Pyrimidine nucleoside phosphorylase mutant and preparation method for 2'-fluoro nucleoside

Abstract

Provided in the present invention are a pyrimidine nucleoside phosphorylase mutant and a preparation method for 2'-fluoro nucleoside. The pyrimidine nucleoside phosphorylase mutant comprises: (a) a mutant protein generated on the basis of a pyrimidine nucleoside phosphorylase wild-type enzyme TtPyNP as shown in SEQ ID NO: 1, the mutation being selected from any one or more of of the following mutations: K80 etc.; or (b) a protein having 70% or more homology with the amino acid sequence defined in (a) and having purine nucleoside phosphorylase activity. The method can solve the problem in the prior art of low yield of 2'-fluoro nucleoside synthesized by enzyme catalysis, and is suitable for the field of enzyme catalysis.

Description

Preparation method of pyrimidine nucleoside phosphorylase mutant and 2'-fluoronucleoside

[0001] This application is based on the Chinese application with CN application number 202311341031.9 and application date October 17, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of enzyme catalysis, and in particular to a preparation method of a pyrimidine nucleoside phosphorylase mutant and a 2'-fluoronucleoside. Background Art

[0003] Small nucleic acid drugs include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and aptamers. Their active ingredients are primarily RNA and DNA, and they can specifically target their targets based on the principle of complementary base pairing. Currently, more than 10 small nucleic acid drugs have been approved for marketing, with applications in ophthalmology and genetic diseases such as cardiovascular and metabolic diseases. Clinical trials have shown that small nucleic acid drugs can also be used in a variety of areas, including cancer, neurological diseases, respiratory diseases, and infectious diseases, thus holding broad application prospects. Small nucleic acid drugs often require specific chemical modifications to improve their stability and extend their in vivo half-life, such as fluorination of the 2' position of the ribose sugar. Therefore, as the market size of small nucleic acid drugs continues to expand, the market demand for 2'-fluorinated nucleoside monomers, an important synthetic precursor for such drugs, has also continued to grow.

[0004] At present, 2'-fluoro nucleoside monomers are produced by chemical methods on the market. However, the chemical synthesis process is relatively cumbersome, involves multiple steps, requires protection and deprotection of multiple functional groups, and requires the use of organic reagents that pollute the environment, resulting in high production costs. In addition, 2'-fluoro modified nucleoside monomers can also be synthesized by biological methods, such as using pyrimidine nucleoside phosphorylase (PyNP) and purine nucleoside phosphorylase (PNP) to synthesize 2'-fluoro-2'-deoxyuridine and adenine in a one-pot method to synthesize 2'-fluoro-2'-deoxyadenosine. However, the reaction of 2'-fluoro-2'-deoxyuridine to 2'-fluoro-2'-deoxyribose-1-phosphate and uracil catalyzed by pyrimidine nucleoside phosphorylase (PyNP) is poor due to the poor activity of the enzyme, thus limiting the commercial production of 2'-fluoro modified nucleoside monomers synthesized by biological methods. Szeker et al. used wild-type pyrimidine nucleoside phosphorylase (GsPyNP) from Geobacillus thermoglucosidasius to hydrolyze only 0.004 mM of 2'-fluoro-2'-deoxyuridine in 0.5 h, while wild-type pyrimidine nucleoside phosphorylase (TtPyNP) from Thermus thermophilus hydrolyzed 0.156 mM of 2'-fluoro-2'-deoxyuridine. Under optimal conditions, wild-type TtPyNP hydrolyzed only 0.65 mM of 2'-fluoro-2'-deoxyuridine after 17 h (Comparative investigations on thermostable pyrimidine nucleosidephosphorylases from Geobacillus thermoglucosidasius and Thermus thermophilus).

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a pyrimidine nucleoside phosphorylase mutant and a preparation method of 2'-fluoronucleosides, so as to solve the problem of low yield of 2'-fluoronucleosides synthesized by enzyme in the prior art.

[0007] To achieve the above object, according to a first aspect of the present invention, a pyrimidine nucleoside phosphorylase mutant is provided, comprising: (a) a protein having a mutation based on the wild-type pyrimidine nucleoside phosphorylase TtPyNP shown in SEQ ID NO: 1, wherein the mutation is selected from any one or more of the following mutations: K80, V4, I7, R8, R17, E19, G27, R30, P34, L47, G49, L50, L58, D61, S91, F105, G112, T119, E135, A151, A157, V173, I179, I181, I190, I201, I213, I224, I236, I247, I250, I261, I273, I284, I291, I309, I310, I321, I330, I341, I351, I360, I371, I380, I391, I403, I411, I423, I431, I443, I454, I461, I471, I483, I491, I491, I492, I493, I494, I495, I50 or (b) a protein that has more than 70% homology to the amino acid sequence defined in (a) and has pyrimidine nucleoside phosphorylase activity.

[0008] Furthermore, in (a), the type of amino acid substituted at each site is independently selected from the following:

[0009] K80Q, K80Y, V4P, V4G, I7G, R8N, R8F, R17E, E19P, G27S, R30K, P34K, P34L, P34G, L47H, G49T, G49V, G49S, L50A, L50D, L50G, L50K, L50 N, L50R, L50S, L58T, L58Q, D61T, S91A, S91V, S91C, F105I, F105T, G112S, G112T, G112A, G112L, G112C, T119N, E135K, E135A, A151S, A157D, 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;

[0010] The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0011] Furthermore, the mutation includes any of the following amino acid mutations:

[0012] 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, K80 Q+G112T+T119N+A157T+V173T+A227D+L281R, K80Y+F105T+G112A+A157T+V173T+A189T+Q225N+L274I, L 50A+K80Q+V173T+I183V+A189T+Q225N+L274I+G285D, L50G+K80Y+T119N+V173T+L215I+L284S+R303K+ D329F、K80Q+G112T+T119N+A157T+V173T+I179V+A227D+E251V、K80Q+G112T+T119N+A157T+V173T+I179 V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+I183V+A227D+L281R, K80Q+G112T+T119N+A157T+V 173T+A189T+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A189V+A227D+L281R, K80Q+G112T+T119N +A157T+V173T+A190T+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A190V+A227D+L281R, K80Q+G11 2T+T119N+A157T+V173T+F206M+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+M207L+A227D+L281R. ,

[0013] Furthermore, pyrimidine nucleoside phosphorylase mutants include proteins having 75% or more, 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology to the amino acid sequence defined in (a).

[0014] In order to achieve the above object, according to a second aspect of the present invention, a DNA molecule is provided, which encodes the above pyrimidine nucleoside phosphorylase mutant.

[0015] In order to achieve the above object, according to the third aspect of the present invention, a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.

[0016] In order to achieve the above object, according to a fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the above DNA molecule or recombinant plasmid.

[0017] Further, the host cell includes a prokaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.

[0018] To achieve the above-mentioned object, according to a fifth aspect of the present invention, a method for preparing 2'-fluoronucleosides is provided, which comprises: a) utilizing the above-mentioned pyrimidine nucleoside phosphorylase mutant to catalyze the cleavage of a substrate nucleoside into a phosphorylated 2'-fluoropentose and a free base, wherein the substrate nucleoside has a 2'-fluoropentose structure; and b) utilizing a purine nucleoside phosphorylase to catalyze the binding of the phosphorylated 2'-fluoropentose and the substrate base to prepare the 2'-fluoronucleoside.

[0019] Furthermore, the substrate nucleoside includes 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine or 2'-deoxy-2'-fluorothymidine.

[0020] Furthermore, the substrate base includes modified or unmodified adenine, guanine, thymine or cytosine, or modified uracil.

[0021] Furthermore, the 2'-fluoro nucleoside includes a nucleoside containing a 2'-fluoro-2'-deoxypentose structure. Preferably, the 2'-fluoro nucleoside includes 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxy-2-aminoadenosine, 2'-fluoro-2'-deoxyguanosine or 2'-fluoro-2'-deoxycytidine; or, the 2'-fluoro nucleoside is selected from a 2'-fluoro-2'-deoxynucleoside having a substituent on the base structure.

[0022] In order to achieve the above-mentioned object, according to the sixth aspect of the present invention, a method for preparing phosphorylated 2'-fluoropentose is provided, which comprises: utilizing the above-mentioned pyrimidine nucleoside phosphorylase mutant to catalyze the cleavage of substrate nucleoside into phosphorylated 2'-fluoropentose and free base, wherein the substrate nucleoside is a 2'-fluoropentose structure.

[0023] Furthermore, the substrate nucleoside includes 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine or 2'-deoxy-2'-fluorothymidine.

[0024] The technical solution of the present invention utilizes the aforementioned pyrimidine nucleoside phosphorylase mutant to catalyze the cleavage of substrate nucleosides containing a 2'-fluoropentose structure into phosphorylated 2'-fluoropentose and a free base, thereby producing the target 2'-fluoronucleoside product. Compared to the wild-type enzyme, the pyrimidine nucleoside phosphorylase mutant exhibits superior enzymatic activity and a higher conversion rate and efficiency in producing 2'-fluoronucleoside products. This significantly reduces enzyme usage, thereby lowering production costs and making it suitable for industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0026] FIG1 shows a schematic diagram of a chemical reaction in which a pyrimidine nucleoside phosphorylase is used to catalyze the cleavage of a substrate nucleoside having a 2'-fluoropentose into a phosphorylated 2'-fluoropentose and a free base according to an embodiment of the present invention.

[0027] FIG2 shows a schematic diagram of a chemical reaction for preparing 2'-fluoronucleosides by using purine nucleoside phosphorylase to catalyze the phosphorylation of 2'-fluoropentose and the combination with the substrate base according to an embodiment of the present invention.

[0028] FIG3 shows an HPLC spectrum of preparing 2'-fluoro-2'-deoxyadenosine using 2'-fluoro-2'-deoxyuridine and adenine as substrates according to Example 6 of the present invention. DETAILED DESCRIPTION

[0029] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0030] Explanation of terms:

[0031] 2'-Fluoronucleoside: A nucleoside in which the hydroxyl group on the 2-carbon atom of the pentose sugar is replaced by a fluorine atom.

[0032] As mentioned in the background art, in the prior art, the yield and production of 2'-fluoronucleosides prepared by enzyme catalysis are both low, which is difficult to meet the requirements of industrial production. Therefore, in this application, the inventors attempted to develop a pyrimidine nucleoside phosphorylase mutant with higher enzyme activity, which can efficiently prepare the target product 2'-fluoronucleoside, and thus proposed a series of protection schemes of this application.

[0033] In a first typical embodiment of the present application, a pyrimidine nucleoside phosphorylase mutant is provided, comprising: (a) a pyrimidine nucleoside phosphorylase having a sequence of SEQ ID NO: 1 is a protein with a mutation based on the wild-type pyrimidine nucleoside phosphorylase TtPyNP, wherein the mutation is selected from any one or more of the following mutations: K80, V4, I7, R8, R17, E19, G27, R30, P34, L47, G49, L50, L58, D61, S91, F105, 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

[0034] (b) A protein having 70% or more homology to the amino acid sequence defined in (a) and having pyrimidine nucleoside phosphorylase activity.

[0035] In a preferred embodiment, in the above (a), the type of amino acid substituted at each site is independently selected from the following:

[0036] 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, A1 57T, 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; among them, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0037] In a preferred embodiment, the mutation includes any of the following amino acid mutations:

[0038] 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+I183V+A189T+Q225N+L274I+G285D, L50G+K80Y+T119N+ V173T+L215I+L284S+R303K+D329F, K80Q+G112T+T119N+A157T+V173T+I179V+A227D+E251 V、K80Q+G112T+T119N+A157T+V173T+I179V+A227D+L281R、K80Q+G112T+T119N+A157T+V173 T+I183V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A189T+A227D+L281R, K80Q+G11 2T+T119N+A157T+V173T+A189V+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+A190T+A2 27D+L281R, K80Q+G112T+T119N+A157T+V173T+A190V+A227D+L281R, K80Q+G112T+T119N+A 157T+V173T+F206M+A227D+L281R, K80Q+G112T+T119N+A157T+V173T+M207L+A227D+L281R. ,

[0039] or a protein having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more homology to the above amino acid sequence.

[0040] In a preferred embodiment, the pyrimidine nucleoside phosphorylase mutant includes a protein having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more homology with the amino acid sequence defined in (a) and a pyrimidine nucleoside phosphorylase mutant.

[0041] The above amino acid mutations were all experimentally explored in the examples of this application, and compared to the parent having the amino acid sequence shown in SEQ ID NO: 1, they all had the activity of catalyzing the cleavage of substrate nucleosides containing 2'-fluoropentose structures into phosphorylated 2'-fluoropentose and free bases. The above mutation sites are all mutations made around the amino acid active site, and such mutations can improve the binding ability and / or catalytic ability of the mutant to the substrate. For mutations away from the active site, the catalytic ability of the enzyme is less affected, so it is possible to obtain proteins having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% or more homology with the above amino acid sequence and the same catalytic activity.

[0042] The term "identity" used herein refers to the "homology" between amino acid sequences, that is, the total ratio of identical amino acid residues in an amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0043] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (e.g., 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% or more, or even 99.9% or more) homology and the same function, whose active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as those of the protein provided by sequence (a), are homologous proteins obtained by amino acid mutations.

[0044] As used herein, amino acid residues are abbreviated 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).

[0045] Substitution and replacement rules generally refer to the fact that amino acids with similar properties will have similar effects when substituted with each other. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0046] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0047] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0048] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0049] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0050] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0051] The "AlphaFold2-Multimer" used in this application is a publicly available artificial intelligence model that can predict the conformation of protein complexes. Its predictions of protein 3D structures are very close to those observed in real experiments using instruments such as cryo-electron microscopy. This allows for the acquisition of relatively realistic protein structures, thus guiding the study of protein structure and activity.

[0052] In a second typical embodiment of the present application, a DNA molecule is provided, which encodes the pyrimidine nucleoside phosphorylase mutant. In a third typical embodiment of the present application, a recombinant plasmid is provided, which is connected to the DNA molecule.

[0053] The DNA can encode the pyrimidine nucleoside phosphorylase mutant and can be linked to a recombinant plasmid to form a circular DNA. Both the DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc. to obtain the pyrimidine nucleoside phosphorylase mutant.

[0054] In a fourth typical embodiment of the present application, a host cell is provided, wherein the host cell contains the above-mentioned DNA molecule or recombinant plasmid.

[0055] In a preferred embodiment, the host cell comprises a prokaryotic cell; preferably, the prokaryotic cell comprises Escherichia coli.

[0056] The host cells described above can replicate recombinant plasmids and transcribe and translate DNA molecules carried on the recombinant plasmids, thereby obtaining a large number of pyrimidine nucleoside phosphorylase mutants. By utilizing existing technologies, host cells can be fragmented for protein purification, followed by crude enzyme catalysis after fragmentation, or other methods to obtain pyrimidine nucleoside phosphorylase mutants, which can then be used to catalyze substrate nucleosides. The host cells are non-plant-derived.

[0057] In a fifth typical embodiment of the present application, a method for preparing a 2'-fluoronucleoside is provided, which comprises: a) using the above-mentioned pyrimidine nucleoside phosphorylase mutant to catalyze the cleavage of the substrate nucleoside into phosphorylated 2'-fluoropentose and a free base, and the substrate nucleoside 2'-fluoropentose structure; b) using purine nucleoside phosphorylase to catalyze the combination of phosphorylated 2'-fluoropentose and the substrate base to prepare the 2'-fluoronucleoside.

[0058] In the above preparation method, the purine nucleoside phosphorylase includes but is not limited to the protein derived from Geobacillus stearothermophilus or its mutant (homology 80% or above) as shown in SEQ ID NO: 3, and also includes purine nucleoside phosphorylase (PNP) from other sources.

[0059] Utilizing the above-mentioned preparation method, the above-mentioned pyrimidine nucleoside phosphorylase mutant (PyNP) can be utilized to first carry out the following catalysis, and the reaction schematic diagram is shown in Figure 1. The substrate nucleoside is prepared as a phosphorylated 2'-fluoropentose and a free base. The phosphate group in the phosphorylated 2'-fluoropentose is derived from the phosphate in the catalytic system. The structure having the substrate nucleoside includes, but is not limited to, the structure in Figure 1.

[0060] Reusing purine nucleoside phosphorylase (PNP) of the prior art, utilize substrate base to replace the phosphate group on phosphorylation 2'-fluoro pentose, thereby obtain being combined with the 2'-fluoro nucleoside of substrate base.PNP used in this step can be flexibly selected from purine nucleoside phosphorylase of the prior art, all can catalyzed reaction carrying out.Reaction schematic diagram as shown in Figure 2, the structure of substrate base and phosphorylation 2'-fluoro pentose includes but not limited to the structure in Fig. 2.

[0061] Furthermore, steps a) and b) can be performed simultaneously in the same container and under the same reaction conditions, enabling a one-pot preparation of 2'-fluoronucleosides. This method offers high conversion rates and efficiency, significantly reducing enzyme usage and thus production costs, making it suitable for industrial scale-up.

[0062] In a preferred embodiment, the substrate nucleoside comprises 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine or 2'-deoxy-2'-fluorothymidine.

[0063] In a preferred embodiment, the substrate base includes modified or unmodified adenine, guanine, thymine or cytosine, or modified uracil.

[0064] In a preferred embodiment, the 2'-fluoro nucleoside includes a nucleoside containing a 2'-fluoro-2'-deoxypentose structure, preferably, the 2'-fluoro nucleoside includes 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxy-2-aminoadenosine, 2'-fluoro-2'-deoxyguanosine or 2'-fluoro-2'-deoxycytidine; or, the 2'-fluoro nucleoside is selected from a 2'-fluoro-2'-deoxynucleoside having a substituent on the base structure.

[0065] The substituents on the above-mentioned base structure or the modification of the above-mentioned substrate base include, but are not limited to, the introduction of a methyl group (-CH3), a carbamoyl group (-CH2NH2), a sugar group, a phosphonic acid group (-PO4), a sulfhydryl group (-SH), a nitro group (-NO2), a hydroxyl group (-OH) or deamination (-NH2) on the base structure, thereby achieving modifications such as methylation, carbamoylation, glycosylation, phosphorylation, thiolation, nitration, oxidation or deamination of the nucleoside.

[0066] Methylation: The modification of a nucleotide base by the addition of a methyl group. For example, in DNA, cytosine can be methylated to form 5-methylcytosine.

[0067] Aminomethylation: A modification of a nucleotide base by the addition of a carbamyl group. This modification can occur on cytosine and adenine bases.

[0068] Glycosylation: The modification of a nucleotide base by the addition of a sugar moiety. For example, adenosine can be glycosylated.

[0069] Phosphorylation: The modification of a nucleotide base by the addition of a phosphate group. Phosphorylation can occur on cytosine, adenine, and guanine bases.

[0070] Sulfuration: A modification of a nucleotide base by the addition of a sulfur group. Sulfuration can occur on cytosine, adenine, and guanine bases.

[0071] Nitration: The modification of a nucleotide base by the addition of a nitrate group. This modification can occur on both cytosine and adenine bases.

[0072] Oxidation: The modification of a nucleotide base by the addition of an oxygen group. For example, in DNA, guanine is oxidized to form 8-oxoguanine (8-oxoG).

[0073] Deamination: A modification of a nucleotide base by the loss of an amino group. This modification can occur on cytosine, adenine, and guanine bases.

[0074] In a sixth typical embodiment of the present application, a method for preparing phosphorylated 2'-fluoropentose is provided, which comprises: utilizing the above-mentioned pyrimidine nucleoside phosphorylase mutant to catalyze the cleavage of substrate nucleosides into phosphorylated 2'-fluoropentose and free bases, wherein the substrate nucleoside is a 2'-fluoropentose structure.

[0075] In a preferred embodiment, the substrate nucleoside comprises 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine or 2'-deoxy-2'-fluorothymidine.

[0076] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0077] Example 1 Construction of TtPyNP and GsPNP expression strains

[0078] The protein sequence of pyrimidine nucleoside phosphorylase (TtPyNP) from Thermus thermophilus was obtained from NCBI (Accession No. BAD71594), as shown in SEQ ID NO: 1. After codon optimization, the DNA sequence encoding the enzyme was obtained, as shown in SEQ ID NO: 2. This sequence was cloned into the expression vector pET28a(+) to generate the plasmid pET28a-TtPyNP. The pET28a-TtPyNP plasmid was transformed into competent Escherichia coli BL21(DE3) to obtain a monoclonal strain.

[0079] The same method was used to obtain the pET28a-GsPNP (GsPNP, NCBI Accession No. BAA13510) monoclonal strain. The GsPNP protein sequence is shown in SEQ ID NO: 3.

[0080] Example 2 Construction of TtPyNP mutants

[0081] Saturation mutagenesis or site-directed mutagenesis was performed using whole-plasmid PCR. The first round of mutagenesis used the pET28a-TtPyNP obtained in Example 1 as a template, and subsequent mutations used the best mutant screened in the previous round as a template. PCR reaction conditions were: pre-denaturation at 94°C for 10 min; 25 amplification cycles (denaturation at 94°C for 30 s, annealing and extension at 68°C for 6.5 min), and a final extension at 68°C for 10 min. The resulting PCR product was digested with DpnI enzyme and transformed into Escherichia coli BL21 (DE3) competent cells, and cultured overnight at 37°C to obtain a single clone.

[0082] Example 3 Expression of TtPyNP mutants and enzyme solution preparation

[0083] Expression of mutants: The mutants were inoculated into a test tube containing 5 mL of Luria-Bertani liquid medium (containing 50 μg / mL kanamycin), cultured at 37°C for 16 h, and then inoculated into a 2 L shake flask containing 500 mL of Luria-Bertani liquid medium at a 1% inoculum size and cultured at 37°C until the OD 600 When the pH value was 0.6, IPTG was added to a final concentration of 0.1 M to induce expression at 20°C for 18 h. The bacterial culture was centrifuged at 7000 rpm for 10 min to collect the cells and stored at -20°C for later use.

[0084] Preparation of enzyme solution: Weigh 0.1 g of the bacterial sludge prepared above, resuspend it in 1 mL of 100 mM potassium phosphate buffer with a pH of 7.5, shake and mix, and then disrupt the bacterial suspension using an ultrasonic disruptor (power: 30%, time: 5 min).

[0085] The expression of GsPNP and the preparation of enzyme solution were the same as above.

[0086] Example 4 HPLC detection method

[0087] HPLC detection method: Atlantise T3 Column, 4.6 mm x 150 mm, mobile phase: 0.1% TFA / methanol, flow rate: 1 mL / min, 40°C, UV detector: 254 nm, detection time: 15 min. These HPLC conditions allow for the determination of 2'-fluoro-2'-deoxyuridine conversion.

[0088] Example 5 Reaction Verification of TtPyNP Mutants

[0089] Using TtPyNP as a template, single- and multi-point mutations were constructed and validated. Reaction conditions were: 20 mM 2'-fluoro-2'-deoxyuridine, 20 mM PBS (pH 7.0), 49 μL enzyme solution, 1 mL total volume, 60°C, and a reaction time of 1 h. Samples were collected and analyzed by HPLC. The conversion rate of 2'-fluoro-2'-deoxyuridine is shown in Table 1. Results showed that the activity of the K80 mutation site was significantly improved, with the final mutant, K80Q+G112T+T119N+A157T+V173T+M207L+A227D+L281R, achieving a conversion rate exceeding 30%.

[0090] Table 1

[0091] Note: In the above table, + 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%.

[0092] Example 6: Application of TtPyNP mutants in the synthesis of 2'-fluoro-2'-deoxyadenosine

[0093] A 10 mL reaction system was prepared in 20 mM phosphate buffer (pH 7.5), including two substrates: 100 mM 2'-fluoro-2'-deoxyuridine and 250 mM adenine, two enzyme solutions: 1.07 mL mutant K80Q+G112T+T119N+A157T+V173T+M207L+A227D+L281R enzyme solution, and 4.3 mL GsPNP enzyme solution. The reaction was carried out at 60°C for 137 h. After the reaction, 1 mL of the reaction solution was dissolved in an equal volume of DMSO, diluted 25-fold, and then sent for HPLC analysis. The HPLC spectrum is shown in Figure 3. The conversion rate of 2'-fluoro-2'-deoxyuridine was >80% (converted to uracil), the conversion rate of adenine was >30%, and the yield of 2'-fluoro-2'-deoxyadenosine was >20 g / L.

[0094] From the above description, it can be seen that the above-mentioned embodiments of the present invention have achieved the following technical effects: utilizing the above-mentioned pyrimidine nucleoside phosphorylase mutant, the substrate nucleoside containing 2'-fluoro pentose structure can be efficiently catalyzed to be cleaved into phosphorylated 2'-fluoro pentose and free base. Further, utilizing the above-mentioned pyrimidine nucleoside phosphorylase mutant and the purine nucleoside phosphorylase in the prior art, 2'-fluoro nucleosides can be prepared by a one-pot method. Compared with the wild-type enzyme, the above-mentioned pyrimidine nucleoside phosphorylase mutant has better enzyme activity, and the conversion rate and efficiency of preparing 2'-fluoro nucleoside products are higher, which can significantly reduce the usage of the enzyme, thereby reducing production costs and being suitable for industrialized amplification production.

[0095] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A pyrimidine nucleoside phosphorylase mutant, characterized in that: include: (a) SEQ ID NO: 1 is a protein with mutations based on the wild-type pyrimidine nucleoside phosphorylase TtPyNP, wherein the mutation is selected from any one or more of the following mutations: K80, V4, I7, R8, R17, E19, G27, R30, P34, L47, G49, L50, L58, D61, S91, F105, 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) A protein having 70% or more homology with the amino acid sequence defined in (a) and having pyrimidine nucleoside phosphorylase activity.

2. The pyrimidine nucleoside phosphorylase mutant according to claim 1, characterized in that In (a), the mutation is selected from any one or more of the following mutations: K80Q, K80Y, V4P, V4G, I7G, R8N, R8F, R17E, E19P, G27S, R30K, P34K, P34L, P34G, L47H, G49T, G49V, G49S, L50A, L50D, L50G, L50K, L50 N, L50R, L50S, L58T, L58Q, D61T, S91A, S91V, S91C, F105I, F105T, G112S, G112T, G112A, G112L, G112C, T119N, E135K, E135A, A151S, A157D, 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; The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

3. The pyrimidine nucleoside phosphorylase mutant according to claim 1, characterized in that: The mutation includes any of the following amino acid mutations: K80Q+G112A, K80Q+G112C, K80Q+G112S, K80Q+G112T, L50A+K80Q+G112T, L50D+K80Q+G112T, L50G+K8 0Q+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+A 190T+L281R, K80Q+G112T+A157T+A190V+L281R, K80Q+G112T+A157T+A271S+L281R, K80Q+G112T+A15 7T+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+L28 1R+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+I183V+A189T+Q225N+L274I+G285D、L50G+K80Y+T119N+V173T+L215I+L284S+R303K+D329F、K80Q+G112T+T119N+A157T+V173T+I179V+A227D+E251V、K80Q+G112T+T119N+A157T+V173T+I179V+A227D+L281R、K80Q+G112T+T119N+A157T+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+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 in that The DNA molecule encodes the pyrimidine nucleoside phosphorylase mutant according to any one of claims 1 to 3.

5. A recombinant plasmid, characterized in that: The recombinant plasmid is connected to the DNA molecule according to claim 4.

6. A host cell, characterized in that The host cell contains the DNA molecule according to claim 4 or the recombinant plasmid according to claim 5.

7. A method for preparing 2′-fluoronucleoside, characterized in that: The preparation method comprises: a) using the pyrimidine nucleoside phosphorylase mutant according to any one of claims 1 to 3 to catalyze the cleavage of substrate nucleosides into phosphorylated 2′-fluoropentose and free bases, wherein the substrate nucleosides are 2′-fluoropentose structures; b) using purine nucleoside phosphorylase to catalyze the combination of the phosphorylated 2′-fluoropentose and substrate base to prepare the 2′-fluoronucleoside.

8. The preparation method according to claim 7, characterized in that: The substrate nucleoside includes 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine or 2'-deoxy-2'-fluorothymidine.

9. The preparation method according to claim 7, characterized in that: The substrate base includes modified or unmodified adenine, guanine, thymine or cytosine, or modified uracil.

10. The preparation method according to any one of claims 7 to 9, characterized in that: The 2'-fluoro nucleosides include nucleosides containing a 2'-fluoro-2'-deoxypentose structure.

11. The preparation method according to claim 10, characterized in that: The 2′-fluoro nucleosides include 2′-fluoro-2′-deoxyadenosine, 2′-fluoro-2′-deoxy-2-aminoadenosine, 2′-fluoro-2′-deoxyguanosine or 2′-fluoro-2′-deoxycytidine; Alternatively, the 2′-fluoro nucleoside is selected from 2′-fluoro-2′-deoxynucleosides having a substituent on the base structure.

12. A method for preparing phosphorylated 2′-fluoropentose, characterized in that: The preparation method comprises: The pyrimidine nucleoside phosphorylase mutant according to any one of claims 1 to 3 is used to catalyze the cleavage of substrate nucleosides into phosphorylated 2'-fluoropentose and free bases, wherein the substrate nucleosides are 2'-fluoropentose structures.

13. The preparation 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.

Images