Deoxycytidine monophosphate hydroxylase and application thereof

Abstract

The invention discloses deoxycytidine monophosphate hydroxylase AfhB and application of the deoxycytidine monophosphate hydroxylase AfhB. The dCMP hydroxylase AfhB obtained by screening from the genome of the bacteriophage HY126 can be used for green synthesis or fermentation production of 5hdCMP, so that the environmental pollution caused by a traditional chemical synthesis method is greatly reduced, and a new enzyme element and way are provided for development of a gene editing technology and fermentation production of 5hdCMP and derivatives thereof in industrial fermentation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a phage-encoded deoxycytidine hydroxylase AfhB and its application in the green synthesis, fermentation production, gene editing, and epigenetic modification of hydroxylated cytosine nucleotides. Background Technology

[0002] Epigenetic modifications are an important class of cellular physiological regulatory mechanisms, enabling cells to finely regulate gene expression. Common epigenetic modifications include 6-methyladenine, 5-methylcytosine, and 5-hydroxymethylcytosine (Rosaura Esteve-Puig et al., 2020). 6-methyladenine, as one of the most widely distributed epigenetic modifications, plays a crucial role in regulating DNA replication initiation, mismatch repair, and phage defense (Beifang Lu et al., 2025). Other epigenetic modifications can also regulate related physiological processes to varying degrees. Under genome replication and various harmful environments, nucleotides can undergo non-natural modifications, most of which are harmful to the cell itself. Cells can eliminate these harmful modifications through specific pathways (Andrea J. Lee et al., 2017). 5-hydroxycytosine is a relatively unique class among these modifications.

[0003] The structural formula of hydroxycytosine is as follows: Cytosine bases in the cytoplasm or DNA can be oxidized or catalyzed by bacterial / phage enzymes to produce 5-hydroxycytosine. When cytosine is oxidized to 5-hydroxycytosine in the cytoplasm, it can be further phosphorylated to form its triphosphate derivatives, which are incorporated into the genome as substrates for DNA synthesis. In-situ oxidation of cytosine on DNA can also produce 5-hydroxycytosine. The base-pairing characteristics of 5-hydroxycytosine differ from those of ordinary cytosine; it tends to pair with dA, thus inducing C→T mutations and posing a potential threat to genome stability. Cells have evolved specific enzymes such as Endonuclease VIII (Nei) and Endonuclease III (Nth) to remove 5-hydroxycytosine. However, for some bacteriophages, 5-hydroxycytosine is a fundamental component of their DNA. For example, in the genome of the rhizobium bacteriophage RL38JI, all cytosines are replaced by overmodified 5-hydroxycytosine, indicating that this modification can be stably maintained in the genome and facilitates the phage's ability to overcome the host's defense system. Furthermore, 5-hydroxycytosine is a naturally occurring modification at position 2501 (ho5C2501) of the 23S rRNA in *E. coli*. The abundance of this modification is dynamic, reaching its peak during the bacterial plateau phase. High abundance of 5-hydroxycytosine can affect translation efficiency and reduce protein synthesis, but it helps *E. coli* adapt to oxidative stress. Related gene deletion mutants cannot grow under millimolecular hydrogen peroxide stress, while strains retaining this modification can grow normally. Recent studies have also found that replacing cytosine in antisense oligonucleotides or siRNAs targeting mRNA with 5-hydroxycytosine can reduce hepatotoxicity while maintaining antisense activity. These studies reveal the importance and complexity of 5-hydroxycytosine in physiological activities.

[0004] Currently, the common method for introducing hydroxyl groups into cytosine nucleosides or deoxynucleosides is bromohydrolysis, which involves substituting elemental bromine at the 5-position of cytosine followed by alkaline hydrolysis to obtain the hydroxylated product. This process is a chemical synthesis that requires the use of toxic and highly irritating elemental bromine and large amounts of organic reagents, resulting in poor safety and economic efficiency. Summary of the Invention

[0005] This invention aims to overcome the defects and shortcomings of existing technologies for synthesizing 5-hydroxycytosine nucleotides (5hdCMP) and provides a 5hdCMP hydroxylase AfhB and its applications, enabling the production of 5hdCMP through in vivo fermentation of *E. coli* or in vitro enzymatic catalysis. To achieve the above objective, this invention adopts the following technical solution: This invention has discovered the AfhB protein and its function. In the genome of the bacteriophage HY126 of Escherichia coli JM109, expression of AfhB can generate 5hCMP in the cytoplasm of JM109 cytoplasm.

[0006] The present invention provides a system for the specific synthesis of 5hCMP, the system comprising AfhB, wherein the nucleotide sequence of the 5hdCMP hydroxylase is shown in SEQ ID NO.1 and the amino acid sequence encoded therein is shown in SEQ ID NO.2.

[0007] This invention provides a vector pWHU5053 for heterologous expression of the afhB gene in Escherichia coli for fermentation production of 5hdCMP.

[0008] The present invention also provides a vector pWHU5054 for expressing recombinant AfhB protein in Escherichia coli BL21(DE3).

[0009] The dCMP hydroxylase can be expressed in *E. coli* for fermentation production of 5hdCMP or purification of recombinant AfhB protein. The host is merely an example; AfhB can also be expressed in cells and corresponding organisms including, but not limited to, *Streptomyces*, *Bacillus subtilis*, *Pseudomonas aeruginosa*, yeast, insect cells, plant cells, mammalian cells, and human cells.

[0010] The dCMP hydroxylase provided by this invention has the activity of catalyzing the formation of 5hdCMP from dCMP.

[0011] The reaction formula for the conversion of dCMP to 5hdCMP is as follows: The advantages of this invention are as follows: This invention discovers a dCMP hydroxylase derived from Escherichia coli bacteriophages, whose structure and function are not documented in the literature, and which possesses the activity to oxidize dCMP to 5hdCMP. This activity can be used to synthesize 5hdCMP via fermentation or protease methods, reducing the use of toxic organic reagents in traditional chemical synthesis methods, lowering production costs, and making it more environmentally friendly. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0013] Figure 1 The distribution of the afhB gene on different bacteriophage genomes; Figure 2 This is a schematic diagram of the structure of plasmid pWHU5053; Figure 3 This is a schematic diagram of the structure of plasmid pWHU5054; Figure 4 This represents the results of heterologous expression of AfhB protein in Escherichia coli JM109; Figure 5 The results of AfhB protein purification; Figure 6 The results are for the in vitro activity assay of AfhB protein; Figure 7 The enzyme kinetics curve for AfhB; Figure 8 This is a schematic diagram of the AfhB catalytic mechanism.

[0014] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention in any way. Unless otherwise stated, the present invention is implemented using conventional molecular biology, biochemistry, and microbiology techniques in the art.

[0016] Materials and general methods used in the examples: Strains and plasmids: Escherichia coli JM109, BL21(DE3) and DH5α were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; plasmids pACYC184 / tac and pET28a(+) were preserved in our laboratory; the genome of phage HY126 was isolated and preserved in our laboratory.

[0017] Enzymes and reagents: Restriction endonucleases, homologous recombinase kits, and high-fidelity DNA polymerase were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; IPTG, FAD, NADH, dCMP, and 5hdCMP standards were purchased from Sigma-Aldrich; HPLC-grade methanol was purchased from Merck; other routine reagents were domestically produced analytical grade.

[0018] Culture conditions: Escherichia coli was cultured in LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride), and appropriate antibiotics were added as needed (ampicillin 100 μg / mL, chloramphenicol 25 μg / mL).

[0019] HPLC-MS and HPLC-MS / MS analytical methods: The chromatographic column was a Waters Atlantis T3 column (2.1 × 150 mm, 3 μm); mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid methanol solution; gradient elution program: 0–5 min, 2% B; 5–24 min, 2%–24% B; 24–26 min, 24%–2% B; 26–36 min, 2% B; flow rate: 0.2 mL / min; column temperature: 30℃; injection volume: 10 μL. HPLC-MS used electrospray ionization (ESI) in negative ion mode, and HPLC-MS / MS used MRM in positive ion mode, with a precursor ion mass-to-charge ratio of 324, a daughter ion mass-to-charge ratio of 128, and a collision energy of 25 V.

[0020] Example 1: The afhB gene and its distribution on different phage genomes This invention first performed whole-genome sequencing on a laboratory-preserved bacteriophage strain HY126 infected with Escherichia coli JM109. Bioinformatics analysis revealed a 717 bp open reading frame (AfhB) in the HY126 genome. The nucleotide sequence of this gene is shown in SEQ ID NO.1, encoding a 238-amino acid protein, the amino acid sequence of which is shown in SEQ ID NO.2. BLASTp alignment analysis showed that the AfhB protein has low homology with proteins of known function, and 40%-60% homology with some presumed phage proteins, suggesting that it may be a novel phage-encoded enzyme.

[0021] 1.2 Distribution of the afhB gene in bacteriophages To understand the distribution of the afhB gene in bacteriophages, this invention retrieved and downloaded 182,364 published E. coli bacteriophage genome sequences from the public database (NCBI GenBank). BLASTn alignment revealed that homologous sequences of the afhB gene exist in 206 bacteriophage genomes, primarily distributed in T4-like and RB49-like bacteriophages, belonging to the Myotail Phage family. Interestingly, the afhB gene is typically located near gene clusters related to DNA modification in the bacteriophage genome, suggesting its potential involvement in bacteriophage DNA modification processes.

[0022] Figure 1 The distribution of the afhB gene in different bacteriophage genomes was shown, revealing that the gene is widely present in bacteriophages, but no previous studies have reported on its function.

[0023] Example 2: Construction of the heterologous expression plasmid pWHU5053 for the afhB gene.

[0024] 2.1 Primer Design Based on the afhB gene sequence and the multiple cloning site sequence of the pACYC184 / tac vector, two pairs of primers were designed: Primers for amplifying the afhB gene fragment: Upstream primer P1: 5'-AGAGGAGAAAGGATCTATGGGAAAACATAACACAATGA-3' Downstream primer P2: 5'-CCGGCGTAGAGGATCCTTAGTCAAGGAATCCTTTAACA-3' where the underlined parts are sequences homologous to the vector and used for homologous recombination.

[0025] Primers for amplifying linearized vectors: Upstream primer P3: 5'-CCATAGATCCTTTCTCCTCT-3' Downstream primer P4: 5'-CTAAGGATCCTCTACGCCGG-3' 2.2 PCR amplification Using phage HY126 genomic DNA as a template, PCR amplification was performed using primers P1 / P2. The reaction mixture (50 μL) consisted of 100 ng template DNA, 0.4 μM each of forward and reverse primers, 0.2 mM dNTPs, 1 U high-fidelity DNA polymerase, 5 μL 10× buffer, and sterile water to a final volume of 50 μL. The amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 30 cycles; and a final extension at 72℃ for 10 min.

[0026] Using pACYC184 / tac plasmid as a template, PCR amplification was performed using primers P3 / P4 to obtain the linearized vector fragment. The amplification program was the same as above, but the extension time was changed to 2 min.

[0027] The PCR products were analyzed by 1% agarose gel electrophoresis, yielding an approximately 750 bp afhB gene fragment and an approximately 4.5 kb linearized vector fragment. The two fragments were purified separately using a gel extraction kit.

[0028] 2.3 Homologous recombination linkage Prepare the homologous recombination reaction system according to Table 1, mix gently, and react at 50°C for 30 min. After the reaction is complete, cool the product on ice for later use.

[0029] Table 1. Homologous recombination reaction system 2.4 Transformation and Identification Add 10 μL of the recombinant product to 100 μL of *E. coli* DH5α competent cells, incubate on ice for 30 min, heat shock at 42℃ for 90 s, and immediately incubate on ice for 2 min. Add 900 μL of LB medium and incubate at 37℃ for 1 h. Spread 100 μL of the bacterial culture onto an LB agar plate containing chloramphenicol (25 μg / mL) and incubate overnight at 37℃.

[0030] Single clones were picked and inoculated into LB liquid medium (containing chloramphenicol) and cultured overnight at 37°C with shaking. The plasmid was then extracted and sequenced for verification. Sequencing results showed that the afhB gene was correctly inserted into the vector without mutation, indicating that the recombinant plasmid pWHU5053 was successfully constructed. Its plasmid map is shown below. Figure 2 As shown. This plasmid drives the expression of the afhB gene via the tac promoter and carries a chloramphenicol resistance marker.

[0031] Example 3: Construction of AfhB recombinant protein expression plasmid pWHU5054.

[0032] 3.1 Primer Design To construct an expression plasmid for the AfhB recombinant protein with a His tag at the C-terminus, the following primers were designed: Primers for amplifying the afhB gene fragment: Upstream primer P5: 5'-CTTTAAGAAGGAGATATACCATGGGAAAACATAACACAATGA-3' Downstream primer P6: 5'-GTGGTGGTGCTCGAGTGCGTCAAGGAATCCTTTAACATAATC-3' where the underlined part is a sequence homologous to the pET28a(+) vector.

[0033] Primers for amplifying linearized vectors: Upstream primer P7: 5'-TGGTATATCTCCTTCTTAAAG-3' Downstream primer P8: 5'-ACGCACTCGAGCACCACCAC-3' 3.2 PCR amplification and homologous recombination The afhB gene was amplified using pWHU5053 plasmid as a template and primers P5 / P6; the linearized vector was amplified using pET28a(+) plasmid as a template and primers P7 / P8. The PCR reaction system and procedure were the same as in Example 2.

[0034] The purified afhB gene fragment and the linearized pET28a(+) vector fragment were ligated according to the homologous recombination reaction system (as in Table 1), transformed into Escherichia coli DH5α competent cells, and plated on LB plates containing kanamycin (50 μg / mL).

[0035] 3.3 Identification and Verification After selecting and culturing single clones, plasmids were extracted and sequenced to obtain the correct recombinant plasmid pWHU5054, as shown in the plasmid map below. Figure 3 As shown in the figure, the afhB gene is expressed in this plasmid with a 6× His tag fused to the C-terminus, controlled by the T7 promoter, and has a kanamycin resistance marker, making it suitable for inducing recombinant protein expression in Escherichia coli BL21(DE3).

[0036] Example 4: Heterologous expression of the afhB gene in Escherichia coli JM109 produces 5hdCMP.

[0037] 4.1 Transformation and Induced Expression The pWHU5053 plasmid was transformed into E. coli JM109 competent cells, plated on LB agar plates containing chloramphenicol, and incubated overnight at 37°C. Single colonies were picked and inoculated into 20 mL of LB medium (containing chloramphenicol), and incubated overnight at 37°C with shaking at 220 rpm.

[0038] Transfer the overnight culture to fresh LB medium (containing chloramphenicol) at a ratio of 1:100 and incubate at 37°C until OD500. 600 Approximately 0.6. Add IPTG to a final concentration of 1 mM and continue incubation at 37°C for 2 h to induce protein expression. Simultaneously, a non-induced group (without IPTG) and an empty vector control group (transformed with pACYC184 / tac empty plasmid) were set up as controls.

[0039] 4.2 Sample Preparation Collect the bacterial culture and centrifuge at 4℃, 6000× g for 10 min, discarding the supernatant. Wash the bacterial pellet twice with pre-chilled PBS buffer (pH 7.4), centrifuging after each wash to collect the bacterial cells. Resuspend the bacterial cells in 2 mL of pre-chilled anhydrous methanol and transfer to a 2 mL centrifuge tube.

[0040] Place centrifuge tubes in an ice-water mixture and use an ultrasonic homogenizer to disrupt the bacterial cells (50% power, 5 s operation, 5 s interval, total operation time 5 min). Incubate the disrupted mixture overnight at -80°C to allow for the full release of intracellular small molecule metabolites.

[0041] The following day, the sample was centrifuged at 4℃ and 10000× g for 10 min, and the supernatant was carefully collected. The supernatant was dried at 37℃ using a nitrogen blower, redissolved in 100 μL of anhydrous methanol, filtered through a 0.22 μm filter membrane, and transferred to a vial for HPLC-MS analysis.

[0042] 4.3 HPLC-MS Detection Results HPLC-MS analysis results are as follows Figure 4As shown. Compared with the standard, in the JM109 strain sample induced to express pWHU5053, a chromatographic peak with the same retention time (6.5 min) and mass spectrometric characteristics as the 5hdCMP standard (m / z 322.0450 [MH)) was detected. - No obvious chromatographic peak was observed at this position in the uninduced group and the empty vector control group samples.

[0043] Quantitative analysis showed that the content of 5hdCMP in the induced expression group was approximately 2.3 μg / g of bacterial wet weight. This result confirms that heterologous expression of the afhB gene in Escherichia coli JM109 can catalyze the intracellular conversion of dCMP to 5hdCMP.

[0044] Example 5: Expression and purification of recombinant AfhB protein.

[0045] 5.1 Protein-induced expression The pWHU5054 plasmid was transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing kanamycin, and incubated overnight at 37°C. Single colonies were picked and inoculated into 10 mL of LB medium (containing kanamycin) and incubated overnight at 37°C.

[0046] Transfer the overnight culture to 1 L LB medium (containing kanamycin) at a ratio of 1:100 and incubate at 37°C until OD500. 600 Approximately 0.6. The culture temperature was lowered to 16℃, and after the temperature stabilized, IPTG was added to a final concentration of 1 mM. Induction culture was continued at 16℃ and 180 rpm for 16 h.

[0047] 5.2 Collection and Disruption of Bacterial Cells Collect bacterial cells by centrifuging the culture at 4°C and 6000 × g for 15 min. Resuspend the bacterial pellet in pre-cooled lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) and add 10 mL of lysis buffer per gram of wet bacterial weight.

[0048] The resuspension was placed in an ice bath and homogenized using a high-pressure homogenizer at 4°C and 1000 bar for 5 min. The homogenized homogenate was centrifuged at 4°C and 10000 × g for 1 h to remove cell debris and insoluble impurities. The supernatant was filtered through a 0.45 μm filter membrane to obtain a clear crude enzyme solution.

[0049] 5.3 Nickel column affinity chromatography The crude enzyme solution was loaded onto a 5 mL HisTrap HP nickel column (GE Healthcare) equilibrated with lysis buffer at a flow rate of 1 mL / min. After loading, the column was washed with lysis buffer for 10 column volumes to remove unbound contaminating proteins.

[0050] To remove products and cofactors that may bind nonspecifically to AfhB protein, five column volumes were washed with lysis buffer containing 2 M urea and 2 M potassium bromide. Subsequently, gradient elution was performed with lysis buffer containing different concentrations of imidazole (50 mM, 100 mM, 200 mM, 300 mM, 500 mM), and each elution peak was collected.

[0051] 5.4 Size Exclusion Chromatography The AfhB protein peaks collected by nickel affinity chromatography were combined and concentrated to 2 mL using an ultrafiltration tube (10 kDa molecular weight cutoff). The concentrate was loaded onto a Superdex 200 Increase 10 / 300 GL column (GE Healthcare) equilibrated with gel filtration buffer (20 mM Tris-HCl, 150 mM NaCl, 5% glycerol, pH 8.0) at a flow rate of 0.5 mL / min, and each elution peak was collected.

[0052] 5.5 SDS-PAGE Validation Samples from each step were analyzed using 12% SDS-PAGE. The results are as follows: Figure 5 As shown, after two-step purification using nickel column affinity chromatography and size exclusion chromatography, a single protein band with a molecular weight of approximately 28 kDa was obtained, consistent with the theoretical molecular weight of AfhB protein (28.3 kDa). The gel filtration chromatogram showed a single symmetrical peak, indicating high protein purity.

[0053] 5.6 Protein Concentration and Preservation The purified AfhB protein was concentrated to approximately 10 mg / mL using ultrafiltration tubes, and the protein concentration was determined (Bradford method). After aliquoting, the protein was flash-frozen in liquid nitrogen and stored at -80°C for later use. Approximately 15 mg of high-purity AfhB protein was ultimately obtained from 1 L of culture.

[0054] Example 6: In vitro activity assay of AfhB.

[0055] 6.1 Preparation of cofactors Since AfhB is a flavin-dependent hydroxylase, it requires reduced FAD (FADH2) as a cofactor. This invention provides reduced FAD in two ways: Method 1 (Enzymatic Reduction): Following the method in Example 5, E. coli NAD(P)H-flavin reductase (Fre) was expressed and purified. The Fre protein can utilize NADH to reduce FAD, generating FADH2.

[0056] Method 2 (Chemical Reduction): Using Ti 3+As a chemical reducing agent, it directly reduces FAD to FADH2.

[0057] 6.2 Reaction System Reaction system A (enzymatic reduction): Prepare a 100 μL reaction mixture in a 1.5 mL centrifuge tube: dCMP: Final concentration 100 μM FAD: Final concentration 50 μM NADH: Final concentration 500 μM AfhB: Final concentration 1 μM (approximately 28 μg / mL) Fre: Final concentration 1 μM (approximately 26 μg / mL) Reaction buffer: 20 mM Tris-HCl, pH 8.0 Add sterile water to a final volume of 100 μL Reaction system B (chemical reduction): FAD (50 μM) was premixed with TiCl3 (500 μM) and incubated at room temperature for 5 min to reduce FAD to FADH2. Then dCMP (100 μM) and AfhB (1 μM) were added, and the volume was brought up to 100 μL with 20 mM Tris-HCl (pH 8.0).

[0058] Simultaneously set the following controls: Comparison 1: Without AfhB Control 2: Without dCMP Comparison 3: Without FAD Control 4: No reducing agent (NADH / Fre or Ti) added 3+ ) 6.3 Reaction conditions and sample preparation The reaction system was placed in a 37°C water bath for 1 h. After the reaction was complete, the reaction tube was immediately cooled on ice, and then added to a 10 kDa ultrafiltration membrane filter tube. The mixture was centrifuged at 12000 × g for 20 min at 4°C to remove proteins and other macromolecules. The filtrate was used directly for HPLC-MS / MS analysis.

[0059] 6.4 Test Results HPLC-MS / MS analysis results are as follows Figure 6 As shown, product peaks with retention times and mass spectrometric characteristics consistent with those of the 5hdCMP standard were detected in both complete reaction systems A and B. However, no 5hdCMP was detected in any of the control reactions (which lacked any key component).

[0060] Quantitative analysis showed that in the enzymatic reduction system (System A), approximately 35% of dCMP was converted to 5hdCMP after 1 h of reaction; in the chemical reduction system (System B), the conversion rate was approximately 28%. This result fully confirms that the AfhB protein can catalyze the production of 5hdCMP using dCMP as a substrate in the presence of FADH2.

[0061] Example 7: Determination of enzyme kinetics of AfhB.

[0062] 7.1 Optimization of Reaction Conditions To accurately determine the enzyme kinetic parameters of AfhB, the reaction conditions were first optimized. Preliminary experiments determined that the reaction rate was linearly related to the amount of enzyme and time under the following conditions: Reaction time: 30 min AfhB dosage: 4.7 μg (approximately 0.17 μM) Fre dosage: 1.8 μg (approximately 0.07 μM) FAD concentration: 50 μM (saturation concentration) NADH concentration: 500 μM (saturation concentration) Reaction temperature: 37℃ Buffer solution: 20 mM Tris-HCl, pH 8.0 7.2 Dynamics Experiment Prepare the reaction solution (100 μL) according to the following system: 20 mM Tris-HCl (pH 8.0) FAD: 50 μM NADH: 500 μM AfhB: 4.7 μg Fre: 1.8 μg dCMP: Add 10, 30, 50, 75, 100, 150, 200, and 250 μM respectively according to the concentration gradient. 7.3 Reaction Termination and Sample Preparation After the reaction was complete, immediately add 4 μL of trichloroacetic acid (100%, w / v), vortex to mix, and incubate on ice for 10 min to allow the protein to denature completely. Centrifuge at 15000 × g for 10 min at 4 °C to remove the protein precipitate, filter the supernatant through a 0.22 μm filter membrane, and transfer it to a vial.

[0063] 7.4 HPLC-UV Analysis Quantitative analysis of the reaction products was performed using HPLC-UV. Chromatographic conditions: C18 reversed-phase column (4.6 × 250 mm, 5 μm); mobile phase: 20 mM potassium phosphate buffer (pH 5.6); flow rate: 1 mL / min; column temperature: 30℃; detection wavelength: 260 nm; injection volume: 20 μL.

[0064] By comparing the 5hdCMP standard curve (which showed good linearity in the 0.5-200 μM range), R... 2 =0.999), calculate the content of 5hdCMP generated in each reaction.

[0065] 7.5 Data Processing The initial reaction rate data at different substrate concentrations were imported into GraphPad Prism software, and nonlinear fitting was performed using the Michaelis-Menten equation: V = Vmax × [S] / (Km + [S]) The calculated enzyme kinetic parameters are as follows: Km = 82.6 ± 6.4 μM Vmax = 0.63 ± 0.02 μM / min kcat = Vmax / [E] = 0.63 / 0.17 = 3.71 min -1 kcat / Km = 3.71 / 82.6 = 0.0449 μM -1 ·min -1 = (7.48 ± 0.44) × 10 -1 μM -1 ·min -1 The results are as follows Figure 7 As shown, AfhB exhibits high affinity and catalytic efficiency for dCMP, with kinetic parameters comparable to those of other reported flavin-dependent hydroxylases.

[0066] 7.6 Substrate Specificity Analysis To further investigate the substrate specificity of AfhB, this invention also tested other possible substrates, including CMP, dCDP, dCTP, cytidine, deoxycytidine, and methylated cytosine nucleotides. The results showed that AfhB exhibited significant catalytic activity only for dCMP, and essentially no activity for other substrates. This result indicates that AfhB possesses high substrate specificity, capable of precisely recognizing and catalyzing the conversion of dCMP to 5hdCMP.

[0067] Example 8: Cofactor Dependency Analysis of AfhB To gain a deeper understanding of the catalytic mechanism of AfhB, this embodiment systematically analyzes its cofactor dependence.

[0068] 8.1 Metal ion dependence Different metal ions (Mg) were added to the standard reaction system respectively. 2+ Mn 2+ Zn 2+ Ca 2+ Co 2+ Ni 2+ Fe 2+ Fe 3+ The concentration was increased to 1 mM, or EDTA (5 mM) was used to chelate any metal ions that might be present. The results showed that the activity of AfhB was not affected by EDTA, and the addition of exogenous metal ions did not have a significant promoting effect, indicating that AfhB is independent of metal ions.

[0069] 8.2 Flavin cofactor dependence FAD was replaced in the standard reaction system with FAD, FMN, or riboflavin, respectively. The results showed that FAD was the most effective cofactor (relative activity 100%), FMN also had some activity (approximately 32%), while riboflavin was essentially inactive. This indicates that AfhB prefers FAD as a cofactor.

[0070] 8.3 Reducing power dependence With different reducing powers (NADH, NADPH, Na2S2O4, Ti) 3+ The NADH / Fre system in the standard reaction system was replaced with ascorbic acid and DTT. The results showed that the NADH / Fre system had the highest efficiency, while the NADPH / Fre system had approximately 78% of the activity of the former. The chemical reducing agent Ti... 3+ Na₂S₂O₄ and Na₂S₂O₄ also effectively supported the reaction (relative activities of 82% and 67%, respectively), while ascorbic acid and DTT showed poor results. This indicates that AfhB requires a strong reducing agent to reduce FAD to FADH₂.

[0071] Example 9: pH and temperature stability of AfhB 9.1 Determination of Optimal pH AfhB activity was determined in different pH buffers: MES buffer for pH 5.0–6.0, MOPS buffer for pH 6.5–7.5, Tris-HCl buffer for pH 8.0–9.0, and CAPS buffer for pH 9.5–10.5. The results showed that the optimal reaction pH for AfhB was 8.0–8.5, with higher activity under neutral and weakly alkaline conditions. Activity decreased significantly at pH below 6.0 or above 9.5.

[0072] 9.2 pH stability AfhB protein was incubated at 4°C for 2 h in different pH buffers, and then the pH was adjusted to 8.0 to determine the remaining activity. The results showed that AfhB had good stability in the pH range of 7.0-9.0 (retaining more than 80% of its activity), while the activity was significantly reduced (less than 50% retained) after treatment with pH 6.0 or 10.0.

[0073] 9.3 Determination of Optimal Temperature The activity of AfhB was determined at different temperatures (4, 16, 25, 30, 37, 42, 50, and 60 °C). The results showed that the optimal reaction temperature for AfhB was 37 °C, with high activity in the range of 30-42 °C, and a sharp decrease in activity when the temperature exceeded 50 °C.

[0074] 9.4 Thermal stability AfhB protein was incubated at different temperatures for 30 min, then rapidly cooled to 4°C, and its remaining activity was measured at 37°C. The results showed that AfhB exhibited good stability below 37°C (retaining over 90% activity), retained approximately 75% activity after treatment at 42°C, and completely lost activity after treatment at 50°C. This result indicates that AfhB is a mesothermal enzyme and is not heat-resistant.

[0075] Example 10: Application of AfhB in the fermentation production of 5hdCMP 10.1 Shake-flask fermentation The JM109 engineered bacteria containing the pWHU5053 plasmid were inoculated into 50 mL of LB medium (containing chloramphenicol) and cultured overnight at 37°C. Then, at a 5% inoculum size, the culture was transferred to 500 mL of fermentation medium (LB medium supplemented with 0.5% glucose) and cultured at 37°C until OD500. 600 Add approximately 0.6 mg of IPTG to a final concentration of 0.5 mM, and induce culture at 25°C for 24 h.

[0076] 10.2 Product Extraction and Quantification After fermentation, the bacterial culture was centrifuged at 6000 × g for 10 min at 4℃ to collect the bacterial cells. The bacterial cells were washed twice with pre-cooled PBS, resuspended in 5 volumes of pre-cooled methanol, sonicated, and then incubated overnight at -80℃. The supernatant was collected by centrifugation, concentrated by nitrogen blowing, and reconstituted in 1 mL of water. The 5hdCMP content was quantitatively analyzed by HPLC-MS / MS.

[0077] The results showed that after 24 h of shake-flask fermentation, the yield of 5hdCMP reached 15.6 mg / L of fermentation broth.

[0078] 10.3 Optimization of Fermentation Conditions To further increase the yield of 5hdCMP, the fermentation conditions were optimized: Induction timing: OD600 IPTG was added at values ​​of 0.4, 0.6, 0.8, and 1.0 respectively. Induction temperatures: 16, 20, 25, 30, 37℃ IPTG concentrations: 0.1, 0.2, 0.5, 1.0, 2.0 mM Induction time: 6, 12, 24, 36, 48 h Culture media: LB, 2× YT, TB, M9 The optimized fermentation conditions are: OD 600 At approximately 0.8 mmol / L, 0.2 mM IPTG was added, and the mixture was induced at 20°C for 36 h using TB medium. Under these conditions, the yield of dCMP reached 42.3 mg / L after 5 h of shake-flask fermentation.

[0079] 10.4 Product Purification The fermentation extract was adsorbed onto an activated carbon column, and impurities were eluted sequentially with water, 10% methanol, and 30% methanol. Finally, the target product was eluted with 50% methanol. The target fraction was collected, concentrated under reduced pressure, and further purified by preparative HPLC to obtain 5hdCMP with a purity >95%, which was used for subsequent research and applications.

[0080] The above embodiments describe in detail the specific implementation of the present invention, fully demonstrating that the AfhB protein has the activity of catalyzing the conversion of dCMP to 5hdCMP, and the catalytic mechanism is as follows: Figure 8 As shown, 5hdCMP can be synthesized in a green manner through fermentation or enzymatic methods.

[0081] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A deoxycytidine monophosphate hydroxylase, characterized in that, Its amino acid sequence is shown below: 1 MGKHNTMKYI KAADFQSAFKAVNREILENP QFVTDSRIGR CNEIGSMTVV VDTPSSFKMT 61 DPRINRISYE YAEDFWKFMI SGGTDAKEAF KAYPNVAKFI SKPKSDALPA NFNTFYGPRI 121 AAQLPALLKE LKEKPNSRRV VFQILESSDQ ALLDSDETLE YPCTDSVTYY IRDGKLYTHC 181 HMRSQNCAVV MQLDFYLQGK LLHYIANECG VEVGDYTHTM VSAHVFERDF DYVKGFLD.

2. The deoxycytidine monophosphate hydroxylase according to claim 1, characterized in that, The deoxycytidine monophosphate hydroxylase is a derived protein of the amino acid sequence shown in claim 1, which has been substituted, deleted, or added with one or more amino acids and has dCMP hydroxylase activity.

3. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the deoxycytidine monophosphate hydroxylase according to claim 1 or 2, and the nucleotide sequence of the nucleic acid molecule is shown below: 1 ATGGGAAAAC ATAACACAAT GAAATATATT AAAGCCGCAG ATTTTCAATC CGCATTTAAA 61 GCTGTCAATC GTGAAATTTT AGAAAATCCA CAGTTCGTTA CTGATTCTCG TATTGGACGT 121 TGTAATGAAA TTTGGGTCTAT GACAGTAGTT GTTGATACCC CTTCATCATT CAAAATGACC 181 GACCCACGTA TTAATCGCAT CTCTTATGAA TATGCTGAAG ATTTCTGGAA ATTTATGATT 241 TCTGGTGGTA CTGATGCAAA AGAAGCATTT AAGGCATATC CTAACGTCGC TAAATTTTATT 301 TCTAAAACCTA AATCAGACGC TTTGCCTGCA AACTTCAACA CTTTCTATGG TCCTCGCATT361GCGGCCCAAT TACCAGCTCT TCTAAAAGAA CTTAAAGAGA AGCCTAACTC CCGAAGAGTT 421 GTGTTCAAA TTCTAGAAAG TTCCGATCAG GCTTTGCTTG ATTCAGACGA AACGCTTGAA 481 TATCCATGTA CTGACTCAGT GACTTATTAT ATTCGTGATG GTAAGCTTTA TACCCACTGT 541 CATATGCGTT CTCAGAACTG TGCTGTTGTT ATGCAGTTGG ATTTTTATCT CCAAGGAAAA 601 TTACTTCATT ATATCGCTAA CGAATGTGGT GTAGAAGTTG GTGATTATAC TCATACCATG 661 GTAAGTGCCC ATGTGTTTGA ACGTGATTTT GATTATGTTA AAGGATTCCT TGACTAA.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 3.

5. An engineered cell, characterized in that, The engineered cells comprise the recombinant expression vector of claim 4.

6. The engineered cell according to claim 5, characterized in that, The host cell of the engineered cells is a prokaryotic cell or a eukaryotic cell.

7. A method for producing 5-hydroxydeoxycytidine monophosphate (5hdCMP), characterized in that, The process includes the following steps: (1) culturing the engineered cells as described in claim 5 or 6 and inducing them to express deoxycytidine monophosphate hydroxylase; (2) collecting and isolating 5hdCMP from the culture.

8. A method for the in vitro enzymatic synthesis of 5-hydroxydeoxycytidine monophosphate (5hdCMP), characterized in that, Using dCMP as a substrate, a reaction is carried out in the presence of the deoxycytidine monophosphate hydroxylase as described in claim 1 or 2 and a cofactor to generate 5hdCMP.

9. The method according to claim 8, characterized in that, The cofactors include flavin adenine dinucleotide (FAD) and reducing power; the reducing power is provided by NADH and NAD(P)H-flavin reductase, or by Ti 3+ supply.

10. The use of the deoxycytidine monophosphate hydroxylase of claim 1 or 2, or the recombinant expression vector of claim 4, or the engineered cells of claim 5 or 6 in the preparation of a biological agent or kit for the production of 5-hydroxydeoxycytidine monophosphate (5hdCMP).

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