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The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.There will be a need for 2Ј-deoxyribonucleoside in the near future due to increasing demand in new medical and biotechnology fields. 2Ј-Deoxyribonucleoside is a building block of promising antisense drugs for cancer therapy. For some recently developed antiviral agents, such as azidothymidine for treatment of human immunodeficiency virus infections, 2Ј-deoxyribonucleoside is a synthesis intermediate. 2Ј-Deoxyribonucleoside is also a precursor of an indispensable material used for widespread PCR applications, 2Ј-deoxyribonucleoside triphosphate. The current 2Ј-deoxyribonucleoside sources include hydrolyzed herring and salmon sperm DNA, which are not suitable sources for sudden high demand. The difficulty in chemical synthesis of 2Ј-deoxyribonucleoside lies in the generation of 2-deoxyribosyl groups. The chemical synthesis of 2-deoxyribosyl groups and the subsequent synthesis of 2Ј-deoxyribonucleoside involve complex protection and deprotection steps (1,8,10,15). It is likely that introduction of biochemical reactions with high selectivity will solve this problem.In general metabolism, there are two reactions involving 2-deoxyriobosyl groups. One of these reactions is reduction of ribonucleotide to 2Ј-deoxyribonuleotide during biosynthesis, and the other is cleavage of 2-deoxyribose 5-phosphate (DR5P) to produce glyceraldehyde 3-phosphate (G3P) and acetaldehyde during degradation of 2Ј-deoxyribonucleoside. The former reaction is regulated in a complicated fashion and is hard to handle for practical purposes (3, 4). We focused on the latter reaction for generation of a 2-deoxyribose frame and found that the reverse reactions of 2Ј-deoxyribonucleoside degradation are promising reactions for synth...

2'-Deoxyribonucleosides are important as building blocks for the synthesis of antisense drugs, antiviral nucleosides, and 2'-deoxyribonucleotides for polymerase chain reaction. The microbial production of 2'-deoxyribonucleosides from simple materials, glucose, acetaldehyde, and a nucleobase, through the reverse reactions of 2'-deoxyribonucleoside degradation and the glycolytic pathway, was investigated. The glycolytic pathway of baker's yeast yielded fructose 1,6-diphosphate from glucose using the energy of adenosine 5'-triphosphate generated from adenosine 5'-monophosphate through alcoholic fermentation with the yeast. Fructose 1,6-diphosphate was further transformed to 2-deoxyribose 5-phosphate in the presence of acetaldehyde by deoxyriboaldolase-expressing Escherichia coli cells via D-glyceraldehyde 3-phosphate. E. coli transformants expressing phosphopentomutase and nucleoside phosphorylase produced 2'-deoxyribonucleosides from 2-deoxyribose 5-phosphate and a nucleobase via 2-deoxyribose 1-phosphate through the reverse reactions of 2'-deoxyribonucleoside degradation. Coupling of the glycolytic pathway and deoxyriboaldolase-catalyzing reaction efficiently supplied 2-deoxyribose 5-phosphate, which is a key intermediate for 2'-deoxyribonucleoside synthesis. 2'-Deoxyinosine (9.9 mM) was produced from glucose, acetaldehyde, and adenine through three-step reactions via fructose 1,6-diphosphate and then 2-deoxyribose 5-phosphate, the molar yield as to glucose being 17.8%.

All life generates deoxyribonucleotides, the building blocks of DNA, via ribonucleotide reductases (RNRs). The complexity of this reaction suggests it did not evolve until well after the advent of templated protein synthesis, which in turn suggests DNA evolved later than both RNA and templated protein synthesis. However, deoxyribonucleotides may have first been synthesised via an alternative, chemically simpler route—the reversal of the deoxyriboaldolase (DERA) step in deoxyribonucleotide salvage. In light of recent work demonstrating that this reaction can drive synthesis of deoxyribonucleosides, we consider what pressures early adoption of this pathway would have placed on cell metabolism. This in turn provides a rationale for the replacement of DERA-dependent DNA production by RNR-dependent production.Electronic supplementary materialThe online version of this article (doi:10.1007/s00239-014-9656-6) contains supplementary material, which is available to authorized users.

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A one-pot enzymatic synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker's yeast (Saccharomyces cerevisiae) generated ATP which was used to produce D: -glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The D: -glyceraldehyde 3-phosphate produced was transformed to 2'-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2'-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2'-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.