N-deoxyribose transferase II mutant and application thereof

Abstract

The invention provides an N-deoxyribose transferase II mutant and an application thereof. Compared with the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence of the N-deoxyribose transferase is shown in SEQ ID NO: 1; the amino acid sequence has substitution mutation at any one position, any two positions, any three positions, any four positions, any five positions, any more than six positions, any more than seven positions, any more than eight positions or all nine positions of the ninth position, the 54th position, the 60th position, the 63rd position, the 68th position, the 88th position, the 91th position and the 154th position. The N-deoxyribose transferase II mutant can obviously promote transfer of deoxyribose between purine and pyrimidine, the catalytic synthesis efficiency of 2 '-fluoro-2'-deoxyguanosine analogues is improved, meanwhile, the action range of substrates can be widened, and the N-deoxyribose transferase II mutant is effective to various substrates.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to an N-deoxyribosyltransferase II mutant and an application thereof. Background Art

[0002] Nucleoside analogs are an important class of anticancer therapeutic agents and specific antiviral drugs, including various purine and pyrimidine nucleoside derivatives, mainly a class of antimetabolites, which are used to destroy viral transcription, interfere with or terminate the synthesis of viral nucleic acids, and are used to fight DNA and RNA viruses such as herpes virus, HIV, HBV, influenza and respiratory viruses. 2'-Fluoro-2'-deoxyguanosine analogs, as a type of nucleoside analog, have antiviral and antitumor activity, and can effectively embed into viral DNA and RNA chains, thereby inhibiting cell division and replication. Therefore, the demand for them in the domestic and foreign pharmaceutical markets and clinical treatments is also steadily increasing.

[0003] The synthesis of 2'-fluoro-2'-deoxyguanosine analogs generally adopts chemical synthesis and enzymatic methods. Chemical synthesis is a multi-step process. In order to achieve regional and stereoselectivity, it is necessary to protect and remove the protecting group. The operation steps are complicated, the cost is high, and it is easy to produce toxic and harmful substances, causing environmental pollution. Enzymatic synthesis is increasingly valued due to its high regional and stereoselectivity. Enzymatic synthesis can be carried out under mild reaction conditions, has high catalytic efficiency, is green and pollution-free, and can effectively reduce purification steps in the post-processing process.

[0004] N-deoxyribosyltransferase (EC.2.4.2.6) can catalyze the transfer of deoxyribose between different bases and can be divided into two categories according to the substrate specificity: N-deoxyribosyltransferase I (Nucleosidedeoxyribosyltransferase I, PDT), which can only catalyze the transfer of deoxyribose between purines; N-deoxyribosyltransferase II (Nucleosidedeoxyribosyltransferase II, NDT), which catalyzes the transfer of deoxyribose between purine and pyrimidine.

[0005] NDT has a wide base recognition range and can recognize a variety of natural and modified bases with a high conversion rate. Therefore, it is used in the synthesis of a variety of deoxynucleoside analogs, such as 5-fluoro-2'-deoxyuridine, 6-chloro-2'-deoxycytidine, etc. However, NDT has a high substrate specificity for the structure of the ribose donor. For nucleoside analogs with sugar modifications, such as cytarabine, stavudine, gemcitabine and zalcitabine, the efficiency of catalytic synthesis is extremely low, which limits the application of NDT.

[0006] Therefore, there is a need in the art to expand the substrate recognition range of NDT and improve the activity of the enzyme through directed evolution modification of the wild-type NDT. Summary of the invention

[0007] The object of the present invention is to provide an N-deoxyribosyltransferase II mutant, wherein the catalytic activity, substrate specificity and / or substrate specificity of the N-deoxyribosyltransferase II mutant are changed, and the transfer of deoxyribose between purine and pyrimidine can be significantly promoted, and the catalytic synthesis efficiency of 2'-fluoro-2'-deoxyguanosine analogs can be improved, and the scope of action of the substrate can be expanded, and the mutant is effective for a variety of substrates.

[0008] Specifically, the present invention provides an N-deoxyribosyltransferase, wherein the N-deoxyribosyltransferase:

[0009] (a) compared to the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence has a substitution mutation at any one position, any two positions, any three positions, any four positions, any five positions, any six or more positions, any seven or more positions, any eight or more positions, or all nine positions among positions 9, 54, 60, 63, 68, 82, 88, 91 and 154; or

[0010] (b) is a protein derived from (a) which is obtained by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of (a), and has the function of N-deoxyribosyltransferase II, and the amino acid residues corresponding to positions 9, 54, 60, 63, 68, 82, 88, 91 and 154 of SEQ ID NO: 1 are as described in (a); or

[0011] (c) is a protein derived from (a) that has more than 80% identity with the amino acid sequence of (a) and has N-deoxyribosyltransferase II function, and the amino acid residues corresponding to positions 9, 54, 60, 63, 68, 82, 88, 91 and 154 of SEQ ID NO:1 are as described in (a).

[0012] The present invention also provides a polynucleotide molecule encoding the N-deoxyribosyltransferase described in any embodiment herein or a complementary sequence thereof.

[0013] The present invention also provides a nucleic acid construct comprising the polynucleotide molecule described herein or a complementary sequence thereof.

[0014] The present invention also provides a host cell, which: (1) expresses the N-deoxyribose transferase described in any embodiment of the present invention; and / or (2) contains the polynucleotide molecule or its complementary sequence or nucleic acid construct described in any embodiment of the present invention.

[0015] The present invention also provides an enzyme preparation, characterized in that the enzyme preparation contains the N-deoxyribosyltransferase described in any embodiment of the present invention.

[0016] The present invention also provides the use of an N-deoxyribosyltransferase selected from any of the embodiments described herein and an N-deoxyribosyltransferase whose amino acid sequence is shown in any of SEQ ID NOs: 1 to 5 in transferring a 2'-fluoro-2'-deoxyribose group to a base, or in preparing a preparation for transferring a 2'-fluoro-2'-deoxyribose group to a base.

[0017] The present invention also provides a method for synthesizing 2'-deoxy-2'-fluorouridine as shown in the following formula III:

[0018]

[0019] The method comprises the step of transferring the 2'-fluoro-2'-deoxyribose group in the 2'-fluoro-2'-deoxyribonucleoside of formula I as a ribose donor to the guanine analog of formula II as a ribose acceptor using an N-deoxyribose transferase selected from any of the embodiments described herein and an N-deoxyribose transferase whose amino acid sequence is shown in any of SEQ ID NOs: 1 to 5; wherein R is a base selected from adenine, hypoxanthine, uracil, cytosine and thymine, preferably adenine or uracil; and X is selected from OH, Cl, S, F or C1-C4 alkoxy, preferably OMe or Cl. DETAILED DESCRIPTION

[0020] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0021] After in-depth research, the inventors have revealed that some N-deoxyribosyltransferases can promote the transfer of deoxyribose between purine and pyrimidine, and improve the catalytic synthesis efficiency of 2'-fluoro-2'-deoxyguanosine analogs. Further, the inventors have also found some N-deoxyribosyltransferase II mutants, whose catalytic activity, substrate specificity and / or substrate specificity have changed, and can also significantly promote the transfer of deoxyribose between purine and pyrimidine, improve the catalytic synthesis efficiency of 2'-fluoro-2'-deoxyguanosine analogs, and can also expand the scope of action of substrates, and are effective for a variety of substrates.

[0022] NDT and its mutants

[0023] The NDT described herein particularly includes an NDT enzyme containing an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 5, and also includes mutants having more than 85%, more than 90%, more than 95%, more than 97%, more than 98%, or more than 99% identity with the amino acid sequence as shown in any one of SEQ ID NOs: 1 to 5 and retaining the biological activity of the NDT as shown in SEQ ID NOs: 1 to 5, wherein the mutants are preferably derived from Lactobacillus helveticus, Lactobacillusdelbrueckii subsp.lactis, Lactobacillus reuteri, Lactobacillus leichmannii or Lactobacillus hilgardii, respectively.

[0024] The biological activities of NDT described in this article include: promoting the transfer of deoxyribose between purine and pyrimidine, and improving the catalytic synthesis efficiency of 2'-fluoro-2'-deoxyguanosine analogs.

[0025] In some embodiments, unless otherwise specified, the "N-deoxyribosyltransferase II mutant", "mutant N-deoxyribosyltransferase II", "mutant NDT", and "NDT mutant" described herein are used interchangeably, and generally refer to a mutant of N-deoxyribosyltransferase II (nucleotide sequence of which is shown in SEQ ID NO: 6) whose amino acid sequence is shown in SEQ ID NO: 1. Preferably, the mutated site is selected from the 9th, 54th, 60th, 63rd, 68th, 82nd, 88th, 91st, and 154th positions of SEQ ID NO: 1. Specifically, the N-deoxyribosyltransferase mutant may have a substitution mutation only at any one of the 9th, 54th, 60th, 63rd, 68th, 82nd, 88th, 91st, and 154th positions relative to SEQ ID NO: 1, or may have a substitution mutation at any two positions, any three positions, any four positions, any five positions, or any six or more positions. In some embodiments, the N-deoxyribosyltransferase mutant is also from a Lactobacillus helveticus strain.

[0026] As used herein, "isolated N-deoxyribosyltransferase II" means that the N-deoxyribosyltransferase II mutant is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated therewith. Those skilled in the art can purify the N-deoxyribosyltransferase II mutant using standard protein purification techniques. Substantially pure protein can produce a single major band on a non-reducing polyacrylamide gel.

[0027] Herein, the mutation at position 9 is a mutation of phenylalanine (F) to a non-polar amino acid, such as alanine, leucine, isoleucine, proline, tryptophan, methionine or glycine, or to an uncharged polar amino acid, such as serine, threonine, cysteine, tyrosine, asparagine or glutamine (Gln). Preferably, the mutation at position 9 is a mutation of phenylalanine to glycine, alanine, leucine or isoleucine, or to serine or threonine. More preferably, the mutation at position 9 is a mutation of phenylalanine to glycine or alanine, or to serine or threonine. More preferably, the mutation at position 9 is not valine. More preferably, the mutation at position 9 is a mutation of phenylalanine to glycine or threonine.

[0028] Herein, the mutation at position 54 is a mutation of aspartic acid (D) to a positively charged polar amino acid, such as lysine, arginine or histidine, or a mutation to a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine. Preferably, the mutation at position 54 is a mutation of aspartic acid to arginine or histidine, or a mutation to leucine or isoleucine. More preferably, the mutation at position 54 is a mutation of aspartic acid to histidine or leucine.

[0029] Herein, the mutation at position 60 is a mutation from leucine (L) to an uncharged polar amino acid, such as serine, threonine, tyrosine, asparagine or glutamine. Preferably, the mutation at position 60 is a mutation from leucine to asparagine or glutamine, more preferably glutamine. More preferably, the mutation at position 60 is not alanine or cysteine.

[0030] Herein, the mutation at position 63 is a mutation of isoleucine (I) to a positively charged polar amino acid, such as lysine, arginine or histidine, or to a negatively charged polar amino acid, such as aspartic acid or glutamic acid. Preferably, the mutation at position 63 is a mutation of isoleucine to arginine or histidine, or to aspartic acid or glutamic acid. Preferably, the mutation at position 63 is a mutation of isoleucine to arginine, histidine or glutamic acid.

[0031] Herein, the mutation at position 68 is a mutation of alanine (A) to a non-polar amino acid, such as valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine, or a mutation to an uncharged polar amino acid, such as serine, threonine, cysteine, tyrosine, asparagine or glutamine. Preferably, the mutation at position 68 is a mutation of alanine to phenylalanine, tryptophan or tyrosine.

[0032] Herein, the mutation at position 82 is a mutation of valine (V) to a positively charged polar amino acid, such as lysine, arginine or histidine, or a mutation to a non-polar amino acid, such as alanine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine. Preferably, the mutation at position 82 is a mutation of valine to arginine or histidine, or a mutation to phenylalanine. More preferably, the mutation at position 82 is a mutation of valine to arginine or phenylalanine.

[0033] Herein, the mutation at position 88 is a mutation of leucine (L) to an uncharged polar amino acid, such as threonine, glutamine, asparagine, tyrosine or cysteine, or a mutation to a negatively charged polar amino acid, such as aspartic acid or glutamic acid, or a mutation to a non-polar amino acid, such as alanine, valine, isoleucine, proline, phenylalanine, methionine or glycine. Preferably, the mutation at position 88 is a mutation of leucine to cysteine, or a mutation to aspartic acid or glutamic acid, or a mutation to valine. More preferably, the mutation at position 88 is a mutation of leucine to cysteine, glutamic acid or valine.

[0034] Herein, the mutation at position 91 is a mutation of glutamic acid (E) to an uncharged polar amino acid, such as serine, threonine, glutamine, asparagine, tyrosine or cysteine, or a mutation to a polar positively charged amino acid, such as lysine, arginine or histidine. Preferably, the mutation of glutamic acid (E) at position 91 is a mutation of serine, threonine or arginine. More preferably, the mutation of glutamic acid (E) at position 91 is a mutation of serine or arginine.

[0035] Herein, the mutation at position 154 is a mutation of aspartic acid to an uncharged polar amino acid, such as serine, threonine, glutamine, asparagine, tyrosine or cysteine, or a mutation to a positively charged polar amino acid, such as lysine, arginine or histidine. Preferably, the mutation at position 154 is a mutation of aspartic acid to glutamine or asparagine, or to lysine or arginine. More preferably, the mutation at position 154 is a mutation of aspartic acid to asparagine, lysine or arginine.

[0036] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribosyltransferase II mutant described herein has the following mutations at one or more of the following amino acid positions:

[0037] (1) Phenylalanine (F) at position 9 mutates to threonine (T) or alanine (A);

[0038] (2) Aspartic acid (D) at position 54 was mutated to leucine (L) or histidine (H);

[0039] (3) leucine (L) at position 60 was mutated to glutamine (Q);

[0040] (4) Isoleucine (I) at position 63 mutated to glutamic acid (E), arginine (R), or histidine (H);

[0041] (5) Alanine (A) at position 68 mutated to phenylalanine (F), tryptophan (W), or tyrosine (Y);

[0042] (6) Valine (V) at position 82 mutated to arginine (R) or phenylalanine (F);

[0043] (7) Leucine (L) at position 88 mutated to cysteine ​​(C), glutamic acid (E) or valine (V);

[0044] (8) Glutamic acid (E) at position 91 is mutated to serine (S) or tyrosine (Y); or

[0045] (9) Aspartic acid (D) at position 154 was mutated to lysine (K), asparagine (N) or arginine (R).

[0046] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribosyltransferase II mutant does not mutate at the 27th alanine (A) and / or the 53rd isoleucine (I) site.

[0047] In one or more preferred embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase II mutant described herein is selected from the group consisting of: F9T, F9A, D54L, D54H, L60Q, I63E, I63R, I63H, A68F, A68W, A68Y, V82R, V82F, L88C, L88E, L88V, E91S, E91Y, D154K, D154N and D154R.

[0048] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribosyltransferase mutant described herein has the substitution mutation described herein at least at position 9, and further has the substitution mutation described in any embodiment of the present invention at any one, any two or any three positions of position 54, position 60, position 63, position 68, position 82, position 88, position 91 and position 154, preferably further has the substitution mutation described in any embodiment of the present invention at any one, any two or all three positions of position 54, position 63 and position 68.

[0049] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribosyltransferase mutant described herein has the substitution mutation described herein at least at position 60, and further has the substitution mutation described in any embodiment of the present invention at any one, any two or any three positions of position 9, 54, 63, 68, 82, 88, 91 and 154, preferably further has the substitution mutation described in any embodiment of the present invention at any one, any two, any three or all four positions of position 54, 88, 91 and 154.

[0050] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribosyltransferase mutant described herein has the substitution mutation described herein at least at position 63, and further has the substitution mutation described in any embodiment herein at any one, any two or any three positions among position 9, 54, 60, 68, 82, 88, 91 and 154, preferably further has the substitution mutation described in any embodiment herein at any one, any two, any three positions, any four positions, any five positions or all six positions among position 9, 54, 82, 88, 91 and 154.

[0051] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribosyltransferase mutant described herein has the substitution mutation described herein at least at position 68, and further has the substitution mutation described in any embodiment herein at any one, any two or any three positions among position 9, 54, 60, 63, 82, 88, 91 and 154, preferably further has the substitution mutation described in any embodiment herein at any one, any two, any three positions, any four positions or all five positions among position 9, 54, 82, 88 and 91.

[0052] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribosyltransferase mutant described herein has the substitution mutation described herein at least at position 82, and further has the substitution mutation described in any embodiment of the present invention at any one, any two or any three positions of position 9, 54, 60, 63, 68, 88, 91 and 154, preferably further has the substitution mutation described in any embodiment of the present invention at any one, any two or all three positions of position 63, 91 and 154.

[0053] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribosyltransferase mutant described herein has the substitution mutation described herein at least at position 88, and further has the substitution mutation described in any embodiment of the present invention at any one, any two or any three positions among position 9, 54, 60, 63, 68, 82, 91 and 154, preferably further has the substitution mutation described in any embodiment of the present invention at any one, any two, any three or all four positions among position 60, 68, 91 and 154.

[0054] In one or more embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase is selected from any one of the following combinations:

[0055] (1) Mutations at positions 9, 54, and 68;

[0056] (2) mutations at positions 9 and 63;

[0057] (3) mutations at positions 9 and 68;

[0058] (4) mutations at positions 60, 88, and 91;

[0059] (5) mutations at positions 54 and 60;

[0060] (6) mutations at positions 54, 63, and 91;

[0061] (7) mutations at positions 60 and 154;

[0062] (8) mutations at positions 63 and 82;

[0063] (9) mutations at positions 63 and 154;

[0064] (10) mutations at positions 68 and 88;

[0065] (11) mutations at positions 68 and 91;

[0066] (12) mutations at positions 82, 91, and 154;

[0067] (13) Mutations at positions 88 and 154.

[0068] In one or more more preferred embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase is selected from any one of the following combinations:

[0069] (1) mutations at positions 9, 54 and 68; wherein the mutation at position 9 is F9T or F9A, the mutation at position 54 is D54L or D54H, and the mutation at position 68 is A68F, A68W or A68Y;

[0070] (2) mutations at positions 9 and 63; wherein the mutation at position 9 is F9T or F9A, and the mutation at position 63 is I63E, I63R, or I63H;

[0071] (3) mutations at positions 9 and 68; wherein the mutation at position 9 is F9T or F9A, and the mutation at position 68 is A68F, A68W or A68Y;

[0072] (4) mutations at positions 60, 88 and 91; wherein the mutation at position 60 is L60Q, the mutation at position 88 is L88C, L88E or L88V, and the mutation at position 91 is E91S or E91Y;

[0073] (5) mutations at positions 54 and 60; wherein position 54 mutates to D54L or D54H, and position 60 mutates to L60Q;

[0074] (6) mutations at positions 54, 63 and 91; wherein position 54 mutates to D54L or D54H, position 63 mutates to I63E, I63R or I63H, and position 91 mutates to E91S or E91Y;

[0075] (7) mutations at positions 60 and 154; wherein the mutation at position 60 is L60Q, and the mutation at position 154 is D154K, D154N, or D154R;

[0076] (8) mutations at positions 63 and 82; wherein the mutation at position 63 is I63E, I63R or I63H, and the mutation at position 82 is V82R or V82F;

[0077] (9) mutations at positions 63 and 154; wherein the mutation at position 63 is I63E, I63R or I63H, and the mutation at position 154 is D154K, D154N or D154R;

[0078] (10) mutations at positions 68 and 88; wherein the mutation at position 68 is A68F, A68W or A68Y, and the mutation at position 88 is L88C, L88E or L88V;

[0079] (11) mutations at positions 68 and 91; wherein the mutation at position 68 is A68F, A68W or A68Y, and the mutation at position 91 is E91S or E91Y;

[0080] (12) mutations at positions 82, 91 and 154; wherein the mutation at position 82 is V82R or V82F, the mutation at position 91 is E91S or E91Y, and the mutation at position 154 is D154K, D154N or D154R;

[0081] (13) Mutations at positions 88 and 154; wherein the mutation at position 88 is L88C, L88E or L88V, and the mutation at position 154 is D154K, D154N or D154R.

[0082] In one or more more preferred embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase is selected from any one of the following combinations:

[0083] (1) F9T, D54H and A68F; (2) F9T and I63E; (3) F9T and A68F; (4) L60Q, L88C and E91S; (5) D54H and L60Q; (6) D54H, I63E and E91S; (7) L60Q and D154K; (8) I63R and V82R; (9) I63E and D154R; (10) A68W and L88V; (11) A68F and E91S; (12) V82R, E91Y and D154K; (13) L88C and D154K.

[0084] The present invention also includes fragments, derivatives and analogs of the N-deoxyribosyltransferase II mutant. As used herein, the terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity as the natural N-deoxyribosyltransferase II mutant of the present invention. The protein fragment, derivative or analog of the present invention may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein having a substitution group in one or more amino acid residues, or (iii) a protein formed by the fusion of an additional amino acid sequence to the protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the protein, or a fusion protein). Preferably, the fragment, derivative and analog still retain at least one mutation described in the present application, i.e., an amino acid substitution corresponding to at least one of positions 9, 54, 60, 63, 68, 82, 88, 91 and 154 in SEQ ID NO:1.

[0085] In the present invention, the term "N-deoxyribosyltransferase II mutant" also includes (but is not limited to): deletion, insertion and / or substitution of several (usually 1-20, preferably 1-10, and even more preferably 1-8, 1-5, 1-3, or 1-2) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. Preferably, the mutant still retains at least one mutation described in the present application, i.e., at least one amino acid substitution corresponding to positions 9, 54, 60, 63, 68, 82, 88, 91, and 154 in SEQ ID NO:1. In some embodiments, the N-deoxyribosyltransferase mutant is also from a Lactobacillus helveticus strain.

[0086] In the present invention, the term "N-deoxyribosyltransferase II mutant" also includes (but is not limited to): a derived protein that retains its protein activity and has an amino acid sequence identity of more than 80%, preferably more than 85%, more preferably more than 90%, further more preferably more than 95%, such as more than 98%, more than 99% with each N-deoxyribosyltransferase II mutant described herein. Similarly, these mutants still retain at least one mutation described herein, i.e., a mutation corresponding to at least one amino acid substitution amino acid at positions 9, 54, 60, 63, 68, 82, 88, 91 and 154 in SEQ ID NO: 1. In some embodiments, such N-deoxyribosyltransferase mutants are also derived from Lactobacillus helveticus strains. The identity between two sequences can be calculated using methods well known in the art, such as BLASTP.

[0087] In the N-deoxyribosyltransferase II mutants, fragments, derivatives and analogs of the present invention, the mutation sites and the mutated amino acids described herein are conservative.

[0088] The protein of the present invention can be a recombinant protein, a natural protein, a synthetic protein, preferably a recombinant protein. The protein of the present invention can be a naturally purified product, or a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects and mammalian cells).

[0089] Polynucleotide molecules

[0090] The present invention also provides a polynucleotide molecule encoding the N-deoxyribosyltransferase II mutant or its conservative variant protein and its complementary sequence.

[0091] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0092] The polynucleotide molecule encoding the mature protein of the mutant includes: a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and optional additional coding sequences) and non-coding sequences.

[0093] A "polynucleotide molecule encoding a protein" may be a polynucleotide that encodes the protein, or may be a polynucleotide that also includes additional coding and / or non-coding sequences.

[0094] The polynucleotide molecules of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0095] Nucleic acid constructs

[0096] The present invention also provides a nucleic acid construct comprising the polynucleotide molecule described herein. The nucleic acid construct may also contain one or more regulatory sequences operably linked to the polynucleotide molecule described herein, such as a promoter, terminator and / or enhancer.

[0097] As used herein, "operably linked" or similar descriptions refer to the arrangement of elements, wherein the components are arranged in a certain shape so as to perform their required functions. Thus, a given promoter operably linked to a coding sequence can effectively express the coding sequence when the correct transcription factors, etc., are present. The promoter does not need to be adjacent to the coding sequence, as long as it functions to direct the expression of the sequence. Thus, for example, a sequence that does not participate in translation but is transcribed can exist between the promoter sequence and the coding sequence, as can a transcribable intron; and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0098] The polynucleotide encoding the present invention can be manipulated in a variety of ways to ensure expression of the NDT mutant or its conservative variant protein. Manipulation of the polynucleotide molecule prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0099] The regulatory sequence may be a suitable promoter, a nucleotide sequence recognized by a host cell for expressing the polynucleotide molecules described herein. The promoter may be any nucleotide sequence showing transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to the host cell.

[0100] In E. coli, suitable promoters include, but are not limited to, lac (lactose promoter), trp (tryptophan promoter), and tac (hybrid promoter of lactose and tryptophan).

[0101] The regulatory sequence also includes a suitable transcription terminator, which is recognized by the host cell to terminate transcription. The terminator sequence is operably connected to the 3' end of the polynucleotide molecule described herein. Any terminator that is functional in the host cell of choice can be used in the present invention. In some embodiments, the transcription terminator sequence is a terminator that is functional in Escherichia coli.

[0102] In some embodiments, the nucleic acid construct is an expression cassette or expression frame comprising a promoter and a transcription terminator operably linked to a polynucleotide molecule described herein.

[0103] Carrier

[0104] The nucleic acid constructs described herein may also be vectors, including but not limited to expression vectors and cloning vectors.

[0105] In expression vectors, various nucleic acid molecules and regulatory sequences can be linked together to produce a recombinant expression vector comprising one or more restriction sites that allow insertion of a polynucleotide molecule or nucleic acid construct described herein at such sites. When making an expression vector, the polynucleotide molecule described herein is located in the vector so that it can be operably linked to an appropriate regulatory sequence.

[0106] The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0107] The recombinant expression vector may be any vector (such as a plasmid or virus) that can be easily subjected to recombinant DNA methods and can result in the expression of the nucleotide sequence of interest. The choice of vector generally depends on the compatibility of the vector with the host cell into which it is introduced. The vector may be a linear or closed circular plasmid.

[0108] The vector can be an autonomously replicating vector, i.e., exists as an extrachromosomal entity, and its replication is independent of the vector of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can include any mode for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicated together with the chromosome into which it has been integrated when being imported into the host cell. In addition, a single vector or plasmid or two or more vectors or plasmids, or a transposon, that together contain the total DNA that will be imported into the host cell genome can be used.

[0109] The vectors described herein may contain one or more selectable markers that allow for easy selection of transformed, transfected, transduced, etc. cells. Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophy to auxotrophy, and the like.

[0110] The vectors described herein may contain elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0111] More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the yield of the gene product. The increase in the number of polynucleotide copies can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selection marker gene and the polynucleotide, wherein the cells containing the amplified copy of the selection marker gene and thus the additional copy of the polynucleotide can be screened by culturing the cells in the presence of an appropriate selection agent.

[0112] The expression vector described herein is more preferably a vector that can be used for expression in E. coli, such as pET-30a.

[0113] The cloning vector containing the polynucleotide sequence described herein can be used to replicate a sufficient number of target plasmids. Therefore, the cloning vector described herein carries a strong self-replication element, such as a replication origin site, etc. Generally, the cloning vector of the present invention does not have an expression element.

[0114] The expression vectors described herein can be constructed using methods well known in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein. Those of ordinary skill in the art are aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0115] Host cells

[0116] The host cell of the present invention can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include: Escherichia coli, Bacillus subtilis, Streptomyces, Agrobacterium; eukaryotic cells such as yeast, plant cells, etc. In a specific embodiment of the present invention, Escherichia coli is used as a host cell.

[0117] The polynucleotide molecules of the present invention can be used to express or produce recombinant NDT mutants by recombinant DNA technology. The expression or production generally includes the following steps: (1) transforming or transducing a suitable host cell with a polynucleotide encoding an NDT mutant of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) culturing the host cell in a suitable culture medium; (3) isolating and purifying the protein from the culture medium or cells.

[0118] In a specific embodiment of the present invention, the NDT mutant is expressed and purified in Escherichia coli.

[0119] Enzyme preparations

[0120] In some embodiments, the present invention provides an enzyme preparation containing the NDT mutant according to any embodiment of the present invention. In some embodiments, the enzyme preparation is a buffer containing the NDT mutant. Preferably, the buffer is a Tris-HCl buffer with a pH of 5 to 10, preferably 6 to 9, and more preferably 7 to 8.

[0121] In some embodiments, the enzyme preparation is a powder, such as a lyophilized powder.

[0122] In some embodiments, the enzyme preparation is the supernatant obtained after lysis of the host cells expressing the NDT mutant described herein, such as Escherichia coli. Lysis can be performed by methods well known in the art. For example, the bacterial solution can be washed twice with 50mmol / L Tris-HCl, and then the bacterial solution can be resuspended in 50mmol / L Tris-HCl (pH7.0) buffer, and the cells are ultrasonically disrupted in an ice bath. The ultrasonically disrupted sample is centrifuged to obtain a supernatant. In some embodiments, the enzyme preparation can be a concentrated solution of the supernatant, and the solvent in the supernatant can be removed by conventional methods. In some embodiments, the enzyme preparation can be a lyophilized product of the supernatant.

[0123] application

[0124] The NDT mutant of the present invention can be used to transfer 2'-fluoro-2'-deoxyribose (group) to add 2'-fluoro-2'-deoxyribose (group) to a substrate; or to prepare a reagent (including a catalytic reagent, such as an enzyme preparation) or a culture medium for catalyzing the transfer of 2'-fluoro-2'-deoxyribose (group).

[0125] In a preferred embodiment, the NDT mutant of the present invention is used to take a guanine analog as a substrate, and add a 2'-fluoro-2'-deoxyribose (group) thereto to form a 2'-deoxy-2'-fluoroguanosine analog.

[0126] The mutant NDT of the present invention or the host cell expressing the mutant NDT can be formulated in a carrier acceptable for industrial synthesis to obtain a composition suitable for catalytic reaction or storage.

[0127] The mutant NDT of the present invention or the host cell containing the same can also be placed in a kit for industrial use or sale. In addition, the kit can also contain a deoxyribose acceptor and a deoxyribose donor involved in the reaction, as well as auxiliary reagents such as a reaction buffer and a reaction termination reagent. In the present invention, the deoxyribose acceptor is cytosine. Generally speaking, the kit also includes instructions for use.

[0128] It should be understood that in the present invention, the "deoxyribose donor" can be a variety of compounds, as long as they can be recognized and acted upon by the NDT enzyme of the present invention to obtain a 2'-fluoro-2'-deoxyribose group.

[0129] Synthesis of 2'-deoxy-2'-fluoroguanosine analogs catalyzed by N-deoxyribosyltransferase II

[0130]

[0131] In the present invention, 2'-fluoro-2'-deoxyribonucleoside and guanine analogs are used as substrates, and N-deoxyribosyltransferase II or its mutant described herein is used to catalyze the transfer of 2'-fluoro-2'-deoxyribose in ribose donor I to guanosine analogs to synthesize 2'-fluoro-2'-deoxyguanosine analogs.

[0132] In the present invention, 2'-fluoro-2'-deoxyribose comes from a deoxyribose donor. The deoxyribose donor is a compound carrying 2'-fluoro-2'-deoxyribose, as shown in Formula I. In Formula I, R is a base, which can be selected from adenine, hypoxanthine, uracil, cytosine and thymine, etc. Exemplary compounds of Formula I include 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxyinosine, 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxythymidine, and 2'-fluoro-2'-deoxycytidine. In some embodiments, the deoxyribose donor I is 2'-fluoro-2'-deoxyuridine.

[0133] In the present invention, the guanine analog is as shown in Formula II, wherein X is selected from: OH, Cl, S, F or C1-C4 alkoxy, preferably OMe or Cl, more preferably OMe.

[0134] The pH value of the reaction system of the above reaction can be 5 to 10, preferably 6 to 9, and more preferably 7 to 8. The reaction temperature can be 30 to 80° C., preferably 40 to 70° C. A Tris-HCl buffer, for example, can be used to provide the pH required for the reaction. The exemplary Tris-HCl buffer preferably has a pH of 7 to 8 and a concentration of 30 to 70 mM.

[0135] The molar ratio of the deoxyribose donor I to the guanine analog II in the reaction system can be 1:(1-5), such as 1:(1-3).

[0136] The mass of N-deoxyribosyltransferase II or its mutant in the reaction system may be 10-80% of the mass of the deoxyribose donor, preferably 40-60%.

[0137] In some embodiments, the amino acid sequence of the N-deoxyribosyltransferase II may be as shown in any one of SEQ ID NOs: 1 to 5. The N-deoxyribosyltransferase II mutant may be the N-deoxyribosyltransferase II mutant described in any embodiment herein. It should be understood that more than one N-deoxyribosyltransferase II and / or its mutants may be used in the same reaction.

[0138] After obtaining the information of the mutant N-deoxyribosyltransferase II described in the present invention, those skilled in the art will know how to use the mutant to catalyze the synthesis of 2'-fluoro-2'-deoxyguanosine analogs using 2'-fluoro-2'-deoxyribonucleoside and guanine analogs as substrates. For example, 2'-fluoro-2'-deoxyuridine and the guanine analog shown in formula II with OMe as the X substituent are used as substrates to catalyze the synthesis of 2'-fluoro-2'-deoxyguanosine analogs. Various intracellular or extracellular catalytic synthesis methods are included in the present invention or can be used in the present invention.

[0139] Therefore, the present invention also provides a method for catalytically synthesizing 2'-fluoro-2'-deoxyguanosine analogs, which comprises using 2'-fluoro-2'-deoxyribonucleoside and guanine analogs as substrates, and using the mutant N-deoxyribosyltransferase II of the present invention to catalyze the synthesis of 2'-fluoro-2'-deoxyguanosine analogs. In this method, a buffer solution can be used as a reaction solvent so that the reaction is carried out under certain pH conditions. In some embodiments, Tris-HCl buffer is used as a reaction solvent. The pH value of the reaction system can be 5 to 10, preferably 6 to 9, and more preferably 7 to 8. The reaction temperature can be 30 to 80°C, preferably 40 to 70°C. The molar ratio of 2'-fluoro-2'-deoxyuridine to guanine analogs can be 1:(1 to 3), preferably 1:(1.5 to 2.5). The mass of N-deoxyribosyltransferase can be 10 to 80% of the mass of 2'-fluoro-2'-deoxyuridine, preferably 40 to 60%.

[0140] Compared with the prior art, the mutant N-deoxyribosyltransferase II obtained in the present invention efficiently catalyzes 2'-fluoro-2'-deoxyribose and guanine analogs in the enzyme reaction. Compared with the wild-type protein, the catalytic efficiency of the mutant is greatly increased, and the action range of the substrate can be expanded, and it is effective for a variety of substrates.

[0141] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0142] The experimental methods in the following examples where specific conditions are not specified are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0143] Unless otherwise indicated, percentages and parts are by weight.

[0144] Unless otherwise specified, the experimental materials involved in the present invention are all commercial reagents and can be obtained from commercial channels.

[0145] Example 1: Preparation of N-deoxyribosyltransferase NDT enzyme powder

[0146] 1. Construction and transformation of recombinant vector

[0147] 3-5 μL of pET-30a-LhNDT recombinant plasmid (commissioned by Jinweizhi Company to synthesize) containing the target gene LhNDT (Genbank: AY064167.1, amino acid sequence as shown in SEQ ID NO: 1) was added to 50-100 μL of E. coli BL21 (DE3) competent cells, placed on ice for 20 minutes, heat shocked at 42°C for 90 seconds, quickly returned to ice for 5 minutes, added 800 μL of resistance-free LB culture medium, incubated at 37°C, 200 rpm, and spread on LB agar plate medium containing kanamycin, and cultured overnight at 37°C for 12 hours to obtain recombinant bacteria E. coli BL21 (DE3) containing pET-30a-LhNDT.

[0148] The formula of the non-resistant LB culture medium is as follows: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L;

[0149] The formula of LB agar plate culture medium containing kanamycin resistance is as follows: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L, agar powder 20g / L, kanamycin 50μg / mL.

[0150] 2. Construction of expression vector and induced expression

[0151] The recombinant E. coli BL21 (DE3) containing pET-30a-LhNDT obtained in 1 was directly spread on a solid LB plate containing 50 μg / mL kanamycin resistance, and cultured at 37°C for 12-14 hours to obtain a monoclonal colony. A single colony of E. coli BL21 (DE3) containing the LhNDT recombinant vector was picked from the solid LB plate containing kanamycin, inoculated into 1 mL of liquid LB medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C for 12 hours. Then, the inoculation amount was 2% (v / v) and inoculated into a fresh 1L liquid LB medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C until OD 600 When the pH value was about 0.6-0.8, IPTG (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to a final concentration of 1.0 mmol / L. Expression was induced at 200 rpm and 25°C for 20 h. The supernatant was removed by centrifugation (4°C, 4000 rpm, 30 min). The obtained bacterial sludge was washed with 30 mL of 0.9% NaCl solution and resuspended for later use.

[0152] 3. Preparation of freeze-dried enzyme powder

[0153] The bacterial solution obtained in 2 was washed twice with 50 mmol / L Tris-HCl, and then resuspended in 50 mmol / L Tris-HCl (pH 7.0) buffer, and the cells were ultrasonically disrupted in an ice bath (amplifier 6, power 500 W, on 2 s, off 5 s, 30 minutes). The ultrasonically disrupted sample was centrifuged at 12000 rpm and 4°C for 30 min, and the supernatant was freeze-dried in a -80°C freeze dryer for 24 h. The freeze-dried sample was ground to prepare freeze-dried enzyme powder.

[0154] The same method was used to prepare N-deoxyribosyltransferase NDT enzyme powder from Lactobacillus delbrueckii subsp. lactis (EGD27012.1, SEQ ID NO: 2), Lactobacillus reuteri (WP_003667788.1, SEQ ID NO: 3), Lactobacillus leichmannii (WP_035184009.1, SEQ ID NO: 4) and Lactobacillus shilgardii (WP_223801675.1, SEQ ID NO: 5).

[0155] NDT-G1 (SEQ ID NO: 1)

[0156] MNKKKTLYFGAGWFNEKQNKAYKEAMAALKENPTVDLENSYVPLENQYKGIRIDEHPEYLHNIEWASATYHNDLVGIKTSDVMLGVYLPEEEDVGLGMELGYALSQGKYILLVIPDEDYGKPINLMSWGVCDNAIKISELKDFDFNKPRYNFYDGAVY.

[0157] NDT-G2 (SEQ ID NO: 2)

[0158] MPKKTIYFGAGWFTDRQNKAYKEAMEALKENPTVDLENSYVPLDNQYKGIRVDEHPEYLHDKVWATATYNNDLNGIKTNDVMLGVYIPDEEDVGLGMELGYALSQGKYVLLVIPDEDYGKPINLMSWGVSDNVIKMSQLKDFNFNKLRFDFYEGAVY.

[0159] NDT-G3 (SEQ ID NO: 3)

[0160] MINQKSKTVYFCAGWFTDKQNKAYEDAMNAIKANPTVDVENSYVPLQHQYKGLRVDEHPELLQDREWSTATYNGDRVGVSTSDMLLAVYIPEEEDVGMGVELGMARALGKYIMVVIPDEDFGKPINLMSWGIADNFIKMSELPNYDFNKPSYNFYDGGVY.

[0161] NDT-G4 (SEQ ID NO: 4)

[0162] MPKKTIYFGAGWFTDRQNKAYKEAMEALKENPTIDLENSYVPLDNQYKGIRVDEHPEYLHDKVWATATYNNDLNGIKTNDIMLGVYIPDEEDVGLGMELGYALSQGKYVLLVIPDEDYGKPINLMSWGVSDNVIKMSQLKDFNFNKPRFDFYEGAVY.

[0163] NDT-G5 (SEQ ID NO: 5)

[0164] MATHQNSVYLASPFFSDGQKDRIATVVSLLKQNSTIDSDRIFIPQDHQFEQEPFGSFKWQDAVFASDMRQVRKADVVVAILDYQLEEGLTEPDSGTIFEIGAAYQANVPVIMVQFGNNGQLNLMLARSYTAFFNGKDDVTDIKNYDFNNLETRYTDKKVL.

[0165] Example 2: Reaction effect of deoxyribosyltransferase NDT on different guanine analogs

[0166] Reaction:

[0167]

[0168] X = OH, Cl, S, F or OMe.

[0169] Add 0.08mM (20mg) of the compound of formula I and 0.16mM of the compound of formula II to a 4mL reaction bottle, and stir evenly with 2mL Tris-HCl buffer (pH 7.0, 50mM), then add N-deoxyribose transferase (lyophilized enzyme powder), the mass of which is 50% (i.e. 10mg) of the mass of the compound of formula I, and place it in a shaker at 60°C and 1000rpm for 20 hours. After the reaction is completed, take 50μL of the reaction solution and add it to a 1.5mL centrifuge tube, then add 1mL of 50% methanol-water post-treatment solution to terminate the reaction, place the centrifuge tube at 14000rpm for 1min, and use HPLC to detect the result of the supernatant after centrifugation. The detection conditions are: the chromatographic column is a Waters Xbridge C18 chromatographic column; the mobile phase is: mobile phase A: 5% acetonitrile solution containing 10mM NH4OAc, mobile phase B: 95% acetonitrile solution; temperature 15°C; flow rate 1.3mL / min. The X substituent can be selected as OH, Cl, S, F or OMe. The reaction results of substrates with different X substituents are shown in Table 1.

[0170] Table 1

[0171]

[0172]

[0173] From the comparison of experiments 1-5, it can be seen that the catalytic effect of deoxyribosyltransferase (Genbank number AY064167.1) on substrates with different X substituents is significantly different. The conversion rate of the enzyme catalytic reaction of deoxyribosyltransferase (Genbank number AY064167.1) on the compound of formula II with X substituent being OMe or Cl is higher than the conversion rate when X substituent is F, S or OH.

[0174] Example 3: Reaction effect of deoxyribosyltransferase NDT on different 2'-fluoro-2'-deoxyribose donors

[0175] Reaction:

[0176]

[0177] R = adenine, uracil, cytosine, hypoxanthine.

[0178] A guanine analogue with a methoxy substituent X was selected, and different 2'-fluoro-deoxyribose donors I were used. The same reaction operation as in Example 2 was performed, with 0.08 mM of the compound of formula I and 0.16 mM of the compound of formula II, and the amount of N-deoxyribosyltransferase (lyophilized enzyme powder) added was 50% of the mass of the compound of formula I. The reaction results of 2'-fluoro-2'-deoxyribose donors I with different R substituents are shown in Table 2.

[0179] Table 2

[0180] Test No. Enzymes Genbank serial number R Conversion rate (%) 1 Deoxyribosyltransferase LhNDT AY064167.1 Adenine 16.25 2 Deoxyribosyltransferase LhNDT AY064167.1 Uracil 24.35 3 Deoxyribosyltransferase LhNDT AY064167.1 Cytosine 5.25 4 Deoxyribosyltransferase LhNDT AY064167.1 Hypoxanthine 9.05

[0181] From the comparison of experiments 1-4, it can be seen that the reaction effect of deoxyribosyltransferase (Genbank No. AY064167.1, and its amino acid sequence is shown in SEQ ID NO: 1) on 2'-fluoro-2'-deoxyribose donor I with different R substituents is significantly different. When the R substituent is uracil or adenine, the conversion rate of the enzyme-catalyzed reaction is higher than the conversion rate when the R substituent is cytosine or hypoxanthine.

[0182] Example 4: Reaction effects of deoxyribosyltransferases from different sources

[0183] Reaction:

[0184]

[0185] Reaction operation: 2'-fluoro-2'-deoxyuridine and a guanine analogue with a methoxy substituent of X were used as substrates, deoxyribosyltransferases from different sources were used, and the same reaction operation as in Example 2 was performed, with 0.08 mM of the compound of formula I and 0.16 mM of the compound of formula II, and the amount of N-deoxyribosyltransferase (lyophilized enzyme powder) added was 50% of the mass of the compound of formula I. The reaction results are shown in Table 3.

[0186] Table 3

[0187]

[0188] Comparison of experiments 1-5 shows that the reaction effects of deoxyribosyltransferases from different sources are different, among which NDT-G1 has the best reaction effect.

[0189] Example 5: Reaction effects of different mutant N-deoxyribosyltransferases

[0190] Reaction:

[0191]

[0192] The sequence of SEQ ID NO:1 (NDT-G1, the coding sequence is shown in SEQ ID NO:6) was used as the parent, and the directed evolution was transformed by roller PCR, iterative saturation mutation, and combination mutation strategies. Then the mutant was transformed into E.coli BL21 (DE3) competent cells, and evenly spread on LB agar plates containing 50μg / mL kanamycin, and placed in a 37℃ incubator for static culture for 18h. The mutants on the transformed plate were picked into a 96-well plate with a sterile toothpick and cultured overnight in a shaker at 37℃ and 250rpm. 50μl of bacterial solution was drawn from the holes of the primary plate and inoculated into the corresponding holes of the secondary plate. After culturing at 37℃ and 250rpm for 2-3h, IPTG with a final concentration of 1mM was added, and the mutants were cultured at 25℃ for 20h to obtain the corresponding mutants for high-throughput screening. Combined with HPLC detection and rescreening, mutants with significantly improved activity and stability were identified for gene sequencing.

[0193] 2'-fluoro-2'-deoxyuridine was used as the deoxyribose donor, a guanine analogue with a methoxy substituent X was selected, and the same reaction operation as in Example 2 was performed, including using 0.08 mM of the compound of formula I and 0.16 mM of the compound of formula II, and the amount of N-deoxyribosyltransferase (lyophilized enzyme powder) added was 50% of the mass of the compound of formula I. Different N-deoxyribosyltransferase mutants were used for the experiment. The reaction results are shown in Table 4.

[0194] Table 4

[0195]

[0196]

[0197]

[0198] From the above results, it can be seen that the mutated NDT-H35 has higher catalytic efficiency and the conversion rate is 2.4 times higher than that of the wild-type NDT-G1.

[0199] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. An N-deoxyribosyltransferase, characterized in that The N-deoxyribosyltransferase: (a) compared to the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence has a substitution mutation at any one position, any two positions, any three positions, any four positions, any five positions, any six or more positions, any seven or more positions, any eight or more positions, or all nine positions among positions 9, 54, 60, 63, 68, 82, 88, 91 and 154; or (b) is a protein derived from (a) which is obtained by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of (a), and has the function of N-deoxyribosyltransferase II, and the amino acid residues corresponding to positions 9, 54, 60, 63, 68, 82, 88, 91 and 154 of SEQ ID NO: 1 are as described in (a); or (c) is a protein derived from (a) that has more than 80% identity with the amino acid sequence of (a) and has N-deoxyribosyltransferase II function, and the amino acid residues corresponding to positions 9, 54, 60, 63, 68, 82, 88, 91 and 154 of SEQ ID NO:1 are as described in (a).

2. The N-deoxyribosyltransferase according to claim 1, characterized in that: The mutation at position 9 is a mutation of phenylalanine to a non-polar amino acid selected from alanine, leucine, isoleucine, proline, tryptophan, methionine or glycine, or a mutation to an uncharged polar amino acid such as serine, threonine, cysteine, tyrosine, asparagine or glutamine; The mutation at position 54 is a mutation of aspartic acid to a positively charged polar amino acid, such as lysine, arginine or histidine, or a mutation to a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine; The mutation at position 60 is a mutation of leucine to an uncharged polar amino acid, such as serine, threonine, tyrosine, asparagine or glutamine; The mutation at position 63 is that isoleucine is mutated to a polar amino acid with a positive charge, such as lysine, arginine or histidine, or is mutated to a polar amino acid with a negative charge, such as aspartic acid or glutamic acid; The mutation at position 68 is a mutation of alanine to a non-polar amino acid, such as valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine, or a mutation to an uncharged polar amino acid, such as serine, threonine, cysteine, tyrosine, asparagine or glutamine; The mutation at position 82 is a mutation of valine to a positively charged polar amino acid, such as lysine, arginine or histidine, or a mutation to a non-polar amino acid, such as alanine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine; The mutation at position 88 is a mutation of leucine to an uncharged polar amino acid, such as threonine, glutamine, asparagine, tyrosine or cysteine, or a mutation to a negatively charged polar amino acid, such as aspartic acid or glutamic acid, or a mutation to a non-polar amino acid, such as alanine, valine, isoleucine, proline, phenylalanine, methionine or glycine; The mutation at position 91 is a mutation of glutamic acid to an uncharged polar amino acid, such as serine, threonine, glutamine, asparagine, tyrosine or cysteine, or a mutation to a polar positively charged amino acid, such as lysine, arginine or histidine; The mutation at position 154 is a mutation of aspartic acid to an uncharged polar amino acid, such as serine, threonine, glutamine, asparagine, tyrosine or cysteine, or a mutation to a positively charged polar amino acid, such as lysine, arginine or histidine.

3. The N-deoxyribosyltransferase according to claim 2, characterized in that: The mutation at position 9 is a mutation of phenylalanine to glycine, alanine, leucine or isoleucine, or a mutation to serine or threonine; The mutation at position 54 is aspartic acid mutated to arginine or histidine, or mutated to leucine or isoleucine; The mutation at position 60 is a mutation from leucine to asparagine or glutamine; The mutation at position 63 is that isoleucine is mutated to arginine or histidine, or is mutated to aspartic acid or glutamic acid; The mutation at position 68 is a mutation of alanine to phenylalanine, tryptophan or tyrosine; The mutation at position 82 is a mutation of valine to arginine, histidine or phenylalanine; The mutation at position 88 is a mutation of leucine to cysteine, aspartic acid, glutamic acid or valine; The glutamic acid at position 91 is mutated to serine, threonine or arginine; The mutation at position 154 is the mutation of aspartic acid to glutamine, asparagine, lysine or arginine.

4. The N-deoxyribosyltransferase according to claim 3, characterized in that: The mutation at position 9 is a mutation of phenylalanine to glycine or threonine; The mutation at position 54 is a mutation of aspartic acid to histidine or leucine; The mutation at position 60 is a mutation from leucine to glutamine; The mutation at position 63 is a mutation of isoleucine to arginine, histidine or glutamic acid; The mutation at position 82 is a mutation of valine to arginine or phenylalanine; The mutation at position 88 is a mutation of leucine to cysteine, glutamic acid or valine; The glutamic acid at position 91 is mutated to serine or arginine; The mutation at position 154 is a mutation from aspartic acid to asparagine, lysine or arginine.

5. The N-deoxyribosyltransferase according to claim 1, wherein Compared with the amino acid sequence shown in SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase is selected from the group consisting of: F9T, F9A, D54L, D54H, L60Q, I63E, I63R, I63H, A68F, A68W, A68Y, V82R, V82F, L88C, L88E, L88V, E91S, E91Y, D154K, D154N and D154R.

6. The N-deoxyribosyltransferase according to any one of claims 1 to 5, characterized in that: Compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribose transferase has the substitution mutation at position 9 at least at position 9, and further has the corresponding substitution mutations at any one, any two or any three of positions 54, 60, 63, 68, 82, 88, 91 and 154, preferably further has the corresponding substitution mutations at any one, any two or all three of positions 54, 63 and 68; or Compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribose transferase has the substitution mutation at position 60 at least at position 60, and further has the corresponding substitution mutation at any one, any two or any three positions among position 9, position 54, position 63, position 68, position 82, position 88, position 91 and position 154, preferably further has the corresponding substitution mutation at any one, any two, any three or all four positions among position 54, position 88, position 91 and position 154; or Compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribosyltransferase has the substitution mutation at least at position 63, and further has the corresponding substitution mutation at any one, any two or any three positions among position 9, 54, 60, 68, 82, 88, 91 and 154, preferably further has the corresponding substitution mutation at any one, any two, any three, any four, any five or all six positions among position 9, 54, 82, 88, 91 and 154; or Compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribose transferase has the substitution mutation at position 68 at least at position 68, and further has the corresponding substitution mutation at any one, any two or any three positions among position 9, position 54, position 60, position 63, position 82, position 88, position 91 and position 154, preferably further has the corresponding substitution mutation at any one, any two, any three, any four or all five positions among position 9, position 54, position 82, position 88 and position 91; or Compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribose transferase has the substitution mutation at position 82 at least at position 82, and further has the corresponding substitution mutation at any one, any two or any three positions among position 9, position 54, position 60, position 63, position 68, position 88, position 91 and position 154, preferably further has the corresponding substitution mutation at any one, any two or all three positions among position 63, position 91 and position 154; or Compared with the amino acid sequence shown in SEQ ID NO: 1, the N-deoxyribose transferase has the substitution mutation at position 88 described herein at least at position 88, and further has the corresponding substitution mutation at any one, any two or any three positions among position 9, position 54, position 60, position 63, position 68, position 82, position 91 and position 154, preferably further has the corresponding substitution mutation at any one, any two, any three or all four positions among position 60, position 68, position 91 and position 154.

7. The N-deoxyribosyltransferase according to claim 6, characterized in that Compared with SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase is selected from any one of the following combinations: (1) Mutations at positions 9, 54, and 68; (2) mutations at positions 9 and 63; (3) mutations at positions 9 and 68; (4) mutations at positions 60, 88, and 91; (5) mutations at positions 54 and 60; (6) mutations at positions 54, 63, and 91; (7) mutations at positions 60 and 154; (8) mutations at positions 63 and 82; (9) mutations at positions 63 and 154; (10) mutations at positions 68 and 88; (11) mutations at positions 68 and 91; (12) mutations at positions 82, 91, and 154; (13) Mutations at positions 88 and 154.

8. The N-deoxyribosyltransferase according to claim 6, characterized in that Compared with SEQ ID NO:1, the mutation of the N-deoxyribosyltransferase is selected from any one of the following combinations: (1) F9T, D54H and A68F; (2) F9T and I63E; (3) F9T and A68F; (4) L60Q, L88C and E91S; (5) D54H and L60Q; (6) D54H, I63E and E91S; (7) L60Q and D154K; (8) I63R and V82R; (9) I63E and D154R; (10) A68W and L88V; (11) A68F and E91S; (12) V82R, E91Y and D154K; (13) L88C and D154K. 9 . A polynucleotide molecule encoding the N-deoxyribosyltransferase according to claim 1 , or a complementary sequence thereof.

10. A nucleic acid construct comprising the polynucleotide molecule according to claim 9 or its complementary sequence.

11. The nucleic acid construct according to claim 10, wherein Also the construct is an expression cassette.

12. The nucleic acid construct according to claim 10, wherein The construct is a vector, preferably an expression vector or a recombinant vector.

13. A host cell, characterized in that The host cell: (1) expressing the N-deoxyribosyltransferase according to any one of claims 1 to 8; and / or (2) A nucleic acid construct comprising the polynucleotide molecule according to claim 9 or its complementary sequence, or the nucleic acid construct according to any one of claims 10 to 12.

14. An enzyme preparation, characterized in that The enzyme preparation contains the N-deoxyribosyltransferase according to any one of claims 1 to 8.

15. The enzyme preparation according to claim 14, characterized in that: The enzyme preparation further contains a buffer; preferably, the buffer is a Tris-HCl buffer with a pH of 5 to 10, preferably 6 to 9, more preferably 7 to 8; or The enzyme preparation is a powder, such as a freeze-dried powder; or The enzyme preparation is the supernatant obtained after the host cells according to claim 13 are lysed, or its concentrated solution or lyophilized product.

16. Use of an N-deoxyribosyltransferase selected from any one of claims 1 to 8 and an N-deoxyribosyltransferase whose amino acid sequence is shown in any one of SEQ ID NOs: 1 to 5 in transferring a 2'-fluoro-2'-deoxyribose group to a base, or in preparing a preparation for transferring a 2'-fluoro-2'-deoxyribose group to a base.

17. A method for synthesizing 2'-deoxy-2'-fluorouridine represented by the following formula III: It is characterized in that The method comprises the steps of transferring the 2'-fluoro-2'-deoxyribose group in the 2'-fluoro-2'-deoxyribonucleoside of formula I as a ribose donor to the guanine analog of formula II as a ribose acceptor using N-deoxyribose transferase; Wherein, the N-deoxyribosyltransferase is selected from the N-deoxyribosyltransferase of any one of claims 1 to 8 and the N-deoxyribosyltransferase whose amino acid sequence is shown in any one of SEQ ID NOs: 1 to 5; R is a base selected from adenine, hypoxanthine, uracil, cytosine and thymine, preferably adenine or uracil; X is selected from: OH, Cl, S, F or C1-C4 alkoxy, preferably OMe or Cl.

18. The method according to claim 17, characterized in that The method has one or more of the following features: The pH value of the reaction system is 5 to 10, preferably 6 to 9, more preferably 7 to 8; The reaction temperature is 30 to 80°C, preferably 40 to 70°C; In the reaction system, the molar ratio of ribose donor I to guanine analog II is 1:(1-5), such as 1:(1-3); In the reaction system, the mass of the N-deoxyribosyl transferase is 10-80% of the mass of the ribose donor I, preferably 40-60%.

19. The method according to claim 17 or 18, characterized in that: The N-deoxyribosyltransferase is as described in any one of claims 1 to 8; In formula I, R is uracil; In formula II, X is a methoxy group.

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