Method for producing pyrimidine nucleoside phosphorylase mutants and 2'-fluoronucleosides
A pyrimidine nucleoside phosphorylase mutant with targeted amino acid mutations addresses the low yield and high cost issues in producing 2'-fluoronucleosides by enhancing enzyme activity and efficiency, enabling cost-effective industrial-scale production.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- JINLIN ASYMCHEM PHARM CO LTD
- Filing Date
- 2023-10-31
- Publication Date
- 2026-07-02
AI Technical Summary
The existing methods for producing 2'-fluoro-modified nucleoside monomers, such as 2'-fluoro-2'-deoxyadenosine, face challenges including low yield, complex chemical synthesis processes, environmental pollution, and high production costs due to the use of organic reagents, as well as the inefficiency of wild-type pyrimidine nucleoside phosphorylase enzymes.
A pyrimidine nucleoside phosphorylase mutant with specific amino acid mutations, such as K80Q+G112T+A157T, is developed to catalyze the decomposition of substrate nucleosides into phosphorylated 2'-fluoropentose and a free base, followed by binding with purine nucleoside phosphorylase to produce 2'-fluoronucleosides, utilizing a one-pot method for improved enzyme activity and efficiency.
The mutant enzyme significantly enhances the conversion rate and reduces production costs, making it suitable for industrial-scale production of 2'-fluoronucleosides with higher yields and reduced enzyme usage.
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Figure 2026521943000001_ABST
Abstract
Description
Technical Field
[0001] (Cross - reference to related applications) This application claims priority based on a Chinese application with a CN application number of 202311341031.9 and a filing date of October 17, 2023. The disclosure content of this CN application is incorporated into this application as a whole again.
[0002] The present invention relates to the field of enzyme catalysis, specifically to a pyrimidine nucleoside phosphorylase mutant and a method for producing 2'-fluoro nucleoside.
Background Art
[0003] [[ID=,18]]Small nucleic acid drugs include antisense nucleic acids (ASO), small interfering RNAs (siRNA), and aptamers, etc. Their active components are mainly RNA and DNA, and they can specifically act on targets based on the principle of base - complementary pairing.
[0004] Currently, more than 10 small nucleic acid drugs have been approved for the market and are applied in the fields of ophthalmology, genetic diseases such as cardiovascular and metabolism, etc., According to clinical trials, small nucleic acid drugs can also be applied in many fields such as cancer, neurological diseases, respiratory systems, and infectious diseases, so they have broad application prospects. Small nucleic acid drugs need to perform specific chemical modifications to improve their stability and extend the half - life in the body. For example, when it is necessary to modify the 2'-position of ribose with fluorine, it is often required.
[0005] Therefore, as the market scale of small nucleic acid drugs continues to expand, the market demand for such important synthetic precursors of drugs, 2'-fluoro - modified nucleoside monomers, is also continuously increasing.
[0006] Currently, 2'-fluoro nucleoside monomers on the market are produced by chemical methods. However, the chemical synthesis process is relatively complicated, involving multiple steps, the protection and de - protection of multiple functional groups are required, and organic reagents are used, which pollutes the environment, etc., resulting in relatively high production costs.
[0007] Furthermore, 2′-fluoro-modified nucleoside monomers can be synthesized by biological methods. For example, 2′-fluoro-2′-deoxyadenosine can be synthesized from 2′-fluoro-2′-deoxyuridine and adenine using a one-pot method with pyrimidine nucleoside phosphorylase (PyNP) and purine nucleoside phosphorylase (PNP).
[0008] However, the reaction in which pyrimidine nucleoside phosphorylase (PyNP) catalyzes 2'-fluoro-2'-deoxyuridine to produce 2'-fluoro-2'-deoxyribose-1-phosphate and uracil is relatively inactive, which limits the commercial production of 2'-fluoro-modified nucleoside monomers using biological methods.
[0009] Szeker et al. found that using wild-type pyrimidine nucleoside phosphorylase (GsPyNP) from Geobacillus thermoglucosidasius, only 0.004 mM of 2′-fluoro-2′-deoxyuridine could be catalyzed within 0.5 h, while wild-type pyrimidine nucleoside phosphorylase (TtPyNP) from Thermus thermophilus could hydrolyze 0.156 mM of 2′-fluoro-2′-deoxyuridine. Under optimal conditions, even after 17 h of reaction using wild-type TtPyNP, only 0.65 mM of 2′-fluoro-2′-deoxyuridine could be catalyzed (Comparative investigations on thermostable pyrimidine nucleoside phosphorylases from Geobacillus thermoglucosidasius and Thermus thermophilus). [Overview of the Initiative] [Problems that the invention aims to solve]
[0010] The main objective of the present invention is to provide a pyrimidine nucleoside phosphorylase mutant and a method for producing 2'-fluoronucleosides, which solves the problem in conventional techniques of low yield of 2'-fluoronucleosides synthesized under catalytic conditions. [Means for solving the problem]
[0011] To achieve the above objective, according to a first aspect of the present invention, a pyrimidine nucleoside phosphorylase mutant is provided, (a) a protein in which a mutation has occurred based on the pyrimidine nucleoside phosphorylase wild-type enzyme TtPyNP represented by SEQ ID NO: 1, wherein the mutations are K80, V4, I7, R8, R17, E19, G27, R30, P34, L47, G49, L50, L58, D61, S91, F105, G112, T119, E135, A151, A157, V173 The proteins include proteins selected from one or more mutations among I179, I183, A189, A190, F206, M207, L215, G224, Q225, A227, V231, E251, A271, L274, R280, L281, L284, G285, R303, D329, V337, G355, and K360, or proteins having 70% or more homology to the amino acid sequences limited in (a) and having pyrimidine nucleoside phosphorylase activity.
[0012] Furthermore, in (a), the types of amino acids substituted at each site are, independently of each other, K80Q, K80Y, V4P, V4G, I7G, R8N, R8F, R17E, E19P, G27S, R30K, P34K, P34L, P34G, L47H, G49T, G49V, G49S, L50A, L50D, L50G, L50K, L50N , L50R, L50S, L58T, L58Q, D61T, S91A, S91V, S91C, F105I, F105T, G112S, G112T, G112A, G112L, G112C, T119N, E135K, E135A, A151S, A15 Select from 7D, A157N, A157T, V173S, V173T, I179V, I183V, A189V, A189T, A190T, A190V, A190I, F206M, M207L, M207R, L215I, G224D, Q225N, A227D, V231D, E251V, A271S, L274K, L274I, R280V, L281R, L284S, L284G, G285D, R303K, R303A, D329F, V337T, G355S, K360L, K360I, Here, the letter before the number represents the original amino acid, and the letter after the number represents the mutant amino acid.
[0013] Furthermore, mutations, K80Q+G112A、K80Q+G112C、K80Q+G112S、K80Q+G112T、L50A+K80Q+G112T、L50D+K80Q+G112T、L50G+K80Q+G112T、L50K+K80Q+G112T、L50N+K80Q+G112T、L50S+K80Q+G112T、K80Q+G112T+A157T、D61T+K80Q+G112T+A157T、E19P+K80Q+G112T+A157T、G27S+K80Q+G112T+A157T+L281R、G49S+K80Q+G112T+A157T、G49T+K80Q+G112T+A157T、G49V+K80Q+G112T+A157T、I7G+K80Q+G112T+A157T、K80Q+G112T+A157T+A190T+L281R、K80Q+G112T+A157T+A190V+L281R、K80Q+G112T+A157T+A271S+L281R、K80Q+G112T+A157T+L215I+L281R、K80Q+G112T+A157T+L274I+L281R、K80Q+G112T+A157T+L274K、K80Q+G112T+A157T+L281R、K80Q+G112T+A157T+L281R+G355S、K80Q+G112T+A157T+L281R+K360I、K80Q+G112T+A157T+L281R+V337T、K80Q+G112T+A157T+L284G、K80Q+G112T+A157T+L284S、K80Q+G112T+A157T+Q225N+L281R、K80Q+G112T+A157T+R280V、K80Q+G112T+A157T+R303K、K80Q+G112T+A157T+V173S、K80Q+G112T+A157T+V173T+A189V+L281R、K80Q+G112T+A157T+V173T+L281R、K80Q+G112T+A189V、K80Q+G112T+A190I、K80Q+G112T+A190T、K80Q+G112T+E135A+A157T+L281R、K80Q+G112T+E135K、K80Q+G112T+K360I、K80Q+G112T+V173T、L47H+K80Q+G112T+A157T、L50A+K80Q+G112T+A157T+V173T+L281R、L50K+K80Q+G112T+A157T+L281R、L50N+K80Q+G112T+A157T+L281R、L50R+K80Q+G112T+A157T、L58Q+K80Q+G112T+A157T、L58T+K80Q+G112T+A157T、P34G+K80Q+G112T+A157T、P34K+K80Q+G112T+A157T、P34L+K80Q+G112T+A157T+L281R、R17E+K80Q+G112T+A157T、V4G+K80Q+G112T+A157T+L281R、V4P+K80Q+G112T+A157T、V4P+L50R+G112C+I183V+A190T+L215I+L281R+K360L、E19P+P34K+F105I+A157N+V173S+F206M+V231D+L281R、K80Q+G112T+A157T+V173T+A227D+L281R、K80Q+G112T+A157T+V173T+G224D+L281R、K80Q+G112T+A157T+V173T+L281R+D329F、K80Q+G112T+A157T+V173T+L281R+G285D、K80Q+G112T+A157T+V173T+L281R+R303A、L47H+L58Q+S91V+T119N+A157T+M207R+G224D+A271S、R17E+R30K+G112L+E135K+A157D+I183V+L284G+K360I、R30K+K80Q+G112T+A157T+V173T+L281R、R8F+G27S+S91A+G112T+A151S+V173T+M207L+V337T、V4G+R8N+P34L+G49V+L50D+V173T+L215I+G355S、K80Q+G112S+I179V+A227D+L274K+R280V+R303A+K360L、K80Q+G112T+A157T+V173T+A189T+A227D+L281R、K80Q+G112T+A157T+V173T+A189V+A227D+L281R、K80Q+G112T+A157T+V173T+A190V+A227D+L281R、K80Q+G112T+A157T+V173T+F206M+A227D+L281R、K80Q+G112T+A157T+V173T+I179V+A227D+L281R、K80Q+G112T+A157T+V173T+I183V+A227D+L281R, K80Q+G112T+A157T+V173T+M207R+A227D+L281R, K80Q+G 112T+T119N+A157T+V173T+A227D+L281R, K80Y+F105T+G112A+A157T+V173T+A189T+Q225N+L274I, L50A+K 80Q+V173T+I183V+A189T+Q225N+L274I+G285D, L50G+K80Y+T119N+V173T+L215I+L284S+R303K+D329F, K8 0Q+G112T+T119N+A157T+V173T+I179V+A227D+E251V, K80Q+G112T+T119N+A157T+V173T+I179V+A227D+L28 1R, K80Q+G112T+T119N+A157T+V173T+I183V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A189T+A22 7D+L281R, K80Q+G112T+T119N+A157T+V173T+A189V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A190 It contains one of the following amino acid mutations: T+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A190V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+F206M+A227D+L281R, or K80Q+G112T+T119N+A157T+V173T+M207L+A227D+L281R.
[0014] Furthermore, the pyrimidine nucleoside phosphorylase mutant has 75% or more, 80% or more, 85% or more, more preferably 95% or more, homology to the amino acid sequence limited in (a), and includes a protein containing the pyrimidine nucleoside phosphorylase mutant.
[0015] To achieve the above objective, according to a second aspect of the present invention, a DNA molecule is provided which encodes the pyrimidine nucleoside phosphorylase mutant.
[0016] To achieve the above objective, according to a third aspect of the present invention, a recombinant plasmid is provided, to which the above-mentioned DNA molecule is ligated.
[0017] To achieve the above objective, according to a fourth aspect of the present invention, a host cell is provided, which contains the above-mentioned DNA molecule or recombinant plasmid.
[0018] Furthermore, the host cells include prokaryotic cells, preferably including E. coli.
[0019] To achieve the above objective, a fifth aspect of the present invention provides a method for producing 2'-fluoronucleosides, which comprises: a) using the pyrimidine nucleoside phosphorylase mutant to catalyze the decomposition of a substrate nucleoside having a 2'-fluoropentose structure into a phosphorylated 2'-fluoropentose and a free base; and b) using purine nucleoside phosphorylase to catalyze the binding of the phosphorylated 2'-fluoropentose to the substrate base, thereby preparing a 2'-fluoronucleoside.
[0020] Furthermore, the substrate nucleoside includes 2′-fluoro-2′-deoxyuridine, 2′-fluoro-2′-deoxycytidine, or 2′-deoxy-2′-fluorothymidine.
[0021] Furthermore, the substrate base contains modified or unmodified adenine, guanine, thymine, or cytosine, or contains modified uracil.
[0022] Furthermore, the 2'-fluoronucleoside includes nucleosides containing a 2'-fluoro-2'-deoxypentose structure. Preferably, the 2'-fluoronucleoside includes 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxy-2-aminoadenosine, 2'-fluoro-2'-deoxyguanosine, or 2'-fluoro-2'-deoxycytidine, or the 2'-fluoronucleoside is selected from 2'-fluoro-2'-deoxynucleosides having a substituent in the base structure.
[0023] To achieve the above object, according to a sixth aspect of the present invention, a method for producing phosphorylated 2'-fluoropentose is provided. This production method uses the above pyrimidine nucleoside phosphorylase mutant to catalyze the decomposition of a substrate nucleoside having a 2'-fluoropentose structure into phosphorylated 2'-fluoropentose and a free base.
[0024] Furthermore, the substrate nucleoside includes 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine, or 2'-deoxy-2'-fluorothymidine.
Effects of the Invention
[0025] When applying the technical solution of the present invention, by using the above pyrimidine nucleoside phosphorylase mutant, it is possible to catalyze the decomposition of a substrate nucleoside containing a 2'-fluoropentose structure into phosphorylated 2'-fluoropentose and a free base, and further prepare the target product 2'-fluoronucleoside. Compared with the wild-type enzyme, the above pyrimidine nucleoside phosphorylase mutant has relatively good enzyme activity, relatively high conversion rate and efficiency for producing 2'-fluoronucleoside products, and can significantly reduce the amount of enzyme used, thereby reducing production costs and being applicable to industrialized scale production.
Brief Description of the Drawings
[0026] The drawings of the specification that form part of this application are for providing a further understanding of the present invention. The exemplary embodiments and the description thereof of the present invention are for explaining the present invention and do not constitute an undue limitation of the present invention. In the drawings, [Figure 1] A schematic diagram of a chemical reaction is shown, which utilizes pyrimidine nucleoside phosphorylase according to an embodiment of the present invention to catalyze the decomposition of a substrate nucleoside having 2'-fluoropentose into phosphorylated 2'-fluoropentose and a free base. [Figure 2] A schematic diagram of a chemical reaction is shown, which utilizes purine nucleoside phosphorylase according to an embodiment of the present invention to catalyze the binding of phosphorylated 2'-fluoropentose and a substrate base to prepare 2'-fluoronucleoside. [Figure 3] An HPLC graph for producing 2'-fluoro-2'-deoxyadenosine using 2'-fluoro-2'-deoxyuridine and adenine as substrates according to Example 6 of the present invention is shown.
Modes for Carrying Out the Invention
[0027] In addition, the examples in this application and the features in the examples can be combined with each other without conflict. Hereinafter, the present invention will be described in detail based on the examples.
[0028] Interpretation of terms: 2'-Fluoronucleoside: A nucleoside in which the hydroxyl group on the carbon atom at the 2-position of pentose is substituted by a fluorine atom.
[0029] As described in the background art, in the prior art, both the production amount and the production volume of producing 2'-fluoronucleoside by utilizing enzyme catalysis are relatively low, and it is difficult to meet the requirements of industrial production. Therefore, in this application, the inventor attempts to develop a pyrimidine nucleoside phosphorylase mutant with higher enzyme activity that can efficiently produce the target product 2'-fluoronucleoside, and a series of protection cases of this application are proposed.
[0030] In a first typical embodiment of this application, a pyrimidine nucleoside phosphorylase mutant is provided, (a) a protein mutated from the pyrimidine nucleoside phosphorylase wild-type enzyme TtPyNP represented by SEQ ID NO: 1, wherein the mutations are K80, V4, I7, R8, R17, E19, G27, R30, P34, L47, G49, L50, L58, D61, S91, F105, A protein selected from one or more mutations among G112, T119, E135, A151, A157, V173, I179, I183, A189, A190, F206, M207, L215, G224, Q225, A227, V231, E251, A271, L274, R280, L281, L284, G285, R303, D329, V337, G355, K360, or (b) Contains a protein that has more than 70% homology to the amino acid sequence limited in (a) and has pyrimidine nucleoside phosphorylase activity.
[0031] In a preferred embodiment, the type of amino acid substituted at each site in (a) above is independently: K80Q, K80Y, V4P, V4G, I7G, R8N, R8F, R17E, E19P, G27S, R30K, P34K, P34L, P 34G, L47H, G49T, G49V, G49S, L50A, L50D, L50G, L50K, L50N, L50R, L50S, L58 T, L58Q, D61T, S91A, S91V, S91C, F105I, F105T, G112S, G112T, G112A, G112 L, G112C, T119N, E135K, E135A, A151S, A157D, A157N, A157T, V173S, V173T, Selected from I179V, I183V, A189V, A189T, A190T, A190V, A190I, F206M, M207L, M207R, L215I, G224D, Q225N, A227D, V231D, E251V, A271S, L274K, L274I, R280V, L281R, L284S, L284G, G285D, R303K, R303A, D329F, V337T, G355S, K360L, K360I, where the letter before the number represents the original amino acid and the letter after the number represents the mutant amino acid.
[0032] In a preferred embodiment, the mutation is K80Q+G112A、K80Q+G112C、K80Q+G112S、K80Q+G112T、L50A+K80Q+G112T、L50D+K80Q+G112T、L50G+K80Q+G112T、L50K+K80Q+G112T、L50N+K80Q+G112T、L50S+K80Q+G112T、K80Q+G112T+A157T、D61T+K80Q+G112T+A157T、E19P+K80Q+G112T+A157T、G27S+K80Q+G112T+A157T+L281R、G49S+K80Q+G112T+A157T、G49T+K80Q+G112T+A157T、G49V+K80Q+G112T+A157T、I7G+K80Q+G112T+A157T、K80Q+G112T+A157T+A190T+L281R、K80Q+G112T+A157T+A190V+L281R、K80Q+G112T+A157T+A271S+L281R、K80Q+G112T+A157T+L215I+L281R、K80Q+G112T+A157T+L274I+L281R、K80Q+G112T+A157T+L274K、K80Q+G112T+A157T+L281R、K80Q+G112T+A157T+L281R+G355S、K80Q+G112T+A157T+L281R+K360I、K80Q+G112T+A157T+L281R+V337T、K80Q+G112T+A157T+L284G、K80Q+G112T+A157T+L284S、K80Q+G112T+A157T+Q225N+L281R、K80Q+G112T+A157T+R280V、K80Q+G112T+A157T+R303K、K80Q+G112T+A157T+V173S、K80Q+G112T+A157T+V173T+A189V+L281R、K80Q+G112T+A157T+V173T+L281R、K80Q+G112T+A189V、K80Q+G112T+A190I、K80Q+G112T+A190T、K80Q+G112T+E135A+A157T+L281R、K80Q+G112T+E135K、K80Q+G112T+K360I、K80Q+G112T+V173T、L47H+K80Q+G112T+A157T、L50A+K80Q+G112T+A157T+V173T+L281R、L50K+K80Q+G112T+A157T+L281R、L50N+K80Q+G112T+A157T+L281R、L50R+K80Q+G112T+A157T、L58Q+K80Q+G112T+A157T、L58T+K80Q+G112T+A157T、P34G+K80Q+G112T+A157T、P34K+K80Q+G112T+A157T、P34L+K80Q+G112T+A157T+L281R、R17E+K80Q+G112T+A157T、V4G+K80Q+G112T+A157T+L281R、V4P+K80Q+G112T+A157T、V4P+L50R+G112C+I183V+A190T+L215I+L281R+K360L、E19P+P34K+F105I+A157N+V173S+F206M+V231D+L281R、K80Q+G112T+A157T+V173T+A227D+L281R、K80Q+G112T+A157T+V173T+G224D+L281R、K80Q+G112T+A157T+V173T+L281R+D329F、K80Q+G112T+A157T+V173T+L281R+G285D、K80Q+G112T+A157T+V173T+L281R+R303A、L47H+L58Q+S91V+T119N+A157T+M207R+G224D+A271S、R17E+R30K+G112L+E135K+A157D+I183V+L284G+K360I、R30K+K80Q+G112T+A157T+V173T+L281R、R8F+G27S+S91A+G112T+A151S+V173T+M207L+V337T、V4G+R8N+P34L+G49V+L50D+V173T+L215I+G355S、K80Q+G112S+I179V+A227D+L274K+R280V+R303A+K360L、K80Q+G112T+A157T+V173T+A189T+A227D+L281R、K80Q+G112T+A157T+V173T+A189V+A227D+L281R、K80Q+G112T+A157T+V173T+A190V+A227D+L281R、K80Q+G112T+A157T+V173T+F206M+A227D+L281R、K80Q+G112T+A157T+V173T+I179V+A227D+L281R、K80Q+G112T+A157T+V173T+I183V+A227D+L281R, K80Q+G112T+A157T+V173T+M207R+A227D+L281R, K80Q+G 112T+T119N+A157T+V173T+A227D+L281R, K80Y+F105T+G112A+A157T+V173T+A189T+Q225N+L274I, L50A+K 80Q+V173T+I183V+A189T+Q225N+L274I+G285D, L50G+K80Y+T119N+V173T+L215I+L284S+R303K+D329F, K8 0Q+G112T+T119N+A157T+V173T+I179V+A227D+E251V, K80Q+G112T+T119N+A157T+V173T+I179V+A227D+L28 1R, K80Q+G112T+T119N+A157T+V173T+I183V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A189T+A22 7D+L281R, K80Q+G112T+T119N+A157T+V173T+A189V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A190 It contains one of the following amino acid mutations: T+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A190V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+F206M+A227D+L281R, or K80Q+G112T+T119N+A157T+V173T+M207L+A227D+L281R.
[0033] Alternatively, it is a protein having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more homology to the above amino acid sequence.
[0034] In preferred embodiments, the pyrimidine nucleoside phosphorylase mutant has 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more homology to the amino acid sequence limited in (a), and includes a protein having the pyrimidine nucleoside phosphorylase mutant.
[0035] All of the above amino acid mutations were experimentally investigated in the examples of this application, and compared to the parent molecule having the amino acid sequence represented by Sequence ID No. 1, all of them have the activity to catalyze the decomposition of substrate nucleosides containing a 2′-fluoropentose structure into phosphorylated 2′-fluoropentoses and free bases. All of the above mutation sites are mutations that occur around the amino acid active site, and these mutations can improve the binding ability and / or catalytic ability between the mutant and the substrate.
[0036] However, because mutations that move away from the active site have a relatively small impact on the catalytic ability of the enzyme, it is possible to obtain proteins that have 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or more homology to the above amino acid sequence, and that have the same catalytic activity.
[0037] In this specification, homology (identity) refers to the "homology" between amino acid sequences, that is, the sum of the proportions of amino acid residues of the same type in the amino acid sequence. The homology of amino acid sequences can be determined using matching programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.
[0038] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more homology and the same function (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, and even 99.9% or more homology and the same function have an active site, active pocket, mechanism of action, protein structure, etc., that are all highly likely to be the same as the protein with sequence (a), and are homologous proteins obtained by amino acid mutation.
[0039] As used in this text, the abbreviations for amino acid residues are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).
[0040] The rules for substitution and replacement generally dictate that the effects of substitutions between amino acids with similar properties are similar. For example, conservative amino acid substitutions can occur in homologous proteins. "Conservative amino acid substitution" refers to... Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are substituted with other hydrophobic amino acids, The substitution of a side-chain hydrophobic amino acid (Phe, Tyr, Trp) with another side-chain hydrophobic amino acid, The substitution of amino acids with positively charged side chains (Arg, His, Lys) with other amino acids with positively charged side chains, This includes, but is not limited to, substitution of an amino acid with a polar, uncharged side chain (Ser, Thr, Asn, Gln) with another amino acid with a polar, uncharged side chain.
[0041] Those skilled in the art can conservatively substitute amino acids based on amino acid substitution rules well known to those skilled in the art, such as the "blosum62 score matrix" in the prior art.
[0042] The "AlphaFold2-Multimer" used in this application is an artificial intelligence model capable of predicting the disclosed protein complex conformation, and its predictions for protein three-dimensional structure can come close to the level observed using instruments such as cryo-electron microscopes in real-world testing. By obtaining relatively true protein structures, it is possible to guide the exploration of protein structure and protein activity.
[0043] A second typical embodiment of this application provides a DNA molecule encoding the pyrimidine nucleoside phosphorylase mutant. A third typical embodiment of this application provides a recombinant plasmid to which the above-mentioned DNA molecule is ligated.
[0044] The above DNA can encode the pyrimidine nucleoside phosphorylase mutant and can be ligated to a recombinant plasmid to form circular DNA. Both the above DNA and the recombinant plasmid can be transcribed and translated by the action of RNA polymerase, ribosomes, tRNA, etc., to obtain the pyrimidine nucleoside phosphorylase mutant.
[0045] A fourth typical embodiment of this application provides a host cell containing the above-mentioned DNA molecule or recombinant plasmid.
[0046] In preferred embodiments, the host cells include prokaryotic cells, and preferably, the prokaryotic cells include Escherichia coli.
[0047] By utilizing the above-mentioned host cells, recombinant plasmids can be replicated in the host cells, the DNA molecules attached to the recombinant plasmids can be transcribed and translated, and a large number of pyrimidine nucleoside phosphorylase mutants can be obtained. Using conventional techniques, the host cells can be purified to obtain lysated proteins, and after lysation, pyrimidine nucleoside phosphorylase mutants can be obtained using crude enzyme catalysis or other methods, and subsequent catalysis on substrate nucleosides can be performed. These host cells are non-plant derived host cells.
[0048] A fifth typical embodiment of this application provides a method for producing a 2'-fluoronucleoside, which includes a) catalyzing the decomposition of a substrate nucleoside having a 2'-fluoropentose structure into a phosphorylated 2'-fluoropentose and a free base using the pyrimidine nucleoside phosphorylase mutant, and b) preparing a 2'-fluoronucleoside by catalyzing the binding of the phosphorylated 2'-fluoropentose to the substrate base using purine nucleoside phosphorylase.
[0049] In the above manufacturing method, the purine nucleoside phosphorylase includes, but is not limited to, that derived from the thermophilic Bacillus stearothermophilus protein represented by Sequence ID No. 3 or its mutants (with homology of 80% or more), and also includes purine nucleoside phosphorylase (PNP) of other origins.
[0050] By utilizing the above manufacturing method, the pyrimidine nucleoside phosphorylase mutant (PyNP) can be used to perform the following catalysis, which is shown in Figure 1. The substrate nucleoside is prepared into a phosphorylated 2'-fluoropentose and a free base. The phosphate group in the phosphorylated 2'-fluoropentose originates from the phosphate in the catalytic system. Structures containing the substrate nucleoside include, but are not limited to, the structures shown in Figure 1.
[0051] By reusing purine nucleoside phosphorylase (PNP) from conventional techniques and substituting the phosphate group on the phosphorylated 2'-fluoropentose with the substrate base, a 2'-fluoronucleoside to which the substrate base is bound is obtained. The PNP used in this step can be flexibly selected from purine nucleoside phosphorylases from conventional techniques, and any of them can catalyze the progress of the reaction. A schematic diagram of the reaction is shown in Figure 2, and the structures of the substrate base and phosphorylated 2'-fluoropentose include, but are not limited to, the structures shown in Figure 2.
[0052] Furthermore, since steps a) and b) above can be carried out simultaneously in the same time, in the same container, and under the same reaction conditions, this manufacturing method enables the production of 2'-fluoronucleoside by a one-pot method. The conversion rate and efficiency of this manufacturing method are relatively high, and by significantly reducing the amount of enzyme used, production costs can be lowered and it can be applied to large-scale industrial production.
[0053] In preferred examples, the substrate nucleoside includes 2′-fluoro-2′-deoxyuridine, 2′-fluoro-2′-deoxycytidine, or 2′-deoxy-2′-fluorothymidine.
[0054] In preferred embodiments, the substrate base contains modified or unmodified adenine, guanine, thymine, or cytosine, or contains modified uracil.
[0055] In preferred embodiments, the 2′-fluoronucleoside comprises a nucleoside containing a 2′-fluoro-2′-deoxypentose structure, preferably the 2′-fluoronucleoside comprises 2′-fluoro-2′-deoxyadenosine, 2′-fluoro-2′-deoxy-2-aminoadenosine, 2′-fluoro-2′-deoxyguanosine, or 2′-fluoro-2′-deoxycytidine, or the 2′-fluoronucleoside is selected from 2′-fluoro-2′-deoxynucleosides having substituents on the base structure.
[0056] The above-mentioned substituents on the base structure or modifications to the substrate base include, but are not limited to, introducing a methyl group (-CH3), a carbamoyl group (-CH2NH2), a glycosyl group, a phosphonic acid group (-PO4), a mercapto group (-SH), a nitro group (-NO2), or a hydroxyl group (-OH) into the base structure, or deaminocing the base structure with a deaminocing group (-NH2).
[0057] Methylation is a modification that occurs when a nucleoside acid base is modified by the addition of a methyl group. For example, in DNA, cytosine bases can be methylated to form 5-methylcytosine.
[0058] Carbamoylation (aminomethylation): This is a modification of nucleoside bases by adding a carbamoyl group. Such modifications can occur with cytosine and adenine bases.
[0059] Glycosylation is a modification that occurs when a nucleoside acid base is modified by adding a glycosyl group (sugar moiety). For example, adenosine on an adenosine nucleoside acid can be glycosylated.
[0060] Phosphorylation is a modification of nucleoside bases by the addition of a phosphate group. Phosphorylation can occur in cytosine, adenine, and guanine bases.
[0061] Thiomination (sulfuration): This is a modification of nucleoside bases by the addition of a sulfur group. Thio modifications can occur in cytosine, adenine, and guanine bases.
[0062] Nitration is a modification that occurs when a nucleoside acid base is modified by adding a nitrate ester group. Such modifications can occur with cytosine and adenine bases.
[0063] Oxidation is a modification that occurs when an oxygen group is added to a nucleoside acid base. For example, in DNA, guanine bases are oxidatively modified to form 8-oxygen guanine (8-oxoG).
[0064] Deamination is a modification that occurs when a nucleoside acid base loses an amino group. Such modifications can occur in cytosine, adenine, and guanine bases.
[0065] A sixth typical embodiment of this application provides a method for producing a phosphorylated 2'-fluoropentose, which involves using the pyrimidine nucleoside phosphorylase mutant to catalyze the decomposition of a substrate nucleoside, which has a 2'-fluoropentose structure, into a phosphorylated 2'-fluoropentose and a free base.
[0066] In preferred examples, the substrate nucleoside includes 2′-fluoro-2′-deoxyuridine, 2′-fluoro-2′-deoxycytidine, or 2′-deoxy-2′-fluorothymidine.
[0067] The beneficial effects of this application will be interpreted in more detail below in conjunction with specific examples. (Example 1) Construction of TtPyNP and GsPNP expressing bacterial strains The protein sequence of pyrimidine nucleoside phosphorylase (TtPyNP) from Thermus thermophilus, obtained by NCBI (Accession No. BAD71594), is shown in SEQ ID NO: 1. After codon optimization, the DNA sequence encoding the enzyme was obtained, shown in SEQ ID NO: 2. This sequence was cloned into the expression carrier pET28a(+) to obtain the plasmid pET28a-TtPyNP. The pET28a-TtPyNP plasmid was transferred to the E. coli BL21(DE3) receiving state to obtain a monoclonal strain.
[0068] A monoclonal strain of pET28a-GsPNP (GsPNP, NCBI Accession No. BAA13510) was obtained using a similar method, and the GsPNP protein sequence is represented by Sequence ID No. 3.
[0069] Sequence ID 1: MNPVAFIREKREGKKHRREDLEAFLLGYLHDEVPDYQVSAWLMAAFLRGLDPEETLWLTETMARSGKVLDLSGLPHPVDQHSSGGVGDKVSLVVGPILAASGCTFA KMSGRTLAHTGGNIDKLESVPGWRGEMTEAEFLERARVGLVIAAQSPDLTPLDGKLYALRDVTATTESVPLIASSIMSKKLAVGARSIVLDVKVGRGAFMKTLEE ARLLAKTMVAIGQGDGRRVRALLTSMEAPLGRAVGNAIEVREAIEALKGEGPGDLLEVALALAEEALRREGLDPALARKALEGGAALEKFRAFLEAQGGDPRAVEDFSLLPLAEEHPLRAEREGVVREVDAYKVGLAVLALGGGRKRKGEPIDHGVGVYLLKKPGDRVERGEALALVYHRRRGLEEALGHLREAYALGEEAHPAPLVLEAI.
[0070] Sequence ID 2:
[0071] Sequence ID 3: MSVHIGAKEHEIADKILLPGDPLRAKYIAETFLEGATCYNQVRGMLGFTGTYKGHRISVQGTGMGVPSISIYITELMQSYNVQTLIRVGTCGAIQKDVKVRDVILAMTSSTDSQMNR MTFGGIDYAPTANFDLLKTAYEIGKEKGLQLKVGSVFTADMFYNENAQFEKLARYGVLAVEMETTALYTLAAKFGRKALSVLTVSDHILTGEETTAEERQTTFNEMIEVALETAIRQ.
[0072] (Example 2) Construction of TtPyNP mutant Saturation mutations or point mutations were performed by whole-plasmid PCR. The first mutation was performed using the pET28a-TtPyNP obtained in Example 1 as a template, and subsequent mutations were performed using the optimal mutants screened previously as templates. The PCR reaction conditions were 10 min pre-denaturation at 94°C, followed by 25 amplification cycles (30 s denaturation at 94°C, 6.5 min annealing extension at 68°C), and final extension at 68°C for 10 min. The obtained PCR products were digested with DpnI enzyme, transferred to E. coli BL21(DE3)-receptor cells, and cultured overnight at 37°C to obtain monoclonals.
[0073] (Example 3) Expression of TtPyNP mutant and preparation of enzyme solution Mutagenic expression: The mutant was inoculated into a test tube containing 5 mL of Luria-Bertani liquid medium (50 μg / mL kanamycin), incubated at 37°C for 16 hours, and then inoculated at a 1% dose into a 2 L vial containing 500 mL of Luria-Bertani liquid medium, and incubated at 37°C. 600 When the culture was maintained until the ratio reached 0.6, expression was finally induced by adding 0.1 M IPTG, and the incubation was carried out at 20°C for 18 hours. The cultured bacterial suspension was centrifuged at 7000 rpm for 10 minutes to collect the cells, which were then stored at -20°C before use.
[0074] Preparation of enzyme solution: 0.1 g of the bacterial sludge prepared as described above was weighed, resuspended in 1 mL of 100 mM potassium phosphate buffer with a pH of 7.5, and after uniform vibration, the bacterial cell suspension was disrupted using an ultrasonic disruptor (power: 30%, time: 5 min).
[0075] The expression of GsPNP and the preparation of the enzyme solution are the same as described above.
[0076] (Example 4) HPLC detection method HPLC detection method: An Atlantise T3 column, 4.6 mm x 150 mm, with a 0.1% TFA / methanol fluid phase, a flow rate of 1 mL / min, at 40°C, a UV detector, a detection wavelength of 254 nm, and a detection duration of 15 min. By utilizing these HPLC conditions, the conversion rate of 2′-fluoro-2′-deoxyuridine can be detected.
[0077] (Example 5) Reaction verification of TtPyNP mutant Using TtPyNP as a template, single-point and multi-point mutations were constructed and their reactions were validated. The reaction conditions were 20 mM 2′-fluoro-2′-deoxyuridine, 20 mM PBS (pH 7.0), 49 μL of enzyme solution, total volume 1 mL, 60°C, and 1 hour. Samples were taken and detected by HPLC, and the conversion rate of 2′-fluoro-2′-deoxyuridine is shown in Table 1. As a result, it was found that the activity containing the K80 mutation site was significantly improved, and the conversion rate to the final mutant K80Q+G112T+T119N+A157T+V173T+M207L+A227D+L281R could reach over 30%.
[0078] [Table 1] TIFF2026521943000003.tif147170
[0079] Note: In the table above, + represents a conversion rate of 1-10% (excluding the endpoint value of 10%), ++ represents a conversion rate of 10-15% (excluding the endpoint value of 15%), +++ represents a conversion rate of 15-20% (excluding the endpoint value of 20%), ++++ represents a conversion rate of 20-25% (excluding the endpoint value of 25%), +++++ represents a conversion rate of 25-30%, and ++++++ represents a conversion rate >30%.
[0080] (Example 6) TtPyNP mutant used in the synthesis of 2′-fluoro-2′-deoxyadenosine A 10 mL reaction system was prepared in 20 mM phosphate buffer (pH 7.5), containing two substrates: 100 mM 2′-fluoro-2′-deoxyuridine and 250 mM adenine, and two enzyme solutions: 1.07 mL of mutant K80Q+G112T+T119N+A157T+V173T+M207L+A227D+L281R enzyme solution and 4.3 mL of GsPNP enzyme solution. The reaction was carried out at 60°C for 137 hours.
[0081] After the reaction was complete, 1 mL of the reaction solution was taken, dissolved in an equal volume of DMSO, and diluted 25-fold before detection by HPLC. The HPLC spectrum is shown in Figure 3. The conversion rate of 2′-fluoro-2′-deoxyuridine was >80% (conversion and production of uracil), the conversion rate of adenine was >30%, and the production amount of 2′-fluoro-2′-deoxyadenosine was >20 g / L.
[0082] From the above, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects and can efficiently catalyze the decomposition of a substrate nucleoside containing a 2'-fluoropentose structure into phosphorylated 2'-fluoropentose and a free base using the pyrimidine nucleoside phosphorylase mutant.
[0083] Furthermore, by utilizing the above-mentioned pyrimidine nucleoside phosphorylase mutant and purine nucleoside phosphorylase from conventional technology, 2′-fluoronucleosides can be prepared in a one-pot method. Compared to the wild-type enzyme, the above-mentioned pyrimidine nucleoside phosphorylase mutant exhibits relatively good enzyme activity, resulting in a relatively high conversion rate and efficiency for producing 2′-fluoronucleoside products. This significantly reduces the amount of enzyme used, thereby lowering production costs and making it applicable to large-scale industrial production.
[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Those skilled in the art will know that various modifications and changes are possible to the present invention. Any modifications, equivalent substitutions, or improvements in the spirit and principles of the present invention must be included within the scope of protection of the present invention.
Claims
1. (a) A protein in which mutations have occurred based on the pyrimidine nucleoside phosphorylase wild-type enzyme TtPyNP represented by Sequence ID No. 1, wherein the mutations are K80, V4, I7, R8, R17, E19, G27, R30, P34, L47, G49, L50, L58, D61, S91, F105, G112, T119, E135, A151, A1 A protein that is one or more mutants selected from 57, V173, I179, I183, A189, A190, F206, M207, L215, G224, Q225, A227, V231, E251, A271, L274, R280, L281, L284, G285, R303, D329, V337, G355, K360, or (b) A pyrimidine nucleoside phosphorylase mutant characterized by containing a protein that has 70% or more homology to the amino acid sequence limited in (a) and has pyrimidine nucleoside phosphorylase activity.
2. In (a) above, the mutation is K80Q, K80Y, V4P, V4G, I7G, R8N, R8F, R17E, E19P, G27S, R30K, P34K, P34L, P34G, L47H, G49T, G49V, G49S, L50A, L50D, L50G, L50K, L50N, L50R , L50S, L58T, L58Q, D61T, S91A, S91V, S91C, F105I, F105T, G112S, G112T, G112A, G112L, G112C, T119N, E135K, E135A, A151S, A157D, A157N, It is one or more mutations selected from A157T, V173S, V173T, I179V, I183V, A189V, A189T, A190T, A190V, A190I, F206M, M207L, M207R, L215I, G224D, Q225N, A227D, V231D, E251V, A271S, L274K, L274I, R280V, L281R, L284S, L284G, G285D, R303K, R303A, D329F, V337T, G355S, K360L, K360I. The pyrimidine nucleoside phosphorylase mutant according to claim 1, characterized in that the alphabet before the number represents the original amino acid, and the alphabet after the number represents the mutant amino acid.
3. The aforementioned mutation is K80Q+G112A、K80Q+G112C、K80Q+G112S、K80Q+G112T、L50A+K80Q+G112T、L50D+K80Q+G112T、L50G+K80Q+G112T、L50K+K80Q+G112T、L50N+K80Q+G112T、L50S+K80Q+G112T、K80Q+G112T+A157T、D61T+K80Q+G112T+A157T、E19P+K80Q+G112T+A157T、G27S+K80Q+G112T+A157T+L281R、G49S+K80Q+G112T+A157T、G49T+K80Q+G112T+A157T、G49V+K80Q+G112T+A157T、I7G+K80Q+G112T+A157T、K80Q+G112T+A157T+A190T+L281R、K80Q+G112T+A157T+A190V+L281R、K80Q+G112T+A157T+A271S+L281R、K80Q+G112T+A157T+L215I+L281R、K80Q+G112T+A157T+L274I+L281R、K80Q+G112T+A157T+L274K、K80Q+G112T+A157T+L281R、K80Q+G112T+A157T+L281R+G355S、K80Q+G112T+A157T+L281R+K360I、K80Q+G112T+A157T+L281R+V337T、K80Q+G112T+A157T+L284G、K80Q+G112T+A157T+L284S、K80Q+G112T+A157T+Q225N+L281R、K80Q+G112T+A157T+R280V、K80Q+G112T+A157T+R303K、K80Q+G112T+A157T+V173S、K80Q+G112T+A157T+V173T+A189V+L281R、K80Q+G112T+A157T+V173T+L281R、K80Q+G112T+A189V、K80Q+G112T+A190I、K80Q+G112T+A190T、K80Q+G112T+E135A+A157T+L281R、K80Q+G112T+E135K、K80Q+G112T+K360I、K80Q+G112T+V173T、L47H+K80Q+G112T+A157T、L50A+K80Q+G112T+A157T+V173T+L281R、L50K+K80Q+G112T+A157T+L281R、L50N+K80Q+G112T+A157T+L281R、L50R+K80Q+G112T+A157T、L58Q+K80Q+G112T+A157T、L58T+K80Q+G112T+A157T、P34G+K80Q+G112T+A157T、P34K+K80Q+G112T+A157T、P34L+K80Q+G112T+A157T+L281R、R17E+K80Q+G112T+A157T、V4G+K80Q+G112T+A157T+L281R、V4P+K80Q+G112T+A157T、V4P+L50R+G112C+I183V+A190T+L215I+L281R+K360L、E19P+P34K+F105I+A157N+V173S+F206M+V231D+L281R、K80Q+G112T+A157T+V173T+A227D+L281R、K80Q+G112T+A157T+V173T+G224D+L281R、K80Q+G112T+A157T+V173T+L281R+D329F、K80Q+G112T+A157T+V173T+L281R+G285D、K80Q+G112T+A157T+V173T+L281R+R303A、L47H+L58Q+S91V+T119N+A157T+M207R+G224D+A271S、R17E+R30K+G112L+E135K+A157D+I183V+L284G+K360I、R30K+K80Q+G112T+A157T+V173T+L281R、R8F+G27S+S91A+G112T+A151S+V173T+M207L+V337T、V4G+R8N+P34L+G49V+L50D+V173T+L215I+G355S、K80Q+G112S+I179V+A227D+L274K+R280V+R303A+K360L、K80Q+G112T+A157T+V173T+A189T+A227D+L281R、K80Q+G112T+A157T+V173T+A189V+A227D+L281R、K80Q+G112T+A157T+V173T+A190V+A227D+L281R、K80Q+G112T+A157T+V173T+F206M+A227D+L281R、K80Q+G112T+A157T+V173T+I179V+A227D+L281R、K80Q+G112T+A157T+V173T+I183V+A227D+L281R, K80Q+G112T+A157T+V173T+M207R+A227D+L281R, K80Q+G112T+ T119N+A157T+V173T+A227D+L281R, K80Y+F105T+G112A+A157T+V173T+A189T+Q225N+L274I, L50A+K80Q+V173T+I 183V+A189T+Q225N+L274I+G285D, L50G+K80Y+T119N+V173T+L215I+L284S+R303K+D329F, K80Q+G112T+T119N+A 157T+V173T+I179V+A227D+E251V, K80Q+G112T+T119N+A157T+V173T+I179V+A227D+L281R, K80Q+G112T+T119N+A 157T+V173T+I183V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A189T+A227D+L281R, K80Q+G112T+T119N+ A157T+V173T+A189V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A190T+A227D+L281R, K80Q+G112T+T119N+ The pyrimidine nucleoside phosphorylase mutant according to claim 1, characterized by containing one amino acid mutation from among A157T+V173T+A190V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+F206M+A227D+L281R, or K80Q+G112T+T119N+A157T+V173T+M207L+A227D+L281R.
4. A DNA molecule characterized by encoding a pyrimidine nucleoside phosphorylase mutant according to any one of claims 1 to 3.
5. A recombinant plasmid characterized by having the DNA molecule described in claim 4 linked to it.
6. A host cell characterized by containing the DNA molecule described in claim 4 or the recombinant plasmid described in claim 5.
7. A method for producing 2'-fluoronucleosides, a) Using the pyrimidine nucleoside phosphorylase mutant described in any one of claims 1 to 3, catalyze the decomposition of a substrate nucleoside having a 2'-fluoropentose structure into a phosphorylated 2'-fluoropentose and a free base, b) A method for producing a 2'-fluoronucleoside, characterized by comprising using a purine nucleoside phosphorylase to catalyze the binding of the phosphorylated 2'-fluoropentose to a substrate base, thereby preparing the 2'-fluoronucleoside.
8. The production method according to claim 7, characterized in that the substrate nucleoside comprises 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine, or 2'-deoxy-2'-fluorothymidine.
9. The manufacturing method according to claim 7, characterized in that the substrate base contains modified or unmodified adenine, guanine, thymine, or cytosine, or contains modified uracil.
10. The method for producing a nucleoside according to any one of claims 7 to 9, characterized in that the 2'-fluoronucleoside includes a nucleoside containing a 2'-fluoro-2'-deoxypentose structure.
11. The 2'-fluoronucleoside includes 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxy-2-aminoadenosine, 2'-fluoro-2'-deoxyguanosine, or 2'-fluoro-2'-deoxycytidine, or The production method according to claim 10, characterized in that the 2'-fluoronucleoside is selected from 2'-fluoro-2'-deoxynucleosides having substituents in the base structure.
12. A method for producing phosphorylated 2'-fluoropentose, A method for producing a product, characterized by using a pyrimidine nucleoside phosphorylase mutant according to any one of claims 1 to 3, and catalyzing the decomposition of a substrate nucleoside having a 2'-fluoropentose structure into a phosphorylated 2'-fluoropentose and a free base.
13. The production method according to claim 12, characterized in that the substrate nucleoside includes 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine, or 2'-deoxy-2'-fluorothymidine.