WO2019066172A1 - N-deoxyribosyl transferase mutant and nucleoside preparing method using same - Google Patents

Abstract

The present invention relates to an N-deoxyribosyl transferase mutant, a polynucleotide coding for the mutant, a vector carrying the polynucleotide, a microorganism containing the polynucleotide, and a method for preparing a nucleoside, using the same vector and microorganism.

Description

N-deoxyribosyltransferase mutant and method for producing nucleoside using the same

The present invention relates to a N-deoxyribosyltransferase mutant, a polynucleotide encoding the mutant, a vector containing the polynucleotide and a microorganism, and a method for producing a nucleoside using the polynucleotide.

The oligonucleotide therapeutic agent directly binds DNA or RNA having genetic information in vivo to block disease-related protein production (antisense drug), aptamer that binds protein, small-interferring RNA functioning as double strand such as siRNA, micro-RNA and antagomir, Decoy acting on transcription factors, and CpG oligonucleotides that cause immune activation.

In order for these oligonucleotide therapeutic agents to be introduced into desired cells and exhibit activity, it is necessary to increase the stability to resist in vivo conditions including rapid degradation by various nucleic acid degrading enzymes. Accordingly, various studies have been made to increase the stability of oligonucleosides by using an unnatural nucleoside (a nucleotide which is a component of nucleic acid or a nucleoside introducing a chemical modification into sugar).

In particular, the 2'-deoxy-2'-fluoronucleosides are excellent in stability against in vivo nucleic acid degrading enzymes, and are thermodynamically stable with an increase in the melting temperature of about 1.8 ° C. per the number of nucleotides in the formation of the RNA duplexes The results have been confirmed and are recently attracting attention as a raw material for an oligonucleotide therapeutic agent. 2'-deoxy-2'-fluoro nucleoside has been known as an industrially difficult substance using an enzyme as a raw material of oligonucleotides so far. Up to now, two kinds of enzymatic production methods using biotransformation of 2'-deoxy-2'-fluoro nucleoside have been reported. One using N-deoxyribosyltransferase derived from Lactobacillus reuteri was confirmed to be reactive with 2'-fluoro-2'-deoxynucleoside and the other was confirmed with purine phosphorylase, And pyrimidine phosphorylase have been known as an enzyme production method using bioconversion. However, the conventional bioconversion process has a difficulty in the manufacturing process because it shows low purity and yield such as a large amount of enzyme use, low substrate concentration and long reaction time.

In addition, the enzyme synthesis method is a method which enables the industrial production of 2'-deoxy-2'-fluoro nucleoside in terms of cost and efficiency, but unlike a chemical synthesis method or a hydrolysis method, an enzyme which is a biological catalyst is used There is a problem in that the yield and stable production of the final product differ greatly depending on differences in the types of enzymes, differences in the origins and amino acid sequences, plasmid constructs, host strains, and culture conditions for gene expression .

Therefore, in order to produce an efficient and stable 2'-deoxy-2'-fluoro nucleoside, an enzyme gene having a strong activity and stable expression is searched, and a gene expression system capable of stably expressing these genes .

Under these technical backgrounds, the present inventors have made intensive efforts to develop novel N-deoxyribosyltransferases having high activity, and have found that they have high substrate reactivity to 2'-deoxy-2'-fluoronucleosides It was found that a novel N-deoxyribosyltransferase mutant can be produced and that an oligonucleoside can be produced with high yield and high purity using this N-deoxyribosyltransferase mutant Respectively.

One object of the present invention is to provide an N-deoxyribosyltransferase mutant having excellent transglycosylation activity and substrate reactivity.

Another object of the present invention is to provide a polynucleotide encoding the mutant, a vector containing the polynucleotide, a microorganism into which the vector is introduced, and a method for producing an N-deoxyribosyltransferase mutant using the microorganism will be.

It is still another object of the present invention to provide a method for producing a nucleoside using the N-deoxyribosyltransferase mutant or a microorganism expressing the mutant.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.

One aspect of the present invention for attaining the above object is to provide a method for producing an N-deoxyribosyltransferase comprising the L59Q mutation in which the 59th leucine in the amino acid sequence of SEQ ID NO: 2 is substituted with glutamine (nucleoside deoxyribosyl transferase (NDT) variant.

The N-deoxyribosyltransferase mutant of the present invention exhibits an activity of highly efficiently catalyzing the transfer or transglycosylation reaction of 2'-deoxy-2'-fluororibos, There is a characteristic of exhibiting high activity and substrate reactivity compared to the known N-deoxyribosyltransferase.

The N-deoxyribosyltransferase mutant may have an increased substrate reactivity to 2'-Deoxy-2'-fluoronucleoside, The 2'-deoxy-2'-fluoro nucleoside may be, but is not limited to, 2'-Deoxy-2'-fluorouridine.

The N-deoxyribosyltransferase having the amino acid sequence of SEQ ID NO: 2 may be derived from Lactobacillus delbrueckii . The NDT mutant may have the amino acid sequence of SEQ ID NO: 10, in which leucine (L), which is the 59th amino acid in SEQ ID NO: 2, is substituted with glutamine (Q).

The term " N-deoxyribosyltransferase (NDT) " in the present invention refers to an enzyme also called trans-N-deoxyribosylase, which is a strain of Lactobacillus helveticus (Biochem. J. 50: 383-397, 1952), two isotopes of DRTase I and II are known. Among them, DRTase I catalyzes the transfer reaction of deoxyribose between two purines and DRTase II catalyzes the transfer of purine, pyrimidine or purine and pyrimidine deoxyribose Catalyzed. Lactobacillus leichmannii DRTase II has the activity of hydrolyzing the nucleoside to base and deoxyribose in the absence of the acceptor base, as well as this transfer reaction. The DRTase II enzyme derived from L. helveticus or L. leichmannii strain has low specificity for the receptor base and stereospecificity for producing only the β-anomer in the sugar transfer reaction, Crude extracts of these two strains are widely used for analog synthesis of antiviral or anticancer nucleosides.

In one embodiment of the present invention, the NDT variant comprising the L59Q mutation in the amino acid sequence of SEQ ID NO: 2 may be a mutant comprising the amino acid sequence of SEQ ID NO: 10.

In one specific embodiment of the present invention, NDT random mutation strain library was obtained by error prone PCR using the NDT gene containing the nucleotide sequence of SEQ ID NO: 1 as a template, The variants of the present invention screened through substrate specificity screening with 2'-Deoxy-2'-Fluorouridine (2FDU) are higher than NDT with the amino acid sequence of SEQ ID NO: 2 Activity and substrate reactivity (Examples 3 and 4).

In another aspect, the present invention provides a polynucleotide encoding the NDT variant, a recombinant vector comprising the polynucleotide, a microorganism into which the recombinant vector is introduced, and a method for producing the NDT mutant using the microorganism.

The NDT mutant is as described above.

As used herein, the term " polynucleotide " has the meaning inclusive of DNA (gDNA and cDNA) and RNA molecules, and the nucleotide, which is the basic constituent unit in the polynucleotide, includes not only natural nucleotides, (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90: 543-584). The polynucleotide may be an isolated polynucleotide.

The polynucleotide encoding the NDT variant may be derived from Lactobacillus delbrueckii . The sequence of a polynucleotide encoding an NDT variant of the present invention may be modified, and the modification includes addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.

The polynucleotides of the present invention are also interpreted to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences described above. The above substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art, , Specifically at least 90% homology, more specifically at least 95% homology.

In one embodiment of the present invention, the NDT variant comprising the amino acid sequence of SEQ ID NO: 10 in which the 59th leucine in the amino acid sequence of SEQ ID NO: 2 is replaced with glutamine may be encoded by the nucleotide sequence of SEQ ID NO:

As used herein, the term " vector " refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cosmeptide vector; And viral vectors such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated viral vector, and specifically, a virus vector prepared by the present applicant and deposited in the Korea Culture Center of Microorganisms (KCCM) pFRPT (Accession No .: KCCM-10320).

A polynucleotide encoding an NDT variant in the vector of the present invention may be operatively linked to a promoter.

As used herein, the term " operably linked " means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The regulatory sequence regulates transcription and / or translation of the different nucleic acid sequences.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) , Which is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (such as a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL 貫 promoter, , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for initiation of decoding, and a transcription / translation termination sequence. The promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., (1984) 158: 1018-8), when E. coli (e.g. HB101, BL21, 1024) and the left promoter of phage lambda (pL? Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980) 14: 399-445). When a Bacillus bacterium is used as a host cell, the promoter of the toxin protein gene of Bacillus subtilis (Appl. Environ. Microbiol. (1998) 64: 3932-3938; Mol. Gen. Genet. (1996) 250: 734-741 ) Or any promoter capable of expressing in Bacillus may be used as a regulatory region.

Meanwhile, the recombinant vector of the present invention can be prepared by using a plasmid (for example, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pGEX series, pET series, pUC19, etc.), phage (e.g., λgt4 · λB, λ-Charon, λΔz1 and M13) or viruses (eg SV40 and the like). For example, the recombinant vector of the present invention can be produced by manipulating an expression vector for E. coli, specifically, pFRPT (Korean Patent Registration No. 10-0449639), but is not limited thereto.

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell such as a metallothionine promoter, a beta -actin promoter, a human hemoglobin promoter, Promoter), or a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse breast tumor virus (MMTV) promoter, The LTR promoter of HIV, the promoter of Moloney virus promoter Epstein bar virus (EBV) and the promoter of rosacekoma virus (RSV)), and generally has a polyadenylation sequence as a transcription termination sequence.

On the other hand, the recombinant vector of the present invention includes an antibiotic resistance gene commonly used in the art as a selection marker, and includes, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The microorganism capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any microorganism known in the art. For example, Escherichia coli , Bacillus subtilis and Bacillus turginensis such as Bacillus sp, Streptomyces (Streptomyces), Pseudomonas (Pseudomonas) (e.g., Pseudomonas footage is (Pseudomonas putida)), Proteus Mira Billy's (Proteus mirabilis) or Staphylococcus (Staphylococcus) (e.g. , Staphylococcus ( Staphylocus carnosus ), and the like).

Suitable eukaryotic microbes of said vector include fungi such as Aspergillus species , Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces , And yeast such as Neurospora crassa , other sub-eukaryotic cells, cells of higher eukaryotes such as insect-derived cells, and cells derived from plants or mammals.

In one embodiment of the present invention, the microorganism into which the recombinant vector is introduced may be Escherichia coli such as E. coli DH5 ?, JM109, specifically E. coli DH5 ?.

In the present invention, " transformation " and / or " transfection " to a microorganism includes any method of introducing the polynucleotide into an organism, cell, tissue or organ, Technology can be selected and performed. Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, Agrobacterium mediated transformation, PEG, dextran sulfate , Lipofectamine, and dry / inhibited mediated transformation methods, and the like.

In one embodiment of the present invention, a microorganism comprising a polynucleotide encoding an NDT variant having the amino acid sequence of SEQ ID NO: 10 is activated or catalyzed by a transglycosylation reaction or a 2'-deoxy-2'-fluoro The substrate reactivity to the nucleoside may be increased and the activity to catalyze the N-glucoside bond between the base and the sugar moiety of the nucleoside may be increased. The polynucleotide may comprise the nucleotide sequence of SEQ ID NO: 9.

The present invention provides a method for producing an NDT variant using the microorganism. Specifically, the production method comprises the steps of: (a) culturing a microorganism transformed with the recombinant vector of the present invention; And (b) recovering the N-deoxyribosyltransferase mutant from the culture.

The cultivation of the transformed microorganism in the NDT variant production can be carried out according to a suitable culture medium and culture conditions known in the art. Such a culturing process can be easily adjusted according to the strain selected by those skilled in the art. Such various culturing methods are disclosed in various publications (e.g., James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). Cell culture can be classified into batch, infusion, and continuous culture depending on the suspension culture, adhesion culture, and culture method according to the cell growth method. The medium used for the culture should suitably meet the requirements of the particular strain.

In animal cell cultures, the medium comprises various carbon sources, nitrogen sources and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, Alcohols such as glycerol and ethanol, and organic acids such as acetic acid, and these carbon sources may be used singly or in combination.

Nitrogen sources that can be used in the present invention include, but are not limited to, organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat, and urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, And the like. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salts as a source. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors and the like may be included.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, foaming can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture. In addition, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain the aerobic state of the culture.

The NDT mutant obtained by culturing the transformed microorganism can be used in an unpurified state and can be further purified and used in high purity by various conventional methods such as dialysis, salt precipitation and chromatography.

In another aspect, the present invention provides a method for producing a nucleoside by forming an N-glucoside bond between a donor and a donor of a sugar residue using a NDT mutant of the present invention or a microorganism expressing the mutant .

The nucleoside preparation method comprises reacting a base donor and a sugar residue donor using a microorganism expressing an NDT variant to form an N-glucoside bond between the base portion of the base donor and the sugar portion of the sugar residue donor, Lt; RTI ID = 0.0 > a < / RTI >

The NDT mutant and the microorganism expressing the NDT mutant are as described above.

As used herein, the term " base donor " refers to a material that provides a base for the N-glucoside binding reaction of a nucleoside. The base donor used in the present invention may be appropriately selected according to the objective nucleoside, and may be a heterocyclic base capable of forming N-glucoside bond with a sugar moiety of the sugar residue donor by the action of NDT But is not limited thereto.

Wherein the heterocyclic base is selected from the group consisting of purine and derivatives thereof, pyrimidine and derivatives thereof, triazole and derivatives thereof, imidazole and derivatives thereof, deazapurine and derivatives thereof, azapurine and derivatives thereof, Derivatives or pyridine and derivatives thereof, and the heterocyclic base itself may be a nucleoside having a heterocyclic base as well.

Specifically, a substituent (for example, an amino group, a substituted amino group, a hydroxyl group, an oxo group, a mercapto group, an acyl group, an alkyl group, a substituted alkyl group, Alkoxy group, halogen atom, etc.), such as adenine, guanine, hypoxanthine, xanthine, 6-mercaptoulinine, 6-thioguanine, N-alkyl or acyladenyl, 2-alkoxyadenine , 2-thioadenine, 2,6-diaminopurine and the like;

Pyrimidine derivatives having substituents similar to those described above at one or two or more positions of 2 ', 4' or 5 'of pyrimidine, such as cytosine, uracil, thymine, 5-halogenothrulose (5-fluorouracil , 5-iodoacyl, etc.), 5-halogenothiocin (such as 5-fluorocytosine), 5-trihalogenomethyluracil (such as 5-trifluoromethyluracil) Uracil, N-acyl cytosine, 5-halogenovinyl uracil, and the like;

1,2,4-triazole derivatives having a substituent at the 3 'position of 1,2,4-triazole such as 1,3,4-triazole-3-carboxamide 1,2,4-triazole 3-carboxylic acid, 1,2,4-triazole-3-carboxylic acid alkyl ester, and the like;

Imidazole derivatives having substituents at 4 ' and 5 ' of imidazole, such as 5-amino-4-imidazolecarboxamide, 4-carbamoyl-imidazol-5-olate, benzimidazole and the like;

Deazapurine derivatives such as 1-deaza adenine, 3-deaza adenine, 3-deaza guanine, 7-deaza adenine, 7-deazaguanine or 7-deaza- Compounds having substituents similar to those of the electrophilic derivatives;

Azaporidine derivatives such as 8-azaadenine, 7-deaza-8-azahepoxatin (azopurinol);

Azapyrimidine derivatives such as 5-azathimine, 5-azasitonin, 6-azauracil and the like; or

Pyridine derivatives such as 3-deazaiacyl, nicotinic acid, nicotinic acid amide and the like.

Specifically, it may be adenine, 2,6-diaminopurine, or cytosine.

As used herein, the term " sugar residue donor " refers to a substance that feeds a sugar residue to the N-glucoside binding reaction of a nucleoside. The sugar residue donor used in the present invention may be appropriately selected according to the objective nucleoside and may be a heterocyclic ribose capable of forming N-glucoside bond with the base portion of the base donor by the action of NDT Compound, or deoxyribose compound, but is not limited thereto.

The sugar residue donor may be a ribose compound such as a ribonucleoside such as inosine, guanosine, uridine, ribofuranosyl thymine, and ribose-1-phosphate; or

Examples of deoxyribose compounds include 2'-deoxyinosine, 2'-deoxyguanosine, 2'-deoxyuridine, thymidine, 2 ', 3'-dideoxyinosine, 2' Deoxyribose-1-phosphate, 2,3-dideoxyribose-1-phosphate and the like, such as deoxyuridine, deoxyuridine, deoxyuridine, deoxyuridine, deoxyuridine, Lt; / RTI >

Specifically, it may be thymidine, 2'-deoxyuridine, 2'-deoxy-2'-fluorouridine.

As an embodiment of the present invention, a method for producing a nucleoside comprises treating the N-deoxyribosyltransferase mutant of the present invention with 2'-deoxy-2'-fluorouridine and adenine, Deoxy-2'-fluoroadenosine, which comprises the step of preparing 2'-deoxy-2'-fluoroadenosine.

The method for producing the above 2'-deoxy-2'-fluoroadenosine can be carried out under the temperature condition of 45 ° C to 55 ° C, preferably 48 ° C to 52 ° C, more preferably about 50 ° C. The preparation of the above 2'-deoxy-2'-fluoroadenosine can be carried out at pH conditions of 5.8 to 6.8, preferably 6.1 to 6.5, more preferably about 6.3. The method for preparing the above 2'-deoxy-2'-fluoroadenosine has a substrate concentration of 1.6 to 2.4 M, preferably 1.8 to 2.2 M, more preferably about 2.0 M, as a substrate concentration condition. In the method for preparing deoxy-2'-fluoroadenosine, the concentration of the dried cells to be fed under the enzyme input conditions is 50 to 150 g / L, preferably 80 to 120 g / L, more preferably about 100 g / L .

The process for preparing 2'-deoxy-2'-fluoroadenosine of the present invention may further comprise a purification process for removing impurities enzymes and uracil after the enzyme is converted. The above purification process comprises filtering under purified water and MeOH; And neutralizing the filtered filtrate to a pH of 7 to 8. Purified water and MeOH may be 1.5 to 3.5 times, preferably 2.5 times, and MeOH may be 9.5 to 11.5 times, preferably 10.5 times, of purified water in comparison to 2FDU doses. It may further include a crystallization step of 2FDA after the purification process.

In one specific embodiment of the present invention, 2'-deoxy-2'-fluoro-uridine and adenine are used as the starting material, using the NDT variant expression strain ( E. coli E206) 2'-fluoroadenosine was prepared, and it was confirmed that high purity 2'-deoxy-2'-fluoroadenosine can be produced with high efficiency (Example 5).

In another embodiment of the present invention, a method for producing a nucleoside comprises contacting an N-deoxyribosyltransferase mutant of the present invention with 2'-deoxy-2'-fluorouridine and 2,6-diaminopurine To produce 2'-deoxy-2'-fluoro-2,6-diaminopurine; And 2'-deoxy-2'-fluoro-2,6-diaminopurine was treated with adenosine deaminase derived from lactococcus lactis to prepare 2'-deoxy-2'-fluoroguanosine 2'-deoxy-2'-fluoro-guanosine.

The method for producing the above 2'-deoxy-2'-fluoroguanosine can be carried out under the temperature condition of 45 ° C to 55 ° C, preferably 48 ° C to 52 ° C, more preferably about 50 ° C. The method of preparing the above 2'-deoxy-2'-fluoroguanosine can be carried out under pH conditions of 5.5 to 6.5, preferably 5.8 to 6.3, more preferably about 6.0. The method for preparing the 2'-deoxy-2'-fluoroadenosine according to the present invention is characterized in that the substrate concentration of 2FDU is 1.4 to 1.8 M, preferably 1.5 to 1.7 M, more preferably about 1.6 M, can do. In the preparation method of the above 2'-deoxy-2'-fluoroadenosine, the amount of the dried cells injected under the enzyme loading condition is 1.5 to 2.5 times, preferably about 2.0 times the 2FDU, have.

The method for preparing 2'-deoxy-2'-fluoro-guanosine of the present invention may further include a purification step for removing an enzyme and URA which are impurities after the enzyme is converted. Wherein the purification process comprises: at least one filtration under purified water and MeOH; And neutralizing the filtered filtrate to a pH of 7 to 8. And may further include a crystallization step of 2FDG after the purification process.

In one specific embodiment of the present invention, 2'-deoxy-2'-fluorouridine and 2,6-diaminopurine are used as a substrate to express N-deoxyribosyltransferase mutant ( E. coli E206 ) Was firstly converted into 2'-deoxy-2'-fluoro-2,6-diaminopurine and then 2'-deoxy-2'-fluoroguanosine was prepared using adenosine deaminase , And it was confirmed that high purity 2'-deoxy-2'-fluoroguanosine can be produced with high efficiency (Example 6).

In another embodiment of the present invention, a method for producing a nucleoside comprises treating the N-deoxyribosyltransferase variant of the present invention with 2'-deoxy-2'-fluorouridine and adenine to produce 2 ' - < / RTI >deoxy-2'-fluoroadenosine; And 2'-deoxy-2'-fluoroadenosine to produce 2'-deoxy-2'-fluoroinosine by treating adenosine deaminase derived from lactococcus lactis. -Deoxy-2 ' -fluoroinosine. ≪ / RTI >

In one specific embodiment of the present invention, 2'-deoxy-2'-fluoroadenosine is converted into 2'-deoxy (2'-deoxy) -2'-fluoroadenosine by using a strain expressing N-deoxyribosyltransferase mutant ( E. coli E206) -2'-fluoroinosine was prepared, and it was confirmed that 2'-deoxy-2'-fluoroinosine could be produced with high purity and high efficiency (Example 7).

The NDT mutant of the present invention or the production method using the microorganism expressing the mutant can mass-produce various nucleosides with high purity and high yield.

The N-deoxyribosyltransferase mutant of the present invention exhibits high activity against a non-natural type 2'-fluoro substrate compared with the conventional N-deoxyribosyltransferase, and has high yield and high purity To produce various nucleosides.

1 shows a process for preparing 2'-Deoxy-2'-fluoroadenosine according to the present invention.

2 shows a process for preparing 2'-Deoxy-2'-fluoroguanosine according to the present invention.

3 shows the conversion of 2'-deoxy-2'-fluoroguanosine according to the present invention.

4 shows the results of HPLC analysis of the secondary crystallization of 2'-deoxy-2'-fluoroguanosine according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1. Random Mutagenesis of NDT Gene by Error-prone PCR Method

The nucleoside deoxyribosyl transferase (NDT) gene derived from Lactobacillus delbrueckii KCCM35470, which is presently owned by the present applicant, is a gene having a total size of 474 bp (SEQ ID NO: 1) and has an amino acid sequence of SEQ ID NO: 2 Lt; / RTI > Since the NDT has a very low activity or no activity of the unnatural nucleoside compared to the natural nucleoside, it has been difficult to use the NDT in the enzymatic production method of the nucleoside.

Thus, Applicants have conducted real-time PCR to increase the reactivity of existing NDTs, particularly 2'-Deoxy-2'-Fluorouridine (2FDU) A random mutagenesis of the NDT gene was performed. Real-time PCR is a method of randomly inducing a mutation in a gene between primers by inducing an error in the polymerase reaction that amplifies the gene. Common mutagenesis methods include random mutagenesis using a mutagenic agent, UV irradiation, and site direct mutagenesis to add, remove, and mutate specific nucleotide sequences. However, Induced mutation of the NDT gene, the real - time PCR method was chosen.

The PCR mutagenesis was performed by using the pFRPT-NDT gene (SEQ ID NO: 3) as a template, and the primer sequences for real-time PCR were as shown in Table 1 below.

NdeI and XbaI were inserted at the start codon and stop codon sites of pFRPT-NDT, respectively. The composition of reaction solution and PCR conditions for error prone PCR are shown in Table 2 below.

The error frequency was controlled by controlling the concentration of MnSO ₄ and dNTP in the PCR reaction solution. The condition used in this example was designed to cause the deformation of 5.8 genes per 1000 bp. In the case of the NDT gene, the modification was designed to occur at 2 to 3 nucleotides, and the Diversify PCR Random Mutagenesis Kit (Cat No. 630703, Real-time PCR was performed using Clontech, www.clontech.com).

Next, 484 bp PCR product amplified by agarose gel electrophoresis was confirmed after PCR reaction, and the extracted PCR product was digested with restriction enzymes XbaI and NdeI . The vector to be inserted with the restriction enzyme digested PCR product was constructed using pFRPT-PUNP (purine nucleoside phosphorylase) (SEQ ID NO: 6) prepared by the applicant and pFRPT-PUNP was digested with restriction enzymes XbaI and NdeI The vector with the PUNP gene removed was treated with CIAP (Calf Intestinal Alkaline Phosphatase) to prevent self ligation. The truncated gene and vector were ligated with PCR products and vectors using T4 ligase, and this product was named pFRPT-mNDT. Using Gene pulser, pFRPT-mNDT was transformed into E. coli DH5α, and 1 mL of fresh SOC medium was added and cultured at 37 ° C for 1 hour. The culture broth was plated on LB agar plate medium containing kanamycin and then further cultured at 37 ° C for 20 hours.

Example 2. Random Mutation Verification

In order to construct the NDT random mutagenesis library, it is first necessary to verify the transformed colonies. In order to isolate colonies in which pFRPT-mNDT was normally inserted in the colonies, PCR screening was performed as follows, and primer sequences for PCR screening were as shown in Table 3 below.

The PCR primers were designed using the RBS (ribosome binding site) region of the pFRPT and the base sequence of MCS (multi cloning site). PCR screening was performed using SapphireAmp Fast PCR Master Mix (Cat No. RR350A, TAKARA, co.) was performed according to the method shown in Table 4 below.

PCR was performed under the conditions shown in Table 4. As a result, 20 to 30 colonies of 50 colonies were found to be normal mutations, and most of the remaining colonies were satellite, and 2 to 3 colonies were PCR ptRPT-NDT bp).

 A total of 214 mutants were obtained through repetitive transformation and PCR screening of pFRPT-mNDT. Among them, 10 colonies were randomly selected to extract the plasmid, and then the plasmid was extracted from Bionics (www.bionicsro.co.kr) .

As a result, it was confirmed that nine mutants were modified in one nucleotide, and one mutation was modified in three nucleotides. Two mutants generated a stop codon, four mutants had no amino acid changes, Of the 10 mutations, only four mutations were found to have the amino acid-modified pFRPT-mNDT as planned. Thus, 80-90 colonies, 40% of 214 mutations, are expected to have normal pFRPT-mNDT.

Example 3. Screening of NDT variants with increased activity from NDT random mutations

A total of 214 mutant strains obtained in Example 2 were cultured in 20 mL of LB containing kanamycin for 4 hours, and 20 μL of 1 M IPTG (isopropyl β-D-1-thiogalactopyranoside) Lt; / RTI > The culture broth was centrifuged and suspended in 0.5 mL of purified water to complete the reaction library. The reaction conditions for the first screening of 2FDU substrate-specific highly active strains using the mutagen library are shown in Table 5 below.

A total of 214 mutants were transfected with pFRPT-NDT / DH5α, which was not mutated in the control reaction, and the activity of the mutant was analyzed based on this.

The conversion rate of 2FDA was calculated as follows.

2FDA Reaction Conversion = 2FDA (mole) x 100 / (2FDA (mole) + 2FDU (mole)

As a result, strains having a conversion ratio of 20% or more were selected, and the plasmid showing the highest action effect was selected from the results. Sequence analysis was performed to confirm changes in the gene and amino acid sequence of NDT.

C71, the most effective screening strain among the primary screening strains, was used to detect 2'-deoxy-2'-fluoro-2,6-diaminopurine riboside -diaminopurineriboside (2FDDAP).

The conversion rate of 2FDDAP was calculated as follows.

* 2FDDAP Reaction Conversion Rate = 2FDDAP (mole) x 100 / (2FDDAP (mole) + 2FDU (mole)

Screening was performed at a minimum substrate concentration of 0.2 M, which is capable of actual industrial production. Four kinds of pFRPT-mNDT in which amino acid changes occurred were transformed with E. coli JM109. The specific composition and conditions of the reaction solution are shown in Table 8, and the results are shown in Table 9. [

As a result of 2FDDAP reaction, it was confirmed that C71 having 59 Leu → Gln mutation among various mutants had the best 2FDU substrate conversion ability as in 2FDA reaction, and named it pFRPT-FNDT / DH5α. Analysis of the genetic information of FNDT revealed that it had the nucleoside sequence of SEQ ID NO: 9 and analyzed the amino acid sequence to confirm that it had the amino acid sequence of SEQ ID NO: 10.

Further, in order to confirm the difference in the selection of the optimal E. coli host and the 2FDDAP reaction according to the enzyme concentration, the conversion rate over time was measured according to the composition of the reaction solution in Table 8, and the results are shown in Table 10.

As shown in Table 10, it was confirmed that FNDT-DH5α showed the best activity for 2FDDAP reaction on the 42-hour reaction standard. Thus, the optimal E. coli host of FNDT was determined to be DH5α, and pFRPT-FNDT / DH5α was designated E. coli E206. In addition, FNDT confirmed that the conversion rate to 2FDU substrate was significantly increased (about 3.7 times as compared with the 42-hour reaction) compared to the conventional NDT.

Example 4. Confirmation of activity of FNDT strain

A potency assay for pFRPT-NDT / JM109, which is an existing NDT-introduced strain, and pFRPT-FNDT / DH5? ( E. coli E206) of Example 3, was performed.

The 1 L fermentation of the strain was carried out by the method of Table 11, and the potency was analyzed using the 2FDA reaction of Table 12 for the produced cells. The results of 5 sets of pFRPT-NDT / JM109 and E. coli E206 cultures are shown in Table 13.

As can be seen in Table 13, pFRPT-NDT / JM109 (E. coli E206) cells showed significantly improved activity (about 290 times) compared to pFRPT-NDT / JM109 cells.

The stomach cells were deposited with the Korean Culture Center of Microorganisms (KCCM) on Aug. 10, 2017, and received the accession number KCCM12092P.

Example 5. Preparation of 2'-deoxy-2'-fluoroadenosine using FNDT

2'-deoxy-2'-fluoroadenosine (2FDA) was prepared using the FNDT of the present invention as a starting material for 2FDU and adenine (ADE) ).

First, 18.0 kg of 2'-deoxy-2'-fluorouridine (2.0 M) and 9.9 kg of adenine (2.0 M) were suspended in 36 L of purified water and 3.6 kg of E. coli E206-dried cells were mixed The reaction was carried out at 48 to 52 ° C and pH 6.1 to 6.5 for 84 hours. The reaction solution was analyzed by HPLC. As a result, it was confirmed that the conversion of 2'-deoxy-2'-fluoroadenosine was 97.6%.

After completion of the reaction, 45 L of purified water and 189 L of methyl alcohol were added, and the pH was adjusted to 1.0-1.3 with 35% hydrochloric acid. Then, 40 kg of celite was added and filtered to produce Gt; uracil < / RTI > was removed in the form of a filtrate. To recover the 2'-deoxy-2'-fluoroadenosine remaining in the filtrate, 27 liters of purified water and 63 L of methyl alcohol were added to the reactor, and the pH was adjusted to 35% using hydrochloric acid 1.0 to 1.3, followed by filtration, and the filtration filtrate was combined twice.

0.36 kg of Acid-charcoal was added to the filtered filtrate from which the cells and uracil were removed, stirred for 1 hour or more, and filtered. The filtrate was concentrated until the total liquid amount reached 63 ~ 81 L while maintaining the external temperature of the reactor at 65 캜 or below. After the concentration was finished, 126 L of purified water was added, and the pH was adjusted to 7.5 to 8.0 using a 10N NaOH aqueous solution.

After the pH was adjusted, the internal temperature of the reactor was raised to 46 ~ 52 캜 and stirred for 1 hour or longer. The internal temperature was gradually cooled to 15 ~ 30 캜 over 2 hours. The resulting filtrate was dried under reduced pressure at an external temperature of 65 DEG C or lower for 12 hours to obtain 16.11 kg of 2'-deoxy-2'-fluoroadenosine (Table 14), and the analysis results of 2FDA produced are shown in Table 15 below.

Based on the above results, it was found that high-purity 2'-deoxy-2'-fluoroadenosine having a purity of 99% or more can be effectively produced with a weight yield of about 90% by using the NDT mutant of the present invention or the cells containing the same Respectively.

Example 6. Preparation of 2'-deoxy-2'-fluoroguanosine using FNDT

2FDU and 2,6-Diaminopurine (DAP) were used as substrates for 2'-deoxy-2'-fluoro-2,6-diaminopurine (2'-Deoxy- 2 '-Fluoro-2,6-Diaminopurine; 2FDDAP), and then transformed with the expression strain of adenosine deaminase (SEQ ID NO: 11) derived from lactococcus lactis (pFRPT-LADD (2'-Deoxy-2'-fluoroguanosine (2FDG)) using a method described in Japanese Patent Application Laid-Open No. 10-2016-0033414 (FIG. 2), followed by a subsequent purification step to produce 2'-deoxy-2'-fluoroguanosine.

First, 20.0 g of 2'-deoxy-2'-fluorouridine (1.62 M) and 6.4 g of 2,6-diaminopurine (0.85 M) were suspended in 50 mL of purified water. deoxy-2'-fluoro-2,6-diaminopurine conversion reaction was carried out at 50 ° C and pH 6.0 under the condition of mixing E. coli E206 cells. The conversion of 2FDDAP was calculated as follows.

* 2FDDAP Reaction Conversion Rate = 2FDDAP (mole) X 100 / 2FDU (mole) + 2FDDAP (mole)

After confirming 40% conversion of 2FDDAP, 4.0 g of 2,6-diaminopurine (0.53M) was added additionally, and after addition of 2.4 g of 2,6-diaminopurine (0.32M) And 70% conversion was confirmed.

Then, after conversion of 70% to 2'-deoxy-2'-fluoro-2,6-diaminopurine was confirmed, deamination was carried out by adding 0.1 g of pFRPT-LADD / JM109 cells, The reaction proceeded. The pH was maintained at 6.0 during the reaction using a 50% aqueous solution of acetic acid. After the final 80% conversion was confirmed, further 10 g of E. coli E206 cells were added to continue the reaction. The 2'-deoxy-2'-fluoro-2,6-diaminopurine reaction and the 2'-deoxy-2'-fluoroguanosine reaction proceeded as one-pot, and the 2FDG conversion was calculated as Respectively.

* 2FDG Reaction Conversion Rate = 2FDG (mole) X 100 / 2FDU (mole) + 2FDDAP (mole) + 2FDG (mole)

After the final 141 hours, the reaction solution was analyzed by HPLC. As a result, it was confirmed that the conversion of 2'-deoxy-2'-fluoroguanosine was 97.5% (FIG. 3).

After completion of the reaction, 260 mL of purified water and 330 mL of methyl alcohol were added, and the pH was adjusted to 1.3 using 35% hydrochloric acid. Then, 20 g of celite was added and filtered to remove the uracil (uracil) was removed in the form of a filtrate. To recover the remaining 2'-deoxy-2'-fluoroguanosine in the filtrate, 80 mL of purified water and 80 mL of methyl alcohol were added to the filtrate, the pH was adjusted to 1.3 using 35% hydrochloric acid Filtered, and the filtrate was combined.

The filtrate, from which the cells and uracil were removed, was adjusted to pH 7.5 with a 10N NaOH aqueous solution and stirred at 70 ° C for 2 hours. After cooling to 0 ~ 5 ℃ over 3 hours, stirring was continued for 3 hours while maintaining the temperature, and 30.87 g of wet crude cake was recovered by filtration.

300 mL of purified water was added to the crude cake, the pH was adjusted to 10.5 using 10N NaOH, the temperature was raised to 75 ° C, and the mixture was stirred for 1 hour. 2 g of Basic-charcoal was added and further stirred for 1 hour and then filtered.

The filtrate was adjusted to pH 7.5 with a 50% acetic acid aqueous solution, and then heated to 75 ° C, stirred for about 3 hours, slowly cooled to 0 to 5 ° C over 3 hours, and stirred for 3 hours or more while maintaining the temperature. The process product was filtered and the obtained filtrate was dried to obtain 2'-deoxy-2'-fluoroguanosine having 16.52 g (weight yield 82.6%), moisture 7.16% and HPLC purity 99.42% .

From the results of this Example, it was confirmed that high-purity 2'-deoxy-2'-fluoroguanosine can be produced at a high yield by using the NDT mutant strain of the present invention.

Example 7. Preparation of 2'-deoxy-2'-fluoroinosine

The adenosine deaminase (LADD) -expressing strain (pFRPT-LADD / JM109) produced by the present applicant with the 2FDA obtained in the above Example 5 as a substrate was treated with 10 μg / ) Was used to prepare 2'-Deoxy-2'-fluoroinosine.

First, 36.7 g of 2FDA (1.36 M) was suspended in 100 ml of purified water, and 0.1 g of pFRPT-LADD / JM109 cells were mixed and agitated for 1.5 hours at 40 ° C and pH 7.0 to 8.0. The reaction solution was analyzed by HPLC and it was confirmed that 2FDA was converted to 2'-deoxy-2'-fluoroinosine by 100%.

After the completion of the reaction, 300 ml of purified water was added, and the pH was adjusted to 12.0 to 12.5 with 10 N NaOH aqueous solution, and the cells were removed by filtration. The filtrate was neutralized with 35% hydrochloric acid and kept at 0 ~ 5 ℃ for more than 3 hours. After stirring, it was filtered and vacuum dried at 55 ℃ for 12 hours.

As a result, 27 g of 2'-deoxy-2'-fluoroinosine having an HPLC purity of 100.0% was obtained. The weight yield based on the starting amount of 2'-deoxy-2'-fluoroadenine as a starting material was 73.6% , And the molar yield was 73.3%.

From the above results, the NDT variant (FNDT) of the present invention shows excellent substrate specificity for high activity, especially 2'-deoxy-2'-fluoro nucleoside, It was confirmed that it was possible to prepare a compound of the present invention.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (18)

1. A N-deoxyribosyltransferase mutant comprising the L59Q mutant in which the 59th leucine in the amino acid sequence of SEQ ID NO: 2 is replaced with glutamine.
2. 2. The N-deoxyribosyltransferase mutant according to claim 1, wherein said mutant has an activity of catalyzing a transglycosylation reaction.
3. The method of claim 1, wherein said variant is an N-deoxyribosyl (2'-deoxy-2'-fluoronucleoside) Transferase mutant.
4. The method of claim 3, wherein the 2'-deoxy-2'-fluoro nucleoside is selected from the group consisting of 2'-Deoxy-2'-fluorouridine, N-deoxy Ribosyltransferase mutant.
5. A polynucleotide encoding the N-deoxyribosyltransferase variant of claim 1.
6. 6. The polynucleotide of claim 5, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
7. A recombinant vector operatively linked to a regulatory sequence of the polynucleotide of claim 5.
8. A microorganism into which the recombinant vector of claim 7 is introduced.
9. 9. The microorganism according to claim 8, wherein the microorganism is E. coli DH5 ?.
10. 10. The microorganism according to claim 9, wherein the microorganism is KCCM12092P.
11. 11. The microorganism according to any one of claims 8 to 10, wherein the microorganism has an activity of catalyzing a transglycosylation reaction.
12. A method for producing a nucleoside by forming an N-glucoside bond between a base donor and a sugar donor using the N-deoxyribosyltransferase mutant of claim 1 or a microorganism expressing the mutant.
13. 13. The method of claim 12, wherein the base donor is selected from the group consisting of adenine, guanine, hypoxanthine, xanthine, 6-mercaptoulinine, 6-thioguanine, N-alkyl or acyladenyl, 2-alkoxyadenine, , 2-thiocytosine, 4-thiouracil, N-acylcytosine, 5-thioguanidine, , 5-halogenovinyluracil, 1,3,4-triazole-3-carboxamide 1,2,4-triazole-3-carboxylic acid, 1,2,4- Imidazol-5-olate, benzimidazole, 1-deazaadenine, 3-deazaadenine, 3-deazaguanine, 7- But are not limited to, deazaadenine, deazaadenine, 7-deazaguanine, 8-azaadenine, 7-deaza-8-azaspiroxan, 5- azathiamine, 5- azasitonin, 6- azauracil, 3- deazauracil, And nicotinic acid amide And any one selected from the group,

    Wherein the sugar donor is selected from the group consisting of inosine, guanosine, uridine, ribofuranosyl thymine, ribose-1-phosphate, 2'-deoxyinosine, 2'-deoxyguanosine, 2'-deoxyuridine, thymidine, 2 2 ', 3'-dideoxyuridine, 3'-deoxythymidine, 2'-deoxyribose-1-phosphate and 2' , 3-dideoxyribose-1-phosphate, and the like.
14. 14. The method of claim 13, wherein the base donor is adenine, 2,6-diaminopurine or cytosine.
15. 14. The method of claim 13, wherein the sugar residue donor is selected from the group consisting of Thymidine, 2'-deoxyuridine, or 2'-deoxy- 2'-fluorouridine. ≪ / RTI >
16. Deoxy-2'-fluoroadenosine is produced by treating the N-deoxyribosyltransferase mutant of claim 1 with 2'-deoxy-2'-fluorouridine and adenine Deoxy-2'-fluoroadenosine. ≪ / RTI >
17. The N-deoxyribosyltransferase mutant of claim 1 is treated with 2'-deoxy-2'-fluorouridine and 2,6-diaminopurine to produce 2'-deoxy-2'-fluoro- Preparing 2,6-diaminopurine; And

    2-deoxy-2'-fluoro-2,6-diaminopurine is treated with adenosine deaminase derived from lactococcus lactis to prepare 2'-deoxy-2'-fluoroguanosine ≪ RTI ID = 0.0 > 2'-deoxy-2'-fluoroguanosine. ≪ / RTI >
18. Preparing 2'-deoxy-2'-fluoroadenosine by treating the N-deoxyribosyltransferase mutant of claim 1 with 2'-deoxy-2'-fluorouridine and adenine; And

    2'-deoxy-2'-fluoroadenosine to produce 2'-deoxy-2'-fluoroinosine by treating the 2'-deoxy-2'-fluoroadenosine with adenosine deaminase derived from lactococcus lactis. Deoxy-2'-fluoroinosine.

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