Cascade biological catalysis-based pyridoxal phosphate synthesis method capable of autonomously supplying ATP (adenosine triphosphate)

Abstract

The invention relates to a pyridoxal phosphate (PLP) synthesis method based on cascade biological catalysis and capable of realizing ATP autonomous regeneration. The method comprises the following steps: constructing engineering bacteria for co-expressing pyridoxal phosphate kinase (PdxK) and polyphosphate kinase (PPK) to prepare a whole-cell catalyst or a crude enzyme solution; under the condition that ATP does not need to be exogenously added, pyridoxal, ADP and polyphosphate serve as substrates, cascade reaction is carried out under the action of a catalyst, ATP is regenerated in situ, and PLP synthesis is driven. A wild enzyme system is adopted, and the yield of PLP exceeds 95% within 4 hours of reaction; after PdxK mutation optimization, the reaction time is shortened to about 2 hours, and conversion can be completed. According to the invention, a polyphosphate-driven ATP regeneration system is coupled with PLP biosynthesis, so that the cofactor cost is remarkably reduced, a feasible technical path is provided for industrial biological manufacturing of PLP, and the application of polyphosphate in cyclic regeneration of cofactors is expanded.

Description

Technical Field

[0001] This invention relates to the field of biocatalytic synthesis technology, and more specifically, to a biocatalytic synthesis method for pyridoxal phosphate, particularly a method for synthesizing pyridoxal phosphate based on a multi-enzyme cascade reaction and achieving autonomous ATP regeneration (self-supply). Background Technology

[0002] Pyridoxal phosphate ( PLP (phospholipid protease), the biologically active form of vitamin B6, is one of the most diverse enzyme cofactors known to date. It participates in hundreds of enzymatic reactions, including transamination, decarboxylation, and racemization, and is crucial for the basic metabolism of organisms. Furthermore, it plays an important role in the regulation of the cardiovascular, nervous, and immune systems. In the field of industrial biocatalysis, PLP-dependent enzymes are key tools for the green and stereoselective synthesis of high-value chemicals such as chiral amines and non-natural amino acids, and have broad application prospects in pharmaceuticals and agrochemicals.

[0003] However, the large-scale production of PLP currently faces significant challenges. Traditional commercial production mainly relies on multi-step chemical synthesis methods using pyridoxine phosphate or pyridoxamine phosphate as precursors. This method generally suffers from problems such as cumbersome process steps, high energy consumption, use of hazardous reagents, and generation of large amounts of difficult-to-treat chemical waste, which contradicts the requirements of environmental sustainability.

[0004] In contrast, enzyme-based biosynthesis routes have the advantages of mild conditions, high selectivity and environmental friendliness, making them an ideal alternative. The natural biosynthesis of PLP mainly relies on two pathways: (1) De novo synthesis: This pathway involves multiple enzymatic steps, has a long metabolic pathway, complex flux regulation, and is prone to low overall efficiency due to the accumulation of intermediate products; (2) Rescue synthesis: Although this pathway is relatively simplified, the catalytic efficiency and stability of its key enzyme, pyridoxine phosphate oxidase (PNPO), are often insufficient, and the initiation of this pathway is strictly dependent on the supply of specific B6 vitamin substrates, which limits both cost and controllability.

[0005] More importantly, whether modifying natural pathways or constructing in vitro biocatalytic systems, the biosynthesis of PLP typically relies on adenosine triphosphate (ATP) as the phosphate group and energy donor. However, ATP is expensive and chemically unstable; its direct addition to the reaction system significantly increases production costs and may introduce impurities and inhibit enzyme activity. This has become one of the core bottlenecks restricting the industrial application of PLP biomanufacturing technology. Therefore, developing a highly efficient and streamlined PLP biosynthesis system that does not rely on exogenous addition and can achieve self-supply of ATP has significant industrial demand and technological value. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for synthesizing pyridoxal phosphate based on cascade biocatalysis and autonomous ATP supply, aiming to solve some of the problems in the prior art or at least alleviate some of the problems in the prior art.

[0007] This invention is achieved by a method for synthesizing pyridoxal phosphate based on cascade biocatalysis and autonomous ATP supply. Pyridoxal phosphate kinase and polyphosphate kinase are co-expressed. The reaction system is constructed using the obtained co-expression strain or its crude enzyme extract, the substrate pyridoxal, ADP and polyphosphate, without the need for exogenous ATP addition.

[0008] Furthermore, the amino acid sequence of the pyridoxal phosphate kinase is shown in SEQ ID NO.1, and the amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO.2.

[0009] Furthermore, the gene fragments of the pyridoxal phosphate kinase and the polyphosphate kinase are derived from the genome of E. coli K12 MG1655.

[0010] Furthermore, the gene fragment of pyridoxal phosphate kinase was ligated into the pET-28a(+) vector to obtain the recombinant plasmid pET28a-pdxK; the polyphosphate kinase was ligated into the pETDuet-1 vector to obtain the recombinant plasmid pETDuet-ppk; the plasmid pET28a-pdxK was transformed into E. coli BL21(DE3) competent cells, and the plasmid pETDuet-ppk was transformed into E. coli BL21(DE3) competent cells that already contained the pET28a-pdxK plasmid to obtain a strain co-expressing the two plasmids.

[0011] Furthermore, the reaction system for synthesizing pyridoxal phosphate using co-expressed strains included: 0.1 g / mL wet cells, 5 mmol / L PL, 20 mmol / L sodium hexapolyphosphate, and 5 mmol / L ADP. The reaction was carried out in a pH 6.5 potassium phosphate buffer at 37°C.

[0012] Furthermore, the reaction system for catalyzing the synthesis of pyridoxal phosphate from pyridoxal using the crude enzyme solution of the co-expressed strain included: 5 mmol / L PL, 20 mmol / L sodium hexapolyphosphate, 5 mmol / L ADP and 10 mmol / L MgCl2. The crude enzyme solution was prepared by ultrasonic disruption and centrifugation of 0.1 g / mL wet cells. The reaction was carried out in a pH 6.5 potassium phosphate buffer at 37°C.

[0013] Furthermore, the amino acid sequence of pyridoxal kinase phosphate shown in SEQ ID NO.1 was mutated as follows: S23A / Y96F / D227E, and the mutated amino acid sequence is shown in SEQ ID NO.7.

[0014] In summary, the advantages and positive effects of this invention are as follows:

[0015] This invention establishes an ATP-autonomous biocatalytic cascade coupled with pyridoxal phosphate kinase (PLK) and polyphosphate kinase (PPK) for sustainable PLP synthesis. The system utilizes inexpensive sodium hexapolymetaphosphate (SHMP) as a phosphate donor to drive continuous ATP regeneration, thus achieving efficient phosphorylation of pyridoxal phosphate to PLP without the need for exogenous ATP input. Recombinant PLK and PPK were co-expressed in *E. coli* BL21(DE3), with specific activities of 50 U / g, respectively. 细胞 and 145 U / g 细胞 Due to mass transfer limitations and intracellular PLP degradation, the whole-cell biocatalyst achieved only about 25% yield. Notably, the optimized cell-free system (45°C, pH 6.5, 25 mmol / L SHMP) achieved a significant 95% PLP yield. This 3.8-fold improvement demonstrates the key advantage of eliminating cell barriers in complex multi-enzyme cascade reactions. Furthermore, after PLK mutation optimization, the reaction was completed in just 2 hours.

[0016] This invention establishes a robust, ATP-independent, and highly efficient PLP biosynthesis platform that seamlessly integrates cofactor regeneration with cascade catalysis, providing a scalable and cost-effective pathway for industrial biomanufacturing. Furthermore, it demonstrates the broader applicability of polyphosphate-driven energy cycling in sustainable biocatalysis. Attached Figure Description

[0017] Figure 1 It is based on the principle of a two-enzyme cascade biocatalytic reaction for the autonomous synthesis of pyridoxal phosphate 5'-phosphate from ATP;

[0018] Figure 2 It involves the construction and expression of the PdxK (PLK) recombinant plasmid;

[0019] A: pET28-pdxK was identified by agarose gel electrophoresis. Lane L: DL15000 DNA Ladder; Lane 1: pET28a; Lane 2: pET28a-pdxK; Lane 3: double digestion of pET28a-pdxK.

[0020] B: SDS-PAGE analysis of E. coli BL21(DE3) / pET28a-pdxK induced expression products. Lane M: Protein Marker; Lane 1: E. coli BL21(DE3) / pET28a; Lane 2: IPTG-induced E. coli BL21(DE3) / pET28a-pdxK.

[0021] Figure 3 It involves the construction and co-representation of PdxK and Ppk;

[0022] A: pDeut-ppk was identified by agarose gel electrophoresis. Lane L: DL15000 DNA Ladder; Lane 1: single digestion of pETDuet-1-ppk; Lane 2: double digestion of pDeut-ppk.

[0023] B: SDS-PAGE analysis of E. coli BL21(DE3) / pET28a-pdxK&pETDuet-1-ppk induced expression products. Lane M: Protein Marker; Lane 1: E. coli BL21(DE3) / pET28a&pETDuet-1; Lane 2: E. coli BL21(DE3) / pET28a&pETDuet-1 induced by IPTG; Lane 3: Uninduced E. coli BL21(DE3) / pETDuet-1-ppk; Lane 4: E. coli BL21(DE3) / pETDuet-1-ppk induced by IPTG; Lane 5: Uninduced E. coli BL21(DE3) / pET28a-pdxK&pETDuet-1-ppk; Lane 6: E. coli BL21(DE3) / pET28a-pdxK&pETDuet-1-ppk induced by IPTG. coliBL21(DE3) / pET28a-pdxK&pETDuet-1-ppk.

[0024] Figure 4 This refers to the enzymatic properties of PdxK;

[0025] A: Optimal reaction temperature of pyridoxal phosphate kinase, B: Optimal reaction pH of pyridoxal phosphate kinase, C: Effect of metal ions on the activity of pyridoxal phosphate kinase, D: Thermal stability of pyridoxal phosphate kinase at different temperatures, E: Stability of pyridoxal phosphate kinase in buffer solutions with different pH values.

[0026] Figure 5 This refers to the enzymatic properties of PPK;

[0027] A: The effect of temperature on PPK activity; B: The effect of pH on PPK activity; C: The thermal stability of PPK; D: The pH stability of PPK.

[0028] Figure 6 It is a comparison of the synthesis of PLP by whole-cell and cell-free dual-enzyme catalysis with co-expression of two plasmids;

[0029] Figure 7 The synthesis of pyridoxal phosphate is achieved by dual enzyme coupling catalysis of PdxK and PPK, using polyphosphate as a phosphate donor.

[0030] A: Effect of reaction temperature on the dual-enzyme coupling catalysis; B: Effect of reaction pH on the dual-enzyme coupling catalysis; C: Effect of enzyme ratio on dual-enzyme coupling catalysis; D: Effect of sodium hexametaphosphate concentration on the dual-enzyme coupling catalysis.

[0031] Figure 8 This is an SDS-PAGE image of the mutant BL21(DE3) / pET28a-pdxK_S23A / Y96F / D227E;

[0032] Figure 9 This is the reaction progress curve of PLP synthesis catalyzed by the mutant cell-free catalytic system. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0034] Based on the information contained in this application, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0035] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "about". Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. In this invention, "about" means within 10%, preferably within 5%, of a given value or range.

[0036] Unless otherwise specified, all embodiments of the present invention are based on ambient temperature conditions. Ambient temperature refers to the natural room temperature during the four seasons, without additional cooling or heating treatment. Generally, ambient temperature is controlled between 10 and 30°C, preferably between 15 and 25°C. The abbreviations are as follows: "min" represents minutes, "s" represents seconds, "U" represents enzyme activity units, "mM" represents millimoles per liter, "M" represents moles per liter, "rpm" represents revolutions per minute, "mol" represents moles, "μg" represents micrograms, "mg" represents milligrams, "g" represents grams, "μL" represents microliters, "mL" represents milliliters, "bp" represents base pairs, and Kan50 indicates that the culture medium contains 50 μg / mL kanamycin.

[0037] In the examples, experimental methods without specific conditions are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (Chinese version) (edited by J. Sambrook and MR. Green, translated by He Fuchu, 4th edition, Beijing: Science Press, 2017) and the methods described in the New England Biolabs (NEB) kit.

[0038] This invention designs an *E. coli* system co-expressing pyridoxal kinase (PLK) and polyphosphate kinase (PPK), creating a catalytic platform in which the phosphorylation of PL to PLP is seamlessly coupled with the continuous regeneration of ATP from ADP and low-cost polyphosphates. This integrated approach eliminates the need for exogenous ATP, significantly reduces production costs, and overcomes key limitations of natural biosynthetic pathways and traditional chemical synthesis. By constructing a highly efficient enzyme-catalyzed cascade reaction system, this invention achieves high-yield synthesis of PLP, successfully establishing a sustainable and scalable enzymatic preparation route, fully demonstrating the powerful capabilities of engineered cascade catalysis in complex cofactor-dependent biotransformations. The catalytic reaction principle is as follows: Figure 1 As shown.

[0039] The Escherichia coli strains K-12 MG1655 and DH5 in this invention BL21(DE3) and BL21(DE3) were used as host strains for plasmid construction and recombinant protein expression, respectively. All strains were cultured in Luria-Bertani (LB) medium. Expression vectors pET-28a(+) and pETDuet-1 were used for the cloning and expression of pyridoxal phosphate kinase (PLK) and polyphosphate kinase (PPK), respectively.

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0041] Example 1: Construction of PLK and PPK expression systems and engineered strains

[0042] 1. Gene cloning and plasmid construction

[0043] Genes encoding pyridoxal phosphate kinase (PLK, gene name: pdxK; Gene ID: 946881, Protein ID: NP_416913.1, SEQ ID NO.1) and polyphosphate kinase (PPK, gene name: ppk; UniProt: P0A7B1.2, SEQ ID NO.2) were amplified from the genomic DNA of *E. coli* K12 MG1655 by PCR. Gene-specific primers were designed to introduce flanking restriction enzyme sites: BamHI and XhoI for pdxK, and BamHI and HindIII for ppk. The primer sequences used are as follows:

[0044] pdxK Forward: 5'-CAGCAAATGGGTCGCG G / GATCC ATGAGTAGTTTGTTGTTGTTTAACG-3' (underlined site is BamHI), SEQ ID NO.3

[0045] pdxK Reverse: 5'-GTGGTGTGTGGTGGTG CTCGA / G TTATGCTTCCGCCAGCGG-3' (underlined site is XhoI); SEQ ID NO.4

[0046] The PCR reaction conditions were as follows: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 45 s, annealing at 61℃ for 50 s, extension at 72℃ for 1 min, 35 cycles, followed by incubation at 72℃ for 10 min at 4℃. After PCR, the band sizes of the PCR products were checked using 1% agarose gel electrophoresis. The PCR products were recovered using a PCR product recovery kit to obtain high-purity target gene fragments.

[0047] ppk cloning

[0048] ppk Forward: 5'-TCATCACCACAGCCAG G / GATCC ATGGGTCAGGAAAAGCTATACATCG-3' (underlined site is BamHI), SEQ ID NO.5

[0049] ppk Reverse: 5'-GCATTATGCGGCCGCA A / AGCTT TTATTCAGGTTGTTCGAGTGATTTG-3' (underlined site is HindIII), SEQ ID NO.6

[0050] PCR conditions were as follows: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 45 s, annealing at 67℃ for 45 s, extension at 72℃ for 2 min, 35 cycles, followed by incubation at 72℃ for 10 min at 4℃. Amplification products were detected by 1% agarose gel electrophoresis.

[0051] Table 1 PCR reaction system

[0052]

[0053] The amplified pdxK and pppk fragments, as well as the plasmid vectors pET-28a(+) and pETDuet-1, were digested with their respective restriction endonucleases.

[0054] The plasmid was double-digested with pdxK and pET-28a(+) restriction endonucleases BamHI and XhoI to obtain a linearized vector. The digestion system is shown in the table below. The digestion conditions were 37℃ for 1.5 h.

[0055] Table 2. Plasmid vector double enzyme digestion system

[0056] Table 2.5 Plasmid vector double enzyme digestion system

[0057]

[0058] Linearized vectors and fragments were obtained by digesting ppk and pETDuet with restriction endonucleases BamHI and Hind III, and the digestion system is shown in Table 2.

[0059] The digested DNA fragments were purified using a DNA gel extraction kit. The pdxK fragment was ligated into the linearized pET-28a(+) vector, and the ppk fragment was ligated into the linearized pETDuet-1 vector, yielding the recombinant plasmids pET28a-pdxK and pETDuet-ppk, respectively. The ligation mixture was then transformed into *E. coli* DH5α. Plasmid amplification was performed in competent cells. Positive clones were screened by colony PCR and subsequently validated by restriction enzyme digestion analysis.

[0060] The pdxK and pET-28a(+) ligation was performed using the Vazyme ClonExpress II one-step homologous recombination kit to ligate the target gene and the linearized vector. The ligation system is shown in Table 3 below. In the ClonExpress II recombination ligation system, the optimal amount of linearized vector was 0.03 pmol, and the optimal amount of target gene fragment was 0.06 pmol. The ligation conditions were 37℃ for 30 min.

[0061] Table 3. Linkage system between target fragment and linearized vector

[0062]

[0063] The ppk and pETDuet ligations were performed using the Vazyme ClonExpress II one-step homologous recombination kit to ligate the target gene and the linearized vector. The ligation system was configured as shown in Table 3.

[0064] The results are as follows Figure 2 A in Figure 3 As shown in Figure A, the pdxK (852 bp) and ppk (2067 bp) genes were successfully amplified from the genomic DNA of E. coli K12 MG1655, and the PCR product sizes were as expected. After sequencing confirmation, these genes were cloned into the pET-28a(+) and pETDuet-1 expression vectors, generating plasmids pET28a-pdxK and pETDuet-ppk, respectively. Restriction endonuclease digestion analysis verified that the released DNA fragments matched the predicted sizes of the vectors and inserts.

[0065] 2. Recombinant protein expression and extraction

[0066] The verified recombinant plasmids pET28a-pdxK and pETDuet-ppk were transformed into E. coli BL21(DE3) competent cells for protein expression, yielding strains E. coli BL21(DE3) / pET28a-pdxK and E. coli BL21(DE3) / pETDuet-ppk, respectively. Single transformed colonies were picked and inoculated into LB medium containing the corresponding antibiotics (50 µg / mL kanamycin for pET28a-pdxK; 100 µg / mL ampicillin for pETDuet-ppk) and cultured overnight at 37°C with shaking at 200 rpm. The overnight culture was then diluted 1:100 and inoculated into fresh LB medium containing the same antibiotics. Cells were cultured at 37°C until the optical density (OD) at 600 nm was reached. 600 The concentration was increased to 0.5-0.6. Then, protein expression was induced by adding IPTG to a final concentration of 0.1 mmol / L, followed by culturing at 16°C with shaking for another 16 hours.

[0067] Following induction, cells were collected by centrifugation (8,000 × g, 10 min, 4 °C), washed twice with ice-cold phosphate-buffered saline (PBS, 50 mmol / L, pH 7.4), and resuspended in the same buffer. Cell lysis was performed on ice using an ultrasonic homogenizer (e.g., sonication for 5 seconds, 10-second intervals, for a total of 15 min). Cell lysates were centrifuged (12,000 × g, 30 min, 4 °C) to remove cell debris, and the resulting supernatant was collected as the crude enzyme extract. The expression of the target protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the enzyme activities of PLK and PPK were determined.

[0068] PLK Activity Assay: PLK enzyme activity was assayed using UV-Vis spectrophotometry. The rate of the enzymatic reaction was quantified by continuously monitoring the increase in absorbance of the product pyridoxal phosphate (PLP) at a characteristic wavelength of 388 nm. The standard reaction system had a total volume of 3.0 mL and contained 50 mmol / L potassium phosphate buffer (pH 6.4), 0.2 mmol / L substrate pyridoxal (PL), 0.2 mmol / L ATP, 0.05 mmol / L ZnCl2, and an appropriate amount of crude enzyme extract. During the assay, all components except the enzyme solution were mixed in a cuvette and equilibrated in a 37°C spectrophotometer for 3–5 minutes. The enzyme solution was then added to initiate the reaction. Immediately after mixing, the absorbance at 388 nm was recorded over time for 3–5 minutes. The initial linear phase of the reaction was used to calculate the rate. A negative control was included in each assay, using an equal volume of buffer solution instead of the enzyme solution to correct for non-enzymatic background changes. Enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol PLP per minute under the stated conditions. In actual calculations, the absorbance change rate is converted into the PLP generation rate based on a pre-established PLP standard curve (concentration range of 0–100 μmol / L), and then the enzyme activity is obtained.

[0069] PPK Activity Assay: PPK activity was determined using a coupled enzyme method to quantify ATP production. This assay couples ATP produced by PPK to a hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) reaction system, leading to the reduction of NADP⁺ to NADPH. The subsequent increase in absorbance at 340 nm due to NADPH formation is proportional to PPK activity. The reaction is initiated by adding an enzyme solution containing PPK, and the initial rate of increase in absorbance at 340 nm is monitored. One unit (U) of PPK activity is defined as the amount of enzyme producing 1 μmol of ATP per minute under the assay conditions.

[0070] The PPK enzyme activity assay was performed in two stages. First, the reaction system for Stage I was prepared as shown in Table 4. Distilled water was used instead of the enzyme solution, and no ADP was added, serving as a control group. The reaction was carried out at 37°C for 15 min. After the reaction, the PPK enzyme was inactivated by boiling in a water bath for 5 min. After cooling, the precipitate was removed by centrifugation, and the supernatant was used for Stage II. The reaction system for Stage II is shown in Table 5, and the reaction was carried out at 30°C for 2 h. The reaction ensured that all ATP generated in Stage I was consumed.

[0071] Table 4. Reaction system for stage I of PPK enzyme activity assay

[0072]

[0073] After the reaction, the reaction solution was appropriately diluted, and the absorbance at 340 nm was measured. The NADPH content was calculated based on the absorbance, and then the ATP content was calculated. PPK enzyme activity is defined as the amount of enzyme required to generate 1 µmol of ATP per minute under the above reaction conditions; one enzyme activity unit is defined as this amount of enzyme.

[0074] Table 5. Reaction system for PPK enzyme activity detection stage II

[0075]

[0076] PLP content determination: Quantitative analysis of the content was performed using high-performance liquid chromatography (HPLC). An XBndge® C18 column (4.6 × 250 mm, 5 μm) was used for analyte separation, with the column temperature controlled at 30℃. Mobile phase: Mobile phase A: containing 0.1 mol / L potassium dihydrogen phosphate, 0.1 mol / L sodium perchlorate, and 0.5 g / L sodium bisulfite, adjusted to pH 3.0 with phosphoric acid; Mobile phase B: 20% acetonitrile. Constant flow rate: 1.0 mL / min. Gradient elution program: Initial stage: isocratic elution with 100% eluent A for 4 minutes; 4.0–13 minutes: linear switching to 10% eluent B; Strong washing stage: washing with 50% acetonitrile for 3 minutes; Reequilibration stage: equilibration with 100% eluent A for 4 minutes, followed by the next injection. Detection conditions: A fluorescence detector was used, with an excitation wavelength of 300 nm and an emission wavelength of 400 nm. Injection volume: 10 μL.

[0077] Experimental Results: After IPTG induction, heterologous expression in E. coli BL21(DE3) successfully produced soluble PdxK and PPK proteins with molecular weights matching theoretical predictions. SDS-PAGE analysis of the PdxK-producing strain (E. coli BL21(DE3) / pET28a-pdxK) showed a distinct band at approximately 31 kDa, consistent with the calculated quality of His-tagged PdxK. Figure 2 In the case of strain B), similarly, for the PPK-producing strain (E. coli BL21(DE3) / pETDuet-ppk), a characteristic band was observed at approximately 80 kDa, corresponding to the expected size of the translated ppk gene product. Figure 3 (Lane B, Lane 4).

[0078] 3. Construction of strains co-expressing two plasmids

[0079] To construct the co-expression engineered strain, the pETDuet-ppk plasmid was first isolated and then transformed into E. coli BL21(DE3) / pET28a-pdxK competent cells. The resulting dual-plasmid strain, named E. coli BL21(DE3) / pET28a-pdxK&pETDuet-ppk, was screened on LB agar plates containing both kanamycin (50 µg / mL) and ampicillin (100 µg / mL). The successful co-expression of PLK and PPK in this engineered strain was confirmed using the same protein induction, extraction, and analysis procedures described above.

[0080] SDS-PAGE analysis confirmed the successful simultaneous expression of the two target proteins, with apparent molecular weights of approximately 31 kDa and 80 kDa, respectively. Figure 3 (lane 6). This validated the establishment of the dual plasmid co-expression system. After optimizing the co-expression conditions, the specific activities of PLK and PPK reached 50 U / g, respectively. 细胞 and 145 U / g 细胞 .

[0081] Example 2 Enzymatic characterization of pyridoxal phosphate kinase and polyphosphate kinase

[0082] This embodiment systematically characterized the key enzymatic properties of recombinant PLK and polyphosphate kinase PPK to determine their optimal reaction conditions and stability characteristics. For PLK, its optimal temperature was determined by measuring activity in a constant 50 mmol / L PIPES buffer (pH 6.4) at a temperature gradient of 25–60 °C, while its optimal pH was determined using a series of buffers covering a wide pH range (e.g., acetate buffer for pH 4.0–6.0, PIPES for pH 6.0–8.0, and Tris-HCl for pH 8.0–10.0) at the optimal temperature. In both cases, the observed maximum activity was defined as 100%. The stability of PLK was assessed by measuring residual activity after pre-incubation at 4 °C for 2 hours in buffers of different pH values, and by measuring residual activity after incubation at different high temperatures (25–60 °C) for the same time. Furthermore, the activity of divalent metal ions (Mn) was also investigated. 2+ Mg 2+ Cu 2+ Zn 2+ Co 2+ Ca 2+ The effect of Mg (provided in the form of 5 mmol / L chloride) on PLK activity, in the presence of Mg 2+ The activity at that time was used as a 100% baseline.

[0083] For the characterization of PPK, the optimal temperature of PPK was determined by measuring its activity at different pH values ​​within a temperature gradient from 25 to 65 °C. The optimal pH was then determined by measuring the activity loss after 2 hours of pre-incubation at different pH values ​​or at different temperatures (25-65 °C). The stability of PPK was similarly assessed by measuring the activity loss after 2 hours of pre-incubation at different pH values ​​or different temperatures (25-65 °C).

[0084] like Figure 4 and Figure 5 As shown, the results reveal the distinct yet compatible biochemical properties of the two enzymes.

[0085] For PLK, a clear temperature optimum of 40°C and an optimum pH of 6.0 were observed. Figure 4 A in Figure 4 (B in the original text). The rapid loss of enzyme activity above 40℃ is a characteristic of thermophilic enzymes undergoing thermal denaturation, and they also prefer a weakly acidic environment. Metal ion dependence (…) Figure 4 The C in the text further emphasizes its catalytic mechanism, Zn 2+ Mg 2+ and Co 2+ It is the most effective cofactor. And Cu 2+ and Ca 2+This inhibits its activity. PLK exhibits moderate thermal stability, retaining most of its activity at 50°C, but losing 68% of its activity after 2.5 hours at 60°C. Figure 4 (D in the text). It exhibits exceptional stability at pH 6.0 (retaining over 95% activity), but significant inactivation at pH 8.0. Figure 4 The difference between its optimal pH (6.0) and the typical physiological pH (7.0–7.5) of bacterial cytoplasm or coupled reaction systems is a key consideration in process integration.

[0086] In comparison, PPK exhibits a higher optimal temperature of 50℃. Figure 5 (A in the text). However, a key trade-off was observed between its high catalytic rate at this temperature and its operational stability. Significant activity loss during prolonged incubation at 50°C is a recognized limitation of type II PPKs, which, despite their high specific activity, typically possess complex oligomeric structures and are prone to thermal dissociation above 50°C. The optimal pH for PPKs is 7.5, with activity decreasing sharply above pH 9.0. Figure 5 (B in the text). The high consistency between its pH-activity and pH-stability curves ( Figure 5 The C in the pH range is very favorable, determining a robust operating window between pH 7.0 and 8.0.

[0087] The compatibility of PLK and PPK provides favorable conditions for designing efficient biocatalytic systems. PLK prefers weakly acidic conditions and moderate temperatures, while PPK performs best under weakly acidic to neutral pH conditions and requires careful temperature management to balance activity and stability.

[0088] Example 3 Synthesis of PLP using whole-cell and cell-free systems

[0089] In this embodiment, whole-cell and cell-free methods derived from the engineered strain E. coli BL21(DE3) / pET28a-pdxK&pETDuet-ppk were used to evaluate the performance of the coupled PLK (PdxK) and PPK enzyme system in catalyzing the phosphorylation of pyridoxal (PL) to PLP.

[0090] In whole-cell biotransformation, induced cells were collected and resuspended at 0.1 g / mL (wet cell weight) in 0.05 mol / L potassium phosphate buffer (pH 6.5). The reaction was carried out at 37 °C with shaking (180 rpm) for 4 hours. The reaction mixture contained 5 mmol / L PL, 20 mmol / L sodium hexametaphosphate (SHMP), and 5 mmol / L ADP.

[0091] Samples were taken every 30 minutes to monitor PLP formation and calculate yield, allowing for a direct comparison of the two synthesis strategies.

[0092] While whole-cell systems are conceptually attractive for process enhancement, experiments have shown that this catalytic approach faces significant operational challenges. For example... Figure 6 As shown, whole-cell biocatalysis achieved a maximum yield of approximately 26.8%, which, while confirming functional activity, is far below the requirements for industrial applications (at least 90% yield). This performance deficiency is mainly attributed to the combined constraints of intracellular diffusion barriers of the macromolecular charged substrate and the degradation of the product PLP by endogenous phosphatases, resulting in a severely insufficient net accumulation.

[0093] Given the limitations of whole-cell methods, this invention develops a cell-free biocatalytic system incorporating recombinantly produced PLK and PPK. This system aims to synthesize PLP from PL while simultaneously regenerating ATP from ADP using the low-cost phosphate donor sodium hexapolyphosphate, thereby addressing the high cost of stoichiometric ATP addition.

[0094] This invention first systematically optimizes key reaction parameters to establish a mutually compatible operating window. Since the optimal temperatures for PLK (40℃) and PPK (50℃) differ, the effect of the temperature gradient was investigated. Figure 7 As shown in Figure A, the PLP yield increases with increasing temperature, reaching over 90% at 45°C, but decreases sharply at higher temperatures. This confirms the thermal instability of PPK-type enzymes above 50°C. The pH curve was subsequently investigated. Figure 7 (B in the original text). This cascade reaction performs optimally under weakly acidic conditions (pH 6.5), with activity significantly decreasing at pH 8.0. The curve is closer to the optimum pH of PLK (pH 6.0) than to the wider optimum pH of PPK (pH 7.5). This indicates that the phosphorylation step catalyzed by PLK is a more pH-sensitive node and a key determinant of the overall system's pH response. Therefore, pH 6.5 was chosen as the optimal compromise.

[0095] Through the above optimization, suitable conditions for the cell-free catalytic system were obtained. Crude enzyme solution was prepared by sonication and centrifugation of 0.1g wet cells in 1mL reaction system. The subsequent cell-free reaction was carried out in potassium phosphate buffer (pH 6.5). The reaction system contained 5 mmol / L PL, 5 mmol / L ADP, 20 mmol / L sodium hexapolyphosphate and 10 mmol / L MgCl2, and was incubated for 4 hours under the same temperature and shaking conditions.

[0096] In this invention, crude enzyme extracts of PLK and PPK were prepared from E. coli BL21(DE3) / pET28a-pdxK and E. coli BL21(DE3) / pETDuet-ppk strains, respectively. The catalytic performance of the co-expression system was evaluated by investigating the catalytic effect under different enzyme activity ratios.

[0097] like Figure 7 As shown in Figure C, the PLP yield is highest (approximately 95%) when the PLK to PPK activity ratio is 1:3, and the yield decreases as the PPK ratio decreases, indicating that ATP regeneration is the rate-limiting step in this cascade reaction. Notably, this optimal ratio closely matches the measured specific enzyme activity in the co-expression system (approximately 50 U / g cells for PLK and 145 U / g cells for PPK, a ratio close to 1:3), demonstrating that the crude enzyme solution produced by the co-expression system constructed in this invention has an endogenous enzyme activity ratio that precisely meets the requirements for efficient PLP synthesis using PL as a substrate.

[0098] Furthermore, this invention also investigated the effect of SHMP concentration. In stark contrast to the inhibition of PPK formation by high SHMP levels in single-enzyme assays, the PLP yield in the two-enzyme cascade increased with increasing SHMP concentration, reaching approximately 95% yield up to 25 mmol / L. Figure 7 (D in the middle).

[0099] Under optimized conditions (45°C, pH 6.5, 25 mmol / L SHMP), the cell-free two-enzyme system achieved approximately 95% final PLP yield within 4 hours. Figure 6 (This is) far superior to whole-cell catalysts.

[0100] Example 4 Construction of high-performance pyridoxal phosphate kinase (PLK) and its catalytic synthesis of PLP

[0101] This embodiment involves in-depth analysis of the PdxK amino acid sequence. A mutant, Ser23Ala / Tyr96Phe / Asp227Glu (S23A / Y96F / D227E), was prepared through site-directed combinatorial mutagenesis to improve catalytic performance. Compared to SEQ ID NO.1, this mutant exhibits only three mutations at S23A, Y96F, and D227E (see SEQ ID NO.7).

[0102] First, primers corresponding to each mutation site were designed, and then site-directed mutagenesis was performed using the NEB Q5® Site-Directed Mutagenesis Kit (Q5 SDM Kit). The primers for each mutation site are as follows (lowercase letters represent the bases at the mutation sites):

[0103] (1) S23A mutant primers

[0104] S23A-F5'-CAGgcgCAGGTGGTTTACGGCAGCGTGGGCAA-3', SEQ ID NO.8

[0105] S23A-R5'-TAAACCACCTGcgcCTGCACGGCGACGATATCC-3', SEQ ID NO.9

[0106] (2) Y96F mutant primers

[0107] Y96F-F5'-TAACCACGGGCttcATGGGAACGGCATCGCAA-3', SEQ ID NO.10

[0108] Y96F-R5'-CATgaaGCCCGTGGTTACAGCACGAAGTTGGC-3', SEQ ID NO.11

[0109] (3) D227E mutant primers

[0110] D227E-F 5'-AAAACCgagCTGAAAGGGACTGGCGACCTGTT-3', SEQ ID NO.12

[0111] D227E-R 5'-CCTTTCAGctcGGTTTTTACCCGTGAATGGGA-3', SEQ ID NO.13

[0112] Primers were synthesized by a nucleic acid synthesis company, then dissolved in sterile water, and the procedure was followed according to the kit instructions. See below:

[0113] Mutation of the corresponding sites using PCR

[0114] PCR reaction system:

[0115] Q5 Hot Start High-Fidelity 2X Master Mix12.5 μL

[0116] 1.25 μL of 10 μM F primer

[0117] 10 μM R primer 1.25 μL

[0118] Template DNA (1–25 ng / μl)1 μL

[0119] 9.0 μL of nuclease-free water

[0120] Total volume 25 μL

[0121] Cyclic program temperature:

[0122]

[0123] For mutants with more than two mutation sites, the PCR product of the previous mutation site is used as a template to perform site-directed mutagenesis at the corresponding sites one after another.

[0124] Kinase, Ligase & DpnI (KLD) (a special mixture of kinase, ligase and DpnI) reaction treatment

[0125] The reaction system is as follows:

[0126]

[0127] React at room temperature for 5 minutes.

[0128] Thermal shock conversion

[0129] Add 5 μL of KLD reaction mixture to 50 μL of chemicompetent E. coli BL21(DE3) cell suspension, incubate on ice for 30 min, subject to heat shock at 42°C for 30 s, incubate on ice for 5 min, add 950 μL of SOC sterile liquid medium, and gently shake at 37°C for 1 h. Spread 40-100 μL of the bacterial suspension onto Kan50 LB agar plates and incubate overnight at 37°C. Single colonies that grow are the corresponding mutant expression strains.

[0130] Mutant identification

[0131] The obtained mutant expression strain was inoculated into 25 mL of Kan50 LB liquid medium and cultured overnight at 37°C. Plasmids were extracted using a plasmid extraction kit. The samples were sent to a third-party biotechnology company for sequencing, confirming that the corresponding product was the target product for site-directed mutagenesis, yielding the mutant BL21(DE3) / pET28a-pdxK_S23A / Y96F / D227E. Expression of this strain was induced, and SDS-PAGE analysis was performed as follows: Figure 8 As shown, there is a distinct band at approximately 31 kDa, consistent with expectations.

[0132] Construction of dual-plasmid co-expression mutant strain: Following the protocol in Example 1, the pETDuet-ppk plasmid was transformed into competent cells of E. coli BL21(DE3) / pET28a-pdxK_S23A / Y96F / D227E. The resulting dual-plasmid strain was named E. coli BL21(DE3) / pET28a-pdxK_S23A / Y96F / D227E&pETDuet-ppk. It was screened on LB agar plates containing both kanamycin (50 µg / mL) and ampicillin (100 µg / mL). After co-expression, the crude enzyme solution was collected for enzyme activity detection and PLP synthesis using a cell-free catalytic system.

[0133] The PLK enzyme activity of the mutant E. coli BL21(DE3) / pET28a-pdxK_S23A / Y96F / D227E&pETDuet-ppk was measured to be approximately 120 U / g. 细胞 Compared to 50 U / g of the original strain. 细胞 It increased by 2.4 times.

[0134] PLP was synthesized using a cell-free system according to the method described in Example 3. The results are as follows: Figure 9 As shown, the experimental results indicate that the mutant significantly improves the performance of catalyzing the synthesis of PLP from PL, and the yield of PLP can reach 96% in just 2 hours.

[0135] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for synthesizing pyridoxal phosphate based on cascade biocatalysis and autonomous ATP supply, characterized in that: Pyridoxal phosphate kinase and polyphosphate kinase were co-expressed, and the resulting co-expressing strain or its crude enzyme extract, substrate pyridoxal, ADP and polyphosphate were used to construct the reaction system.

2. The method for synthesizing pyridoxal phosphate based on cascade biocatalysis and autonomous ATP supply according to claim 1, characterized in that: The amino acid sequence of the pyridoxal phosphate kinase is shown in SEQ ID NO.1, and the amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO.

2.

3. The method for synthesizing pyridoxal phosphate based on cascade biocatalysis and autonomous ATP supply according to claim 1, characterized in that: The gene fragments of the pyridoxal phosphate kinase and the polyphosphate kinase are derived from the genome of E. coli K12MG1655.

4. The method for synthesizing pyridoxal phosphate based on cascade biocatalysis and autonomous ATP supply according to claim 1, characterized in that: The gene fragment of pyridoxal phosphate kinase was ligated into the pET-28a(+) vector to obtain the recombinant plasmid pET28a-pdxK; the polyphosphate kinase was ligated into the pETDuet-1 vector to obtain the recombinant plasmid pETDuet-ppk; the plasmid pET28a-pdxK was transformed into E. coli BL21(DE3) competent cells, and the plasmid pETDuet-ppk was transformed into E. coli BL21(DE3) competent cells that already contained the pET28a-pdxK plasmid to obtain a strain co-expressing the two plasmids.

5. The method for synthesizing pyridoxal phosphate based on cascade biocatalysis and autonomous ATP supply according to claim 1, characterized in that: The reaction system for synthesizing pyridoxal phosphate using co-expressed strains consisted of 0.1 g / mL wet cells, 5 mmol / L PL, 20 mmol / L sodium hexapolyphosphate, and 5 mmol / L ADP. The reaction was carried out in a pH 6.5 potassium phosphate buffer at 37°C.

6. The method for synthesizing pyridoxal phosphate based on cascade biocatalysis and autonomous ATP supply according to claim 1, characterized in that: The reaction system for synthesizing pyridoxal phosphate using the crude enzyme solution of the co-expressed strain included: 5 mmol / L PL, 20 mmol / L sodium hexapolyphosphate, 5 mmol / L ADP and 10 mmol / L MgCl2. The crude enzyme solution was prepared by sonication and centrifugation of 0.1 g / mL wet cells. The reaction was carried out in a pH 6.5 potassium phosphate buffer at 37°C.

7. The method for synthesizing pyridoxal phosphate based on cascade biocatalysis and autonomous ATP supply according to claim 2, characterized in that: The amino acid sequence of pyridoxal kinase phosphate shown in SEQ ID NO.1 was mutated as follows: S23A / Y96F / D227E, and the mutated amino acid sequence is shown in SEQ ID NO.7.

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