most likely safe mutant amino acid sites are selected and confirmed.

[0084] Based on mutation frequency analysis, positions 92 and 191 were predicted to be high-potential mutation hotspots. Subsequent experiments confirmed that K92A and S191E mutations can significantly improve enzyme activity.

[0085] (3) Predicting mutation sites based on molecular docking and virtual saturation mutation

[0086] Using the substrate-binding pocket as the research object, the ResidueScanning module in Schrödinger Maestro software was employed to conduct virtual saturation mutation analysis on key amino acid residues within a 5 Å radius centered on the substrate, systematically evaluating the impact of different sites and their mutation forms on substrate binding affinity. Molecular docking calculations were performed for each mutant, with changes in docking affinity used as the evaluation index. Based on the combined trends of docking score changes and the spatial distribution characteristics of each residue within the substrate-binding pocket, amino acids at positions 205, 121, 71, 93, 67, and 72 were screened as potential key functional sites for further experimental validation. Among these, position 205 exhibited the strongest substrate binding affinity change during molecular docking and was selected as one of the core mutation sites.

[0087] (4) Construction of mutant expression strains

[0088] Site-directed mutagenesis was performed using a one-step PCR amplification plasmid. The PCR product was digested with the rapid restriction endonuclease Dpn I at 37°C for 30 min, followed by incubation at 80°C for 20 min to thermally inactivate the enzyme. The degraded product was transformed into E. coli DH5α competent cells, and after positive verification and correct DNA sequencing, it was transformed into E. coli BL21(DE3) competent cells to construct the mutant expression strain.

[0089] (5) Mutant expression and purification

[0090] The pET-28a (+) plasmid carrying the BsPPK gene was introduced into competent Escherichia coli BL21 (DE3), and single colonies were obtained by plating. The obtained single colonies were inoculated into 5 mL of LB medium (containing 50 mg / L Kan resistance) and cultured at 37°C for 12 h. Then, 1% (v / v) was inoculated into 100 mL of LB medium (containing 50 mg / L Kan resistance). When the cell density reached OD600 = 0.6-0.8, 100 mM isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM, and the cells were cultured at 18°C ​​and 200 rpm for 16-18 h to induce protein expression. The fermented bacterial broth was centrifuged at 8000 rpm and 4℃ for 10 min to collect the bacterial cells, and resuspended in 20-30 mL of 25 mM Tris-HCl (pH 8.0) buffer. The resuspended broth was then sonicated under ice-water bath conditions (400W power, on for 3 s, off for 5 s). The cell lysate was centrifuged at 8000 rpm and 4℃ for 10 min to remove cell debris, and the crude enzyme solution was collected. The crude enzyme solution was purified using a Ni-NTA affinity chromatography column, and the target protein with the His tag was collected. The protein was eluted sequentially with imidazole concentrations of 20 mM, 50 mM, 100 mM, and 200 mM, and the 200 mM eluent was collected. The purified protein was desalted and concentrated using an ultrafiltration tube (Millipore, 10 kDa). The purified wild-type BsPPK and the mutant Mut-K92A / T95A / S205R were analyzed by SDS-PAGE, and the results are as follows: Figure 7 and Figure 8 As shown in the figure. The results show that the molecular weight of the target protein is consistent with the theoretical value (approximately 35 kDa), indicating that the protein has been successfully expressed and purified to homogeneity.

[0091] (6) Construction of combined mutants

[0092] Based on the aforementioned single-site mutants, combined mutants containing two or three mutation sites are constructed using multiple rounds of site-directed PCR or overlap extension PCR. Specifically, using the constructed single-site mutant plasmid as a template, a second mutation site is introduced to obtain a two-site combined mutant; further, using the two-site mutant plasmid as a template, a third mutation site is introduced to obtain a three-site combined mutant. Using the same method, mutants such as... Figure 15 All two-point and three-point combination mutants are shown. After the constructed mutants were verified to be correct by sequencing, they were transformed into E. coli BL21(DE3) competent cells to construct the combination mutant expression strain. The expression and purification methods of the mutants were the same as those for the single-point mutants.

[0093] For combined mutants with four or five mutation sites, refer to the above method.

[0094] Example 2: Enzyme activity assay of polyphosphate kinase BsPPK mutant

[0095] The reaction system was as follows: 5 mM MgSO4, 1 mM polyP6, 1 mM AMP, 50 mM Tris-HCl (pH 8.0), 1 µg / mL BsPPK. The reaction was carried out at 37℃ for 30 min, then terminated with 0.1% trichloroacetic acid. HPLC analysis conditions were: 20 mmol / L potassium dihydrogen phosphate-dipoxatol phosphate (pH 6.0) as the mobile phase, flow rate 1.0 mL / min, column temperature 30℃, UV detection wavelength 254 nm, and column: AQ-C18 (250 mm × 4.6 mm, 5 µm). Different concentrations of ATP standards were prepared, and a standard curve was plotted. The BsPPK enzyme activity was defined as the amount of enzyme required to generate 1 µmol of ATP per minute, defined as one polyphosphate kinase activity unit (U). The HPLC analysis results of the reaction products are as follows: Figure 14 As shown, the specific enzyme activity is determined by the ratio of enzyme activity (U) to the amount of enzyme used (mg). Experimental results are as follows... Figure 1 and Figure 15 As shown, the most beneficial single-point mutation mutants are A68G (26.25 U / mg), K92A (18.27 U / mg), T95A (22.51 U / mg), S191E (19.50 U / mg), and S205R (35.54 U / mg), which, compared with the wild type (16.61 U / mg), show relative enzyme activities increased by 58%, 10%, 35%, 17%, and 114%, respectively. The specific enzyme activities of the two-point combination mutants are 95%-295% higher than the wild type, with A68G / T95A exhibiting the highest relative enzyme activity at approximately 48.95 U / mg, representing a 195% (48.95 / 16.61-1)*100 increase compared to the wild type. The specific enzyme activity of the three / four / five-point combination mutants is 100%-325% higher than that of the wild type. Among them, the most beneficial combination mutant is K92A / T95A / S205R, with an enzyme activity of 70.5 U / mg, which is 324.4% higher than that of the wild type.

[0096] Example 3: The purified protein obtained in Example 1 (i.e., the polyphosphate kinase BsPPK mutant K92A / T95A / S205R) was tested for enzyme activity under different temperature conditions. The enzyme activity assay method was the same as in Example 2. The results are as follows: Figure 2 As shown, the results indicate that the optimal temperature for the polyphosphate kinase BsPPK mutant of the present invention is 30°C.

[0097] Example 4: The purified protein obtained in Example 1 (i.e., the polyphosphate kinase BsPPK mutant K92A / T95A / S205R) was tested for enzyme activity under different pH conditions. The enzyme activity assay method was the same as in Example 2. The results are as follows: Figure 3 As shown, the results indicate that the optimal pH for the polyphosphate kinase BsPPK mutant of the present invention is 8 (Tris-HCl).

[0098] Example 5: The purified proteins obtained in Example 1 (i.e., the BsPPK mutant K92A / T95A / S205R and the wild-type BsPPK) were incubated for 24 h under different pH conditions, and their enzyme activity was tested. The enzyme activity assay method was the same as in Example 2. The results are as follows: Figure 4 As shown, the results indicate that the mutant exhibits superior stability compared to the wild type within a pH range of 4.0 to 8.0. Particularly under acidic conditions at pH 4.0, the residual enzyme activity of the mutant reached 88%, an increase of 83% compared to the wild type (48%), indicating a significant enhancement in the mutant's acid stability.

[0099] Example 6: The purified protein obtained in Example 1 (i.e., wild-type polyphosphokinase BsPPK) was reacted with solutions of different concentrations of ADP (0.0625 mM - 30 mM), AMP (0.0625 mM - 50 mM), and polyP6 (0.0625 mM - 1.25 mM) to obtain the kinetic parameters of the BsPPK mutant K92A / T95A / S205R. These parameters were calculated by measuring the rate of the enzymatic reaction over 30 min, expressed as enzyme activity. Nonlinear regression software was used to calculate... K m (Mi constant) and V max (Maximum catalytic rate). Results are as follows: Figure 5 As shown in Table 1, ADP and polyP6 are the substrates for ATP phosphorylation. k cat / K m ADP It is 25.2 s -1 mM -1 For ATP phosphorylation, AMP and polyP6 are the substrates. k cat / K m AMP It is 2.5 s -1 mM -1The purified protein obtained in Example 1 (i.e., the polyphosphokinase BsPPK mutant K92A / T95A / S205R) was reacted with solutions of different concentrations of ADP (0.0625 mM - 30 mM), AMP (0.0625 mM - 50 mM), and polyP6 (0.0625 mM - 1.5 mM) to obtain the kinetic parameters of the polyphosphokinase BsPPK mutant K92A / T95A / S205R. These parameters were calculated by measuring the rate of the enzymatic reaction over 10 min, expressed as enzyme activity. Nonlinear regression software was used for calculation. K m (Mi constant) and V max (Maximum catalytic rate). Results are as follows: Figure 6 As shown in Table 1. The results indicate that ADP and polyP6 are the substrates for the ATP phosphorylation reaction. k cat / K m ADP It is 51.4 s -1 mM -1 For ATP phosphorylation, AMP and polyP6 are the substrates. k cat / K m AMP It is 40.8 s -1 mM -1 The results showed that, compared with wild-type BsPPK, the mutant... k cat / K m AMP It is approximately 20 times better than BsPPK. k cat / K m ADP It is about twice as good as BsPPK.

[0100] Table 1 Kinetic parameters of wild-type BsPPK and mutants K92A / T95A / S205R

[0101]

[0102] Example 7: The purified protein obtained in Example 1 (i.e., the polyphosphokinase BsPPK mutant K92A / T95A / S205R, denoted as Mut-K92A / T95A / S205R or Mut) was applied to the LNT in vitro enzymatic synthesis system. The UDP-Gal de novo synthesis pathway was used to synthesize the protein derived from... Streptococcus pneumoniaegalactokinase (SpGalK, NCBI accession number: AE005672, see literature: M. Chen et al. / Carbohydrate Research 346 (2011) 2421–2425), Bifidobacterium longum UDP-pyrophosphorylase (BlUSP, NCBI accession number: AAN24556, see literature: Muthana et al., 2012, Chem Commun, 48:2728-2730), Pasteurella multocida The inorganic pyrophosphatase (PmPpA, NCBI accession number: AE004439, see reference: Kam Lau et al., 2010, ChemCommun, 46, 6066–6068) was expressed and purified according to the method described in the above examples, and the purified enzyme was used for the next reaction.

[0103] The reaction system consisted of 200 µL of 100 mM pH 8.0 Tris-HCl buffer containing a final concentration of 0.5 mg / mL UDP-Gal de novo synthesis-related enzyme and 0.5 mg / mL of [unspecified ingredient]. Chromobacterium violaceum Galactosyltransferase (Cvβ3GalT, see literature: McArthur JB et al., ACS Catal. 2019, 9(12):10721-10726; NCBI accession number: WP_080969100.1), 0.5 mg / mL mutant K92A / T95A / S205R, 30 mM galactose, 22.5 mM lactose-N-trisaccharide, 30 mM polyP6, 30 mM UTP, and 15 mM AMP or 30 mM AMP. The reaction temperature was 37℃ and the reaction time was 20 h.

[0104] The reaction products were detected by HPLC (Agilent 1260 II). The analytical conditions were: mobile phase acetonitrile-water (70:30), flow rate 0.5 mL / min, column temperature 45℃, UV detection wavelength 200 nm, and column: BEH Xbridge Amide (250 mm × 4.6 mm, 5 µm). Different concentrations of LNT standards were prepared, and standard curves were plotted. The HPLC analysis results are as follows: Figure 10 As shown. LNT production is as follows. Figure 9As shown, the in vitro one-pot reaction with 0.5 mg / mL mutant K92A / T95A / S205R and 15 mM AMP resulted in a LNT yield of 13.79 g / L after 20 h. In comparison, the wild-type in the comparative example yielded 14.56 g / L with 30 mM AMP (twice the amount of the mutant). This indicates that even with half the AMP dosage, the mutant can still achieve a yield level comparable to the wild-type. The product was subsequently identified by positive ion mode electrospray ionization mass spectrometry (ESI-MS). The identification results are as follows. Figure 13 As shown, with Figure 11 The results were consistent with those of the standard, and [LNT + Na] of LNT (MW 707) was detected in the positive ion ESI mass spectrum. + ion peak ( m / z 730.24).

[0105] Comparative Example

[0106] The purified protein obtained in Example 1 (i.e., polyphosphate kinase BsPPK) was applied to the LNT in vitro enzymatic synthesis system. The protein derived from... Streptococcus pneumoniae galactokinase (SpGalK, NCBI accession number: AE005672, see literature: M. Chen et al. / Carbohydrate Research 346 (2011) 2421–2425), Bifidobacterium longum UDP-pyrophosphorylase (BlUSP, NCBI accession number: AAN24556, see literature: Muthana et al., 2012, Chem Commun, 48:2728-2730), Pasteurella multocida The inorganic pyrophosphatase (PmPpA, NCBI accession number: AE004439, see reference: Kam Lau et al., 2010, ChemCommun, 46, 6066–6068) was expressed and purified according to the method described in the above examples, and the purified enzyme was used for the next reaction.

[0107] The reaction system consisted of 200 µL of 100 mM pH 8.0 Tris-HCl buffer containing a final concentration of 0.5 mg / mL of the UDP-Gal de novo synthesis-related enzyme. The 0.5 mg / mL concentration was derived from... Chromobacterium violaceumGalactosyltransferase (Cvβ3GalT, see literature: McArthur JB et al., ACS Catal. 2019, 9(12):10721-10726; NCBI accession number: WP_080969100.1), 0.5 mg / mL wild-type BsPPK, 30 mM galactose, 22.5 mM lactose-N-trisaccharide, 30 mM polyP6, 30 mM UTP, and 15 mM AMP or 30 mM AMP. The reaction temperature was 37℃ and the reaction time was 20 h. The reaction products were detected by HPLC (Agilent 1260 II) under the following conditions: mobile phase acetonitrile-water (70:30), flow rate 0.5 mL / min, column temperature 45℃, UV detection wavelength 200 nm, and column: BEH Xbridge Amide (250 mm × 4.6 mm, 5 µm). Prepare LNT standards at different concentrations and plot standard curves. HPLC analysis results are as follows: Figure 10 As shown. LNT production is as follows. Figure 9 As shown, the in vitro one-pot reaction with 0.5 mg / mL BsPPK and 30 mM AMP resulted in a LNT yield of 14.56 g / L after 20 h; when the AMP concentration was reduced to 15 mM, the LNT yield decreased to 8.5 g / L. The product was subsequently identified by positive ion mode electrospray ionization mass spectrometry (ESI-MS). The identification results are as follows. Figure 12 As shown, with Figure 11 The results were consistent with those of the standard, and [LNT + Na] of LNT (MW 707) was detected in the positive ion ESI mass spectrum. + ion peak ( m / z 730.24).

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polyphosphate kinase mutant, characterized in that, The mutant is a mutant obtained by substituting amino acids into the amino acid sequence of polyphosphokinase as shown in SEQ ID NO.

1. The mutant is as follows: (a) The lysine at position 92 is mutated to alanine; (b) Threonine at position 95 is mutated to alanine; (c) Serine at position 205 is mutated to arginine; (d) The threonine at position 95 is mutated to alanine, and the lysine at position 92 is mutated to alanine; (e) The threonine at position 95 is mutated to alanine, and the serine at position 205 is mutated to arginine; (f) The 92nd lysine is mutated to alanine, the 95th threonine is mutated to alanine, and the 205th serine is mutated to arginine.

2. The gene encoding the polyphosphate kinase mutant of claim 1.

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

4. Microbial cells expressing the polyphosphate kinase mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3.

5. A method for increasing polyphosphate kinase activity, characterized in that, The method involves substituting amino acids into the polyphosphokinase shown in SEQ ID NO.1, wherein the substitution is as follows: (a) The lysine at position 92 is mutated to alanine; (b) Threonine at position 95 is mutated to alanine; (c) Serine at position 205 is mutated to arginine; (d) The threonine at position 95 is mutated to alanine, and the lysine at position 92 is mutated to alanine; (e) The threonine at position 95 is mutated to alanine, and the serine at position 205 is mutated to arginine; (f) The 92nd lysine is mutated to alanine, the 95th threonine is mutated to alanine, and the 205th serine is mutated to arginine.

6. The use of the polyphosphate kinase mutant of claim 1 in the preparation of adenosine triphosphate (ATP).

7. The use of the polyphosphate kinase mutant of claim 1 in the preparation of uridine diphosphate-galactose (UDP-Gal).

8. The use of the polyphosphate kinase mutant of claim 1 in the preparation of lactosyl-N-tetrasaccharide (LNT) and / or lactosyl-N-neotetrasaccharide (LNnT).

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