Thymidine phosphorylase ancestor enzyme based on ancestor sequence reconstruction and application of thymidine phosphorylase ancestor enzyme

Abstract

The thymidine phosphorylase progenitor enzyme is provided on the basis of a strategy of combining progenitor enzyme reconstruction and catalytic efficiency prediction, and compared with a wild type, the thermal stability and catalytic activity of the thymidine phosphorylase progenitor enzyme are remarkably improved. The thymidine phosphorylase ancestor enzyme and purine nucleoside phosphorylase can be synthesized into 2 ' / 3'-modified purine nucleoside through a cascade transglycosylation reaction, and the thymidine phosphorylase ancestor enzyme has important significance in promoting the enzymatic synthesis industrialization process of the 2 ' / 3'-modified purine nucleoside.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and technology, specifically relating to the ancestral enzyme of thymidine phosphorylase reconstructed from ancestral sequence and its application. Background Technology

[0002] Nucleoside analogs are key small molecules in the life sciences field, widely used in the research and development of drugs for the treatment of cancer and viral infections. Nucleosides modified at the C2' / C3' position of the pentafuranose ring are core raw materials for the synthesis of therapeutic oligonucleotides, with modifications including amino, methoxy, fluorine, and hydroxyl (ara) atoms. Currently, the chemical synthesis of modified 2' / 3'-pyrimidine nucleosides has been industrialized; however, the chemical synthesis of 2' / 3'-modified purine nucleosides requires cumbersome protection / deprotection steps and inevitably generates α / β isomers, significantly increasing separation costs and limiting industrial applications.

[0003] In contrast, biological methods, due to their advantages such as mild reaction, single product, and environmental friendliness, are gradually replacing chemical methods as the preferred approach for the synthesis of 2' / 3'-modified purine nucleosides. The current mainstream pathway is a cascade transglycosylation reaction involving thymidine phosphorylase (TP) and purine nucleoside phosphorylase (PNP). Figure 1 Several patent documents have reported on this technology (such as CN111534493A, CN115851694A, CN 115725676A, CN 116855471A, CN 117070491A, CN 117106744A, CN120138086A, and CN 121182923A). Although enzymatic synthesis significantly reduces production costs, existing technologies still face key bottlenecks: wild-type TP enzymes have inherent defects such as poor thermostability and low catalytic activity, resulting in the need for large amounts of enzyme solution and reaction cycles exceeding one week in actual production, severely restricting production efficiency. Furthermore, from a cost control perspective, using crude enzyme preparations is more economical than purified enzymes, but the adenosine deaminase (ADA) naturally present in the host *E. coli* can trigger side reactions: catalyzing the irreversible conversion of 2' / 3'-modified adenosine to 2' / 3'-modified inosine, interfering with the synthesis of the target product. Although ADA has poor thermal stability, its interference can be eliminated by heating treatment, and PNP has excellent thermal stability and can still maintain high catalytic activity after heating treatment, wild-type TP enzyme has very poor thermal stability. The crude enzyme loses more than half of its enzyme activity after heating at 50°C for a few hours. At the same time, wild-type TP enzyme also has very low catalytic activity for non-natural substrates 2' / 3'-pyrimidine nucleosides.

[0004] Wild-type TP enzymes have catalytic performance that is insufficient to meet the needs of large-scale industrial production. Therefore, obtaining TP enzymes with both high thermal stability and high catalytic activity is crucial for promoting the industrialization of enzymatic synthesis of 2' / 3'-modified purine nucleosides. Summary of the Invention

[0005] To address the issues of poor thermal stability and low catalytic activity of thymidine phosphorylase, this invention provides a thymidine phosphorylase progenitor enzyme based on a strategy combining progenitor enzyme reconstruction and catalytic efficiency (Kcat / Km) prediction, along with its preparation method and applications. Compared to wild-type TP, its thermal stability and catalytic efficiency are significantly improved.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention uses thymidine phosphorylase (EcTP) from Escherichia coli as a starting template and employs FireProt... ASRThe ancestral thymidine phosphorylase sequence was generated using (https: / / loschmidt.chemi.muni.cz / fireprotasr / ), and candidate sequences with significantly enhanced catalytic activity were identified using Kcat / Km virtual screening (https: / / turnup.cs.hhu.de / Kcat_single_input). The top ten candidate ancestral TP sequences were heterologously expressed in *E. coli*. The amino acid sequence of the wild-type thymidine phosphorylase is shown in SEQ ID: NO.1, and the nucleotide sequence is shown in SEQ ID: NO.13. The thymidine phosphorylase progenitor enzymes include ASR76 (amino acid sequence as shown in SEQ ID: NO.3, nucleotide sequence as shown in SEQ ID: NO.15), ASR100 (amino acid sequence as shown in SEQ ID: NO.4, nucleotide sequence as shown in SEQ ID: NO.16), ASR102 (amino acid sequence as shown in SEQ ID: NO.5, nucleotide sequence as shown in SEQ ID: NO.17), ASR106 (amino acid sequence as shown in SEQ ID: NO.6, nucleotide sequence as shown in SEQ ID: NO.18), ASR113 (amino acid sequence as shown in SEQ ID: NO.7, nucleotide sequence as shown in SEQ ID: NO.19), ASR114 (amino acid sequence as shown in SEQ ID: NO.8, nucleotide sequence as shown in SEQ ID: NO.20), ASR115 (amino acid sequence as shown in SEQ ID: NO.9, nucleotide sequence as shown in SEQ ID: NO.21), and ASR120 (amino acid sequence as shown in SEQ ID: NO.10, nucleotide sequence as shown in SEQ ID: NO.22). The ancestral enzymes shown in SEQ ID NO: 8, ASR140 (amino acid sequence as shown in SEQ ID NO: 11, nucleotide sequence as shown in SEQ ID NO: 23), and ASR148 (amino acid sequence as shown in SEQ ID NO: 12, nucleotide sequence as shown in SEQ ID NO: 24) were screened for by evaluating their thermal stability and catalytic activity. An ancestral enzyme with significantly improved catalytic activity and thermal stability was obtained and named EcTP-ASR114, with an amino acid sequence as shown in SEQ ID NO: 8. This invention provides the gene encoding the ancestral enzyme of thymidine phosphorylase described in this invention, a recombinant vector containing the ancestral enzyme gene of thymidine phosphorylase, and a transformant of the recombinant vector.

[0008] This invention provides a method for preparing the recombinant vector, wherein the thymidine phosphorylase ancestor enzyme gene of this invention is prepared by artificial synthesis or gene cloning, an expression vector is constructed to obtain a recombinant plasmid, and the plasmid is transformed into a host cell. The expression vector can be a plasmid, bacteriophage, virus, or host cell, preferably pET28a-EcTP-ASR114. The host cell can be a prokaryotic or eukaryotic cell, and can be Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, or Trichoderma, preferably E. coli BL21(DE3).

[0009] This invention provides a high-density fermentation method for the ancestral enzyme of thymidine phosphorylase.

[0010] This invention provides a method for obtaining, processing, and preserving the crude enzyme solution of the thymidine phosphorylase ancestor enzyme.

[0011] The present invention also provides the application of the thymidine phosphorylase progenitor enzyme in the catalytic decomposition or synthesis of 2' / 3'-modified pyrimidine nucleosides.

[0012] The structure of the 2' / 3'-modified pyrimidine nucleoside is shown below:

[0013] R1 is selected from H, -OH or -NH2, and R2 is selected from -H, C1-C6 alkoxy groups, and halogens.

[0014] R1 is selected from H, -OH or -NH2, R2 is selected from -H, -OCH3, -F, -OH(ara), and R3 is selected from H or -CH3.

[0015] In a specific example of the present invention, the pyrimidine deoxynucleoside is selected from uridine, 2-deoxyuridine, 2',3'-dideoxyuridine, 3'-amino-2',3'-dideoxythymidine, vidaruridine, or 2'-fluoro-2'-deoxyuridine.

[0016] This invention also provides the application of the thymidine phosphorylase progenitor enzyme in the catalytic synthesis of 2' / 3'-modified purine nucleosides. The thymidine phosphorylase and purine nucleoside phosphorylase synthesize 2' / 3'-modified purine nucleosides through a cascade transglycosylation reaction. Specifically, the 2' / 3'-modified pyrimidine nucleosides are catalyzed and decomposed into the corresponding pyrimidine bases and 2' / 3'-modified ribose-1-phosphate by the thymidine phosphorylase progenitor enzyme in the presence of phosphate. The 2' / 3'-modified ribose-1-phosphate and purine bases are then catalyzed by purine nucleoside phosphorylase to synthesize 2' / 3'-modified purine nucleosides.

[0017] The 2' / 3'-modified purine nucleoside structure is shown below:

[0018] R1 is selected from H, -OH or -NH2, R2 is selected from -H, C1-C6 alkoxy, halogen or hydroxyl, and R4 is selected from H or -NH2. Preferably, R1 is selected from H, -OH or -NH2, and R2 is selected from -H, -OCH3, -F, -OH(ara).

[0019] In a specific example of the present invention, the 2' / 3'-modified purine nucleoside is selected from vidarabine, 2-aminovidarabine, 2',3'-dideoxyadenosine, 2-amino-2',3'-dideoxyadenosine, 3'-amino-2',3'-dideoxyadenosine, 2-amino-3'-amino-2',3'-dideoxyadenosine, 2'-fluoro-2'-deoxyadenosine, or 2-amino-2'-fluoro-2'-deoxyadenosine.

[0020] In the above applications, the thymidine phosphorylase progenitor enzyme is used in the form of enzyme solution, lyophilized enzyme powder, enzyme-containing cells, immobilized enzyme, or immobilized enzyme-containing cells. Preferably, it is the crude enzyme solution described in this invention.

[0021] The beneficial effects of this invention compared to the prior art are as follows:

[0022] Compared with existing technologies, the present invention has the following advantages: The present invention employs a strategy combining ancestral enzyme reconstruction and catalytic efficiency (Kcat / Km) prediction to obtain a thymidine ancestral enzyme, EcTP-ASR114, with significantly superior thermal stability and catalytic activity compared to wild-type thymidine phosphorylase. Compared to the wild-type enzyme, the thymidine phosphorylase ancestral enzyme EcTP-ASR114 exhibits a 2-fold increase in activity against the substrate 2-fluoro-2'-deoxyuridine, and a half-life at 50°C greater than 386 h, which is 386 times greater than the wild-type. The excellent catalytic efficiency and thermal stability of the thymidine ancestral enzyme EcTP-ASR114 obtained by the present invention make it more suitable for large-scale industrial production. Attached Figure Description

[0023] Figure 1 Pathway diagram for the synthesis of purine nucleosides via the cascade reaction of thymidine phosphorylase and purine nucleoside phosphorylase.

[0024] Figure 2 The results show the expression and purification of thymidine phosphorylase and its ancestral enzyme.

[0025] Figure 3 The results are the thermostability analysis results of the ancestral enzyme of thymidine phosphorylase. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0027] Example 1: Obtaining the progenitor enzyme sequence of thymidine phosphorylase

[0028] The amino acid sequence of thymidine phosphorylase from Escherichiacoli (LOCUS: UJY27368, amino acid sequence as shown in SEQ ID No. 1) was obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Using this sequence as a starting template, 74 thymidine phosphorylase progenitor sequences (EcTP-ASR76—EcTP-ASR149) were generated by inputting them into the FireProtASR website (https: / / loschmidt.chemi.muni.cz / fireprotasr / ). These 74 thymidine phosphorylase progenitor sequences were then input into the website (https: / / turnup.cs.hhu.de / Kcat_single_input), using 2'-fluoro-2'-deoxyuridine as a substrate and 2'-fluoro-2'-deoxynucleoside-1-phosphate as the product, and the corresponding Km and Kcat values ​​were calculated. Subsequently, the Kcat / Km ratios were sorted to obtain the top ten candidate thymidine phosphorylase progenitor enzymes (EcTP-ASR76, ASR100, ASR102, ASR106, ASR113, ASR114, ASR115, ASR120, ASR140, and ASR148), as shown in SEQ ID No. 3-No. 12. Then, the gene sequences of wild-type thymidine phosphorylase, wild-type purine nucleoside phosphorylase from *E. coli* (LOCUS: KIH37227, amino acid sequence shown in SEQ ID No. 2), and the ten thymidine phosphorylase progenitor enzymes were optimized using *E. coli* codons (nucleotide sequences shown in SEQ ID No. 13-No. 24). The optimized gene sequences were sent to Anhui General Biosystems Co., Ltd. for chemical synthesis, and the genes were constructed between the NdeI and XhoI restriction sites of the plasmid vector pET28(a)+.

[0029] Example 2: Construction of engineered Escherichia coli expressing thymidine phosphorylase, thymidine phosphorylase progenitor enzyme, and purine nucleoside phosphorylase.

[0030] The synthesized plasmid was transformed into competent E. coli BL21(DE3) cells using the calcium chloride heat shock method (42℃). Transformants were plated on LB agar plates containing 50 μg / mL kanamycin and cultured for 12 h. Recombinants were initially screened by colony PCR, followed by sequencing verification to obtain the correct genetically engineered strains: E. coli BL21(DE3) / pET28a(+)-EcTP expressing thymidine phosphorylase; E. coli BL21(DE3) / pET28a(+)-EcTP-ASR76, ASR100, ASR102, ASR113, ASR114, ASR115, ASR106, ASR120, ASR140, and ASR148 expressing thymidine phosphorylase progenitor enzymes; and E. coli expressing purine nucleoside phosphorylase. BL21(DE3) / pET28a(+)-EcPNP.

[0031] Example 3: Induced expression of recombinant thymidine phosphorylase, thymidine phosphorylase progenitor enzyme, and purine nucleoside phosphorylase by engineered bacteria

[0032] The engineered bacteria E. coli BL21(DE3) / pET28a(+)-EcTP, E. coli BL21(DE3) / pET28a(+)-EcTP-ASR76, ASR100, ASR102, ASR106, ASR113, ASR114, ASR115, ASR120, ASR140, ASR148 and E. coli BL21(DE3) / pET28a(+)-EcPNP constructed in Example 2 were inoculated into sterile tubes containing 5 mL of antibiotic-free LB culture medium. Kanamycin was added to a final concentration of 50 μg / mL, and the tubes were cultured in a shaker at 37°C and 200 rpm for more than 12 h to prepare seed culture. Take 4 mL of seed culture and inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of LB medium. Add kanamycin to a final concentration of 50 μg / mL. After culturing at 37℃ and 200 rpm for 2 h in a shaker, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.1 mmol / L for induction. Set the induction temperature to 25℃ and the induction time to 8 h. Centrifuge the induced bacterial culture at 10000 rpm for 10 min, remove the supernatant, wash off the medium, add 20 mL of 20 mM phosphate buffer (pH 7.0), resuspend, and sonicate to disrupt the cells. Set the sonication instrument power to 400 W, sonicate for 3 seconds, pause for 5 seconds, and disrupt the cells for 15 min. Centrifuge the disrupted bacterial culture at 12000 rpm for 10 min, and collect the supernatant as the crude enzyme. Store at 4℃ for later use.

[0033] Example 4: Purification of thymidine phosphorylase, thymidine phosphorylase progenitor enzyme, and purine nucleoside phosphorylase

[0034] The obtained crude enzyme solution was filtered through a 0.22 μm pore size filter and stored at 4°C as a purified sample. The nickel column was equilibrated by washing with at least 10 column volumes of binding buffer (25 mM imidazole, 50 mM PBS, pH 7.0) at a flow rate of 1.0 mL / min. The sample was then loaded onto the nickel column at a flow rate of 1.0 mL / min. Impurities were eluted with 5 column volumes of washing buffer (50 mM imidazole, 50 mM PBS, pH 7.0), followed by elution with 10 column volumes of elution buffer (250 mM imidazole, 50 mM PBS, pH 7.0). The collected protein solution was desalted and concentrated using a 10 kDa ultrafiltration tube. The purity of the purified enzyme was detected by SDS-PAGE. Finally, the protein concentration was measured using a BCA kit. The SDS-PAGE electrophoresis results are shown below. Figure 2 As shown: Recombinant proteins EcTP-ASR100 and EcTP-ASR148 did not show any expression of the target protein; recombinant proteins EcTP-ASR76, EcTP-ASR102, EcTP-ASR120, and EcTP-ASR140 could be expressed, with the products mainly being insoluble inclusion bodies and no significant soluble protein; recombinant proteins EcTP-ASR106, EcTP-ASR113, EcTP-ASR114, and EcTP-ASR115 could be expressed efficiently and solublely. Therefore, EcTP-ASR106, EcTP-ASR113, EcTP-ASR114, and EcTP-ASR115 were selected as candidate enzymes for subsequent enzyme activity and stability studies.

[0035] Example 5: Activities of thymidine phosphorylase and its progenitor enzyme on different substrates

[0036] The activities of the pure enzymes EcTP, EcTP-ASR106, EcTP-ASR113, EcTP-ASR114, and EcTP-ASR115 obtained in Example 4 were tested using different 2' / 3'-modified pyrimidine nucleosides (substrates: uridine, 2-deoxyuridine, 2',3'-dideoxyuridine, 3'-amino-2',3'-dideoxythymidine, vidaruridine, and 2'-fluoro-2'-deoxyuridine).

[0037] A 1 mL reaction system contained 10 mM of pyrimidine nucleoside substrates (uridine, 2'-deoxyuridine, 2',3'-dideoxyuridine, vidaruridine, 2'-fluoro-2'-deoxyuridine, 3'-amino-2',3'-dideoxyuridine), 1 / 15 M phosphate buffer (pH 7.0), and a certain amount of enzyme (2.5 µg of enzyme was added for substrates uridine, 2-deoxyuridine, 2',3'-dideoxyuridine, and 3'-amino-2',3'-dideoxyuridine; and 50 µg of enzyme was added for substrates vidaruridine and 2'-fluoro-2'-deoxyuridine). After incubation at 50 °C in a metal bath at 600 rpm for 10 min, samples were taken and incubated at 100 °C in a metal bath for 5 min to inactivate the enzymes. The activity of thymidine phosphorylase and its progenitor enzyme against different pyrimidine nucleoside substrates was calculated using high performance liquid chromatography (HPLC) to detect the amount of pyrimidine produced.

[0038] The high-performance liquid chromatography (HPLC) detection conditions were as follows: mobile phase: 15% methanol; flow rate: 1.0 mL / min; detection wavelength: 254 nm; detection time: 10 min; detection temperature: room temperature; column: C18 5 μm 250-4.6 mm.

[0039] Enzyme activity is defined as the amount of enzyme required to catalyze the phosphorylation of 1 μm pyrimidine nucleoside into 1 μm corresponding pyrimidine per minute at 50°C and with a 1 / 15 M phosphate buffer (pH 7.0).

[0040] The activity of thymidine phosphorylase and its progenitor enzyme against different pyrimidine nucleoside substrates is shown in Table 1.

[0041] Table 1. Activities of thymidine phosphorylase and its progenitor enzyme for different 2' / 3'-modified pyrimidine nucleoside substrates.

[0042] .

[0043] The results showed that the ancestral enzymes EcTP-ASR106, EcTP-ASR113, EcTP-ASR114, and EcTP-ASR115 were active for different 2' / 3'-modified pyrimidine nucleoside substrates. Among them, EcTP-ASR114 showed the best performance, with significantly improved phosphorylation activity for uridine, 2-deoxyuridine, 2',3'-dideoxyuridine, 3'-amino-2',3'-dideoxythymidine, ardaruridine, and 2'-fluoro-2'-deoxyuridine substrates compared to wild-type EcTP.

[0044] Example 6: Thermal stability of the progenitor enzyme of thymidine phosphorylase

[0045] The EcTP, EcTP-ASR106, EcTP-ASR113, EcTP-ASR114, and EcTP-ASR115 enzymes obtained in Example 4 were incubated in a metal bath at 50°C. Samples were taken at different times, and their remaining enzyme activity was measured using uridine as a substrate. The detection results are as follows: Figure 2 As shown, the thymidine progenitor enzymes EcTP-ASR113, EcTP-ASR114, and EcTP-ASR115 exhibited excellent thermal stability and thermal activation. Among them, the progenitor enzyme EcTP-ASR114 had a half-life exceeding 386 h at 50 °C, which is 386 times longer than that of EcTP (approximately 1 h). The progenitor enzyme EcTP-ASR114 showed significantly higher catalytic activity and thermal stability than wild-type EcTP, meeting industrial-scale catalytic performance requirements. Therefore, the subsequent synthesis of 2' / 3'-modified purine nucleosides was carried out using a cascade reaction of EcTP-ASR114 and EcPNP.

[0046] Example 7 High-density fermentation of thymidine phosphorylase progenitor enzyme EcTP-ASR114 and purine nucleoside phosphorylase

[0047] The engineered strains EcTP-ASR114 and EcPNP were inoculated into 2L of LB medium in a clean bench and cultured in a shaker at 37℃ and 200rpm for more than 12 hours to prepare seed culture.

[0048] The prepared seed culture was inoculated into a sterilized 50L fermenter, with 40L of fermentation broth containing 12 g / L peptone, 24 g / L yeast extract, 10 g / L glycerol, 0.5 g / L MgSO4·7H2O, 2.3 g / L KH2PO4, 9.5 g / L K2HPO4, 50 mg / L kanamycin, and 10 mL antifoaming agent. The pH was adjusted to 7.0 with ammonia. The following parameters were controlled during the culture process: the temperature was maintained at 37℃ using the machine, the pH was controlled at approximately 7.0 using ammonia, dissolved oxygen (DO) was maintained at 20%-30% by adjusting the stirring speed and aeration rate, and supplemented with fed culture medium (500 g / L glycerol, 12 g / L MgSO4·7H2O) until the OD reached approximately 600 to 10. The temperature was lowered to 25℃, and IPTG was added to a final concentration of 0.1 mmol / L for overnight induction (16-20 h). 600 Around 30 minutes, the induction process ends, and the bacterial cells are collected for tube centrifugation.

[0049] Example 8 Preparation of thymidine phosphorylase progenitor enzyme EcTP-ASR114 and crude purine nucleoside phosphorylase

[0050] Take one portion each of the EcTP-ASR114 and EcPNP wet bacterial sludge obtained in Example 7, and resuspend them in 1 / 15 M phosphate buffer (pH 7.0) at a ratio of 1 g wet bacterial sludge to 10 mL buffer. Hypolyze the resuspended bacterial solutions using a high-pressure homogenizer. It is recommended to set an appropriate pressure (e.g., 800-1000 bar) and cycle the homogenization 2-3 times. Transfer the resulting cell lysate to a water bath and heat at 50°C for 1 h. This step aims to selectively precipitate some heat-sensitive proteins and remove adenosine deaminase, which may affect subsequent reactions, by utilizing the difference in thermostability between the target enzyme and contaminating proteins. After heating, slowly add polyethyleneimine (PEI) stock solution to the sample until the final concentration is 0.1% (v / v). Gentle but continuous stirring is required during addition to ensure uniform dispersion of PEI. After addition, continue stirring at room temperature or allow to stand for 10 minutes to allow nucleic acids and acidic contaminating proteins to fully bind with PEI and form a precipitate.

[0051] Transfer the above mixture to suitable centrifuge tubes and perform solid-liquid separation using a tube centrifuge. The supernatant obtained after centrifugation is the crude enzyme solution containing EcTP-ASR114 and EcPNP. This crude enzyme solution is best used within one week; if used after one week, DTT and glycerol should be added and stored at 4°C.

[0052] Example 9: One-pot two-enzyme synthesis of vidarabine (120L) using EcTP-ASR114 and EcPNP

[0053] In a 150L reactor, 120L of reaction solution containing the following was added: a final concentration of 20mM phosphate buffer (pH 7.0); 1.5KU of EcTP-ASR114 and 4KU of crude EcPNP enzyme solution; 4395.6 g of vidarabine (150 mM) and 1621.6 g of adenine (100 mM). After 72 h of reaction, samples were taken for analysis, and the yield of vidarabine reached 2440.5 g (Table 2).

[0054] Example 10: One-pot two-enzyme synthesis of 2-aminoarabinoside (120L) using EcTP-ASR114 and EcPNP.

[0055] In a 150L reactor, 120L of reaction solution containing the following was added: a final concentration of 20mM phosphate buffer (pH 7.0); 1.5KU of EcTP-ASR114 and 4KU of crude EcPNP enzyme solution; 4395.6 g of uridine (150 mM) and 1801.7 g of 2-aminoadenosine (100 mM). After 72 h of reaction, samples were taken for analysis, and the yield of 2-aminoadenosine reached 2889.1 g (Table 2).

[0056] Example 11: One-pot two-enzyme synthesis of 2',3'-dideoxyadenosine (120 L) using EcTP-ASR114 and EcPNP.

[0057] In a 150L reactor, 120L of reaction solution was added containing the following: a final concentration of 20mM phosphate buffer (pH 7.0); 1.5KU of EcTP-ASR114 and 4KU of crude EcPNP enzyme solution; 3819.6 g of 2',3'-dideoxyuridine (150 mM) and 1621.6 g of adenine (100 mM). After 72 h of reaction, samples were taken for analysis, and the yield of 2',3'-dideoxyadenosine reached 2272.0 g (Table 2).

[0058] Example 12: One-pot two-enzyme synthesis of 2-amino-2',3'-dideoxyadenosine (120 L) using EcTP-ASR114 and EcPNP.

[0059] In a 150L reactor, 120L of reaction solution was added containing the following: a final concentration of 20mM phosphate buffer (pH 7.0); 1.5KU of EcTP-ASR114 and 4KU of crude EcPNP enzyme solution; 3819.6 g of 2',3'-dideoxyuridine (150 mM) and 1801.7 g of 2-amino-2',3'-dideoxyadenosine (100 mM). After 72 h of reaction, samples were taken for analysis, and the yield of 2-amino-2',3'-dideoxyadenosine reached 2645.1 g (Table 2).

[0060] Example 13: One-pot two-enzyme synthesis of 3'-amino-2',3'-dideoxyadenosine (120L) using EcTP-ASR114 and EcPNP.

[0061] In a 150L reactor, 120L of reaction solution containing the following was added: a final concentration of 20mM phosphate buffer (pH 7.0); 1.5KU of EcTP-ASR114 and 4KU of crude EcPNP enzyme solution; 4342.3 g of 3'-amino-2',3'-dideoxythymidine (150 mM) and 1621.6 g of adenine (100 mM). After 72 h of reaction, samples were taken for analysis, and the yield of 3'-amino-2',3'-dideoxythymidine reached 2543.0 g (Table 2).

[0062] Example 14: One-pot two-enzyme synthesis of 2-amino-3'-amino-2',3'-dideoxyadenosine (120L) using EcTP-ASR114 and EcPNP.

[0063] In a 150L reactor, 120L of reaction solution was added containing the following: a final concentration of 20mM phosphate buffer (pH 7.0); 1.5KU of EcTP-ASR114 and 4KU of crude EcPNP enzyme solution; 4342.3 g of 3'-amino-2',3'-dideoxythymidine (150 mM) and 1801.7 g of 2-aminoadenine (100 mM). After 72 h of reaction, samples were taken for analysis, and the yield of 2-amino-3'-amino-2',3'-dideoxyadenine reached 2620.4 g (Table 2).

[0064] Example 15: One-pot two-enzyme synthesis of 2'-fluoro-2'-deoxyadenosine (120 L) using EcTP-ASR114 and EcPNP.

[0065] In a 150L reactor, 120L of reaction solution containing the following was added: a final concentration of 20mM phosphate buffer (pH 7.0); 1.5KU of EcTP-ASR114 and 4KU of crude EcPNP enzyme solution; 4431.4 g of 2'-fluoro-2'-deoxyuridine (150 mM) and 1621.6 g of adenine (100 mM). After 72 h of reaction, samples were taken for analysis, and the yield of 2'-fluoro-2'-deoxyadenosine reached 2665.0 g (Table 2).

[0066] Example 16: One-pot two-enzyme synthesis of 2-amino-2'-fluoro-2'-deoxyadenosine (120L) using EcTP-ASR114 and EcPNP.

[0067] In a 150L reactor, 120L of reaction solution was added containing the following: a final concentration of 20mM phosphate buffer (pH 7.0); 1.5KU of EcTP-ASR114 and 4KU of crude EcPNP enzyme solution; 4431.4 g of 2'-fluoro-2'-deoxyuridine (150 mM) and 1801.7 g of 2-aminoadenine (100 mM). After 72 h of reaction, samples were taken for analysis, and the yield of 2-amino-2'-fluoro-2'-deoxyadenine reached 2857.8 g (Table 2).

[0068] Table 2. One-pot two-enzyme synthesis of different purine nucleosides using EcTP-ASR114 and EcPNP (120L)

[0069] .

[0070] The above results indicate that the present invention provides a thymidine phosphorylase ancestor enzyme EcTP-ASR114 based on a strategy combining ancestor enzyme reconstruction and catalytic efficiency (Kcat / Km) prediction. Compared with wild-type TP, it has significantly improved thermal stability and catalytic efficiency, and can meet the industrial kg-scale production requirements of 2' / 3'-modified purine nucleosides.

[0071] It should be noted that this embodiment is only used to clearly illustrate the technical solution of this application and is not intended to limit the scope of protection of this application. Within the scope of the technical concept disclosed in this application, those skilled in the art can deduce or make several adjustments and improvements to the above embodiments according to actual needs, and these modifications should all be considered to fall within the protection scope of the claims of this application.

Claims

1. A ancestral enzyme of thymidine phosphorylase reconstructed from an ancestral sequence, characterized in that, The amino acid sequence is shown in SEQ ID NO:

8.

2. Encoding the thymidine phosphorylase ancestor enzyme gene according to claim 1.

3. A recombinant expression vector comprising the gene as described in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is a plasmid or a virus.

5. A host cell comprising the gene of claim 2, characterized in that, The host cells are selected from Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, or Trichoderma.

6. The application of the thymidine phosphorylase progenitor enzyme according to claim 1 in the catalytic decomposition or synthesis of 2' / 3'-modified pyrimidine nucleosides, wherein the structure of the 2' / 3'-modified pyrimidine nucleosides is shown below. R1 is selected from H, -OH or -NH2, R2 is selected from H, -OH, C1-C6 alkoxy or halogen, and R3 is selected from H or C1-C6 alkyl.

7. The application according to claim 6, characterized in that... R1 is selected from -OH or -NH2, and R2 is selected from -H or -OCH. 3, -F, -OH(ara), R3 is selected from H, -CH3.

8. The application of the thymidine phosphorylase progenitor enzyme according to claim 1 in the catalytic synthesis of 2' / 3'-modified purine nucleosides, wherein the thymidine phosphorylase and purine nucleoside phosphorylase synthesize 2' / 3'-modified purine nucleosides through a cascade transglycosylation reaction, specifically, the 2' / 3'-modified pyrimidine nucleosides are catalyzed and decomposed into the corresponding pyrimidine bases and 2' / 3'-modified ribose-1-phosphate by the thymidine phosphorylase progenitor enzyme in the presence of phosphate, and the 2' / 3'-modified ribose-1-phosphate and purine bases are catalyzed by purine nucleoside phosphorylase to synthesize 2' / 3'-modified purine nucleosides.

9. The application according to claim 9, characterized in that... The 2' / 3'-modified purine nucleoside structure is shown below. R1 is selected from H, -OH or -NH2, R2 is selected from H, -OH, C1-C6 alkoxy or halogen, and R4 is selected from H or -NH2.

10. The application according to claim 9, characterized in that... R1 is selected from H, -OH or -NH2, and R2 is selected from -H, -OCH3, -F, -OH (ara).

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