Enzymatic synthesis of deoxyribonucleosides

Abstract

The present invention relates to a method for the in vitro synthesis of deoxyribonucleosides and enzymes suitable for this method.

1 - 45 . (canceled) 
     
     
         46 . A method for in vitro enzymatic synthesis of a deoxyribonucleoside comprising reacting deoxyribose 1-phosphate (dR1P) and a nucleobase to form a deoxyribonucleoside and an inorganic phosphate. 
     
     
         47 . The method of  claim 46 , further comprising removing the inorganic phosphate. 
     
     
         48 . The method of  claim 46 , wherein said reacting comprises catalyzing said dR1P and said nucleobase with a thymidine phosphorylase (TP, EC 2.4.2.4.) or a purine nucleoside phosphorylase (PNP, EC 2.4.2.1.). 
     
     
         49 . The method of  claim 47 , comprising removing the inorganic phosphate by one of: (i) converting the inorganic phosphate to inorganic pyrophosphate, (ii) precipitating the inorganic phosphate, (iii) complexing the inorganic phosphate, or (iv) phosphorylating a substrate with the inorganic phosphate. 
     
     
         50 . The method of  claim 47 , comprising removing the inorganic phosphate by phosphorylating a substrate with the inorganic phosphate. 
     
     
         51 . The method of  claim 46 , wherein the nucleobase is selected from the group consisting of thymine, uracil, adenine, guanine, hypoxanthinine and an analog thereof. 
     
     
         52 . The method of  claim 51 , wherein said analog is selected from the group consisting of: 2-thio-uracil, 6-aza-uracil, 5-carboxy-2-thio-uracil, 6-aza-thymine, 6-aza-2-thio-thymine and 2,6-diamino-purine. 
     
     
         53 . The method of  claim 46 , further comprising reacting said inorganic phosphate with fructose-diphosphate (FDP) to form pyrophosphate and fructose-6-phosphate (F6P). 
     
     
         54 . The method of  claim 53 , wherein said reacting comprises catalyzing said inorganic phosphate with said FDP with a Ppi-dependent phosphofructokinase (PFK-Ppi, EC 2.7.1.90). 
     
     
         55 . The method of  claim 46 , further comprising reacting said inorganic phosphate with a disaccharide to form a monosaccharide and a phosphorylated monosaccharide. 
     
     
         56 . The method of  claim 55 , wherein the disaccharide is sucrose or maltose. 
     
     
         57 . The method of  claim 56 , wherein said reacting comprises catalyzing said inorganic phosphate and said disaccharide with a sucrose phosphorylase (EC 2.4.1.7) or a maltose phosphorylase (EC 2.4.1.8). 
     
     
         58 . The method of  claim 46 , further comprising generating dR1P by isomerizing deoxyribose 5-phosphate (dR5P) prior to reacting said dR1P with a nucleobase. 
     
     
         59 . The method of  claim 58 , comprising isomerizing said dR5P with a phosphopentose mutase (PPM, EC 5.4.2.7). 
     
     
         60 . The method of  claim 58 , further comprising forming the dR5P by condensing glyceraldehyde 3-phosphate (GAP) with acetaldehyde prior to isomerizing said dR5P. 
     
     
         61 . The method of  claim 60 , wherein said condensing comprises catalyzing condensing of GAP with acetaldehyde with a phosphopentose aldolase (PPA, EC 4.1.2.4). 
     
     
         62 . The method of  claim 60 , further comprising enzymatically generating said GAP from fructose 1,6-diphosphate, dihydroxyacetone (DHA) or glycerolphosphate (GP) prior to said condensing. 
     
     
         63 . The method of  claim 58 , comprising generating said deoxyribose 5-phosphate by phosphorylating deoxyribose prior to isomerizing said dR5P. 
     
     
         64 . The method of  claim 63 , wherein said phosphorylating comprises catalyzing of deoxyribose with a deoxyribokinase (dRK, EC 2.7.1.15.). 
     
     
         65 . The method of  claim 64 , wherein said dRK is encoded by (a) the nucleotide sequence of SEQ ID NO: 11, (b) a nucleotide sequence encoding the protein encoded by SEQ ID NO: 11 or (c) a nucleotide sequence which hybridizes under stringent conditions to the complementary sequence of (a) or (b). 
     
     
         66 . The method of  claim 62 , comprising reacting fructose 1,6-diphosphate with an FDP-aldolase I or an FDP-aldolase II to form said GAP. 
     
     
         67 . The method of  claim 62 , comprising forming said GAP by reacting DHA with ATP to form dihydroxyacetone phosphate (DHAP), followed by catalyzing isomerization of said DHAP to GAP with a glycerokinase (GK, EC 2.7.1.30) and a triose phosphate isomerase (TIM, EC 5.3.1.1). 
     
     
         68 . The method of  claim 62 , comprising forming said GAP by reacting GP with O 2  to form dihydroxyacetone phosphate (DHAP) and H 2 O 2 , followed by catalyzing isomerization of said DHAP to GAP with a glycerophosphate oxidase (GPO, EC 1.1.3.21) and a triose phosphate isomerase (TIM, EC 5.3.1.1). 
     
     
         69 . The method of  claim 46 , further comprising reacting said deoxyribonucleoside with a second nucleobase to form a second deoxyribonucleoside containing the second nucleobase. 
     
     
         70 . The method of  claim 69 , comprising catalyzing said reacting with a nucleoside 2-deoxyribosyl transferase (NdT, EC 2.4.2.6). 
     
     
         71 . The method of  claim 70 , wherein said NdT is encoded by (a) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 13, (b) a nucleic acid molecule consisting of a nucleotide sequence encoding the protein encoded by SEQ ID NO: 13 or (c) a nucleic acid molecule which hybridizes under stringent conditions to the nucleic acid molecule of (a) or (b). 
     
     
         72 . The method of  claim 69 , wherein said second nucleobase is selected from the group consisting of cytosine and a cytosine analog. 
     
     
         73 . The method of  claim 69 , wherein said second nucleobase is selected from the group consisting of 5-aza-cytosine, 2,6-dichloro-purine, 6-aza-thymine and 5-fluoro-uracil. 
     
     
         74 . A method for the in vitro enzymatic synthesis of a deoxyribonucleoside comprising:
 (i) condensing glyceraldehyde 3-phosphate (GAP) with acetaldehyde to form deoxyribose 5-phosphate (dR5P),   (ii) isomerizing said dR5P to deoxyribose 1-phosphate (dR1P), and   (iii) reacting said dR1P and a nucleobase, to form said deoxyribonucleoside and an inorganic phosphate.   
     
     
         75 . The method of  claim 74 , further comprising removing the inorganic phosphate. 
     
     
         76 . The method of  claim 75 , comprising removing the inorganic phosphate by phosphorylation of a substrate with the inorganic phosphate. 
     
     
         77 . The method of  claim 74 , comprising carrying out the complete reaction of steps (i) to (iii) without isolating intermediate products. 
     
     
         78 . The method of  claim 74 , comprising generating said GAP from fructose 1,6-diphosphate (FDP), dihydroxy-acetone (DHA) or glycerolphosphate (GP) prior to said condensing of GAP. 
     
     
         79 . The method of  claim 74 , further comprising removing excess acetaldehyde before step (ii). 
     
     
         80 . The method of  claim 78 , further comprising generating said GAP and removing excess starting materials or by-products before step (ii). 
     
     
         81 . The method of  claim 80 , wherein said excess starting material is fructose 1,6-diphosphate and said excess by-product is deoxyxylulose 1-phosphate (dX1P). 
     
     
         82 . The method of  claim 78 , comprising generating GAP from FDP, and generating DXP1 as an excess by-product thereby. 
     
     
         83 . A method for the in vitro enzymatic synthesis of a deoxyribonucleoside comprising:
 (i) phosphorylating deoxyribose to deoxyribose 5-phosphate (dR5P),   (ii) isomerizing said dR5P to deoxyribose 1-phosphate (dR1P), and   (iii) reacting said dR1P and a nucleobase to form said deoxyribonucleoside and an inorganic phosphate.   
     
     
         84 . The method of  claim 83 , further comprising removing the inorganic phosphate. 
     
     
         85 . The method of  claim 84 , comprising removing the inorganic phosphate by phosphorylating a substrate with the inorganic phosphate. 
     
     
         86 . The method of  claim 83 , comprising conducting the complete reaction of steps (i) to (iii) without isolating intermediate products. 
     
     
         87 . A method for preparing an enzyme for an in vitro method for enzymatic synthesis of a deoxyribonucleoside, comprising reacting (i) an isolated nucleic acid molecule encoding a nucleoside 2-deoxyribosyl transferase (NdT, EC 2.4.2.6) with (ii) a deoxyribonucleoside containing a first nucleobase, wherein said nucleic acid molecule comprises (a) the nucleotide sequence shown in SEQ ID NO: 13, (b) a nucleotide sequence encoding the protein encoded by SEQ ID NO: 13 or (c) a nucleotide sequence hybridizing under stringent conditions to the complementary sequence of (a) or (b), and wherein said deoxyribonucleoside containing a first nucleobase is further reacted with a second nucleobase to form a deoxyribonucleoside containing said second nucleobase. 
     
     
         88 . The method of  claim 87 , wherein the second nucleobase is selected from the group consisting of cytidine and a cytidine analog. 
     
     
         89 . The method of  claim 88 , wherein the analog is selected from the group consisting of: 6-methyl purine, 2-amino-6-methylmercaptopurine, 6-dimethylaminopurine, 5-azacytidine, 2,6-dichloropurine, 6-chloroguanine, 6-chloropurine, 6-azathymine, 5-fluorouracil, ethyl-4-amino-5-imidazole carboxylate, imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide. 
     
     
         90 . The method of  claim 87 , wherein the first nucleobase is selected from the group consisting of adenine, guanine, thymine, uracil and hypoxanthine. 
     
     
         91 . The method of  claim 87 , comprising containing the nucleic acid molecule in a recombinant vector in operative linkage with an expression control sequence. 
     
     
         92 . The method of  claim 81 , comprising containing the nucleic acid in a recombinant cell. 
     
     
         93 . A method for preparing an enzyme for an in vitro method for enzymatic synthesis of a deoxyribonucleoside, comprising reacting (i) an isolated nucleic acid molecule encoding a deoxyribokinase (dRK, EC 2.7.1.5) with (ii) deoxyribose, further comprising phosphorylating said deoxyribose to deoxyribose 5-phosphate, wherein said nucleic acid molecule comprises (a) the nucleotide sequence shown in SEQ ID NO: 11, (b) a nucleotide sequence encoding the protein encoded by SEQ ID NO: 11 or (c) a nucleotide sequence hybridizing under stringent conditions to the complementary sequence of (a) or (b). 
     
     
         94 . A method for synthesizing a deoxyribonucleoside in vitro, comprising contacting a mixture containing deoxyribose and phosphate with an enzyme having NdT activity to form deoxyribose 5-phosphate and obtaining deoxyribose 5-phosphate therefrom.