Microbial Production of 2-Deoxyribose 5-Phosphate from Acetaldehyde and Triosephosphate for the Synthesis of 2′-Deoxyribonucleosides

Ogawa, Jun; Saito, Kyota; Sakai, Takafumi; Horinouchi, Nobuyuki; Kawano, Takako; Matsumoto, Seiichiro; Sasaki, Makiko; Mikami, Yoichi; Shimizu, Sakayu

doi:10.1271/bbb.67.933

Cited by 32 publications

(27 citation statements)

References 10 publications

(7 reference statements)

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“…K. pneumoniae B-4-4 (ϭ IFO 16579) was used as the source of genomic DNA (14). E. coli strain JM109 and the alkaline phosphatase-defective mutant 10B5 (20) were kindly provided by Hideyuki Suzuki, Graduate School of Biostudies, Kyoto University (Kyoto, Japan).…”

Section: Enzymes and Chemicalsmentioning

confidence: 99%

“…Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a 12.5% polyacrylamide slab gel was performed as described previously (11) with the following standard proteins (Daiichi Chemicals Co., Tokyo, Japan): phosphorylase b (molecular weight, 97,400), bovine serum albumin (66,267), aldolase (42,400), carbonic anhydrase (30,000), trypsin inhibitor (20,100), and lysozyme (14,400).…”

Section: Enzymes and Chemicalsmentioning

confidence: 99%

“…A reaction mixture containing DR5P produced from glucose and acetaldehyde was centrifuged (10,000 ϫ g, 20 min), and the resultant supernatant was used directly for 2Ј-deoxyribonucleoside production. The reaction mixture (total volume, 0.5 ml) contained the 25% (vol/vol) DR5P solution produced as described above (DR5P concentration, 25 mM), 50 mM adenine, 200 mM acetaldehyde, 0.1 mM glucose 1,6-diphosphate, 1 mM MnCl 2 ⅐ 4H 2 O, 150 mM NH 4 Cl-NH 4 OH buffer (pH 9.0), 1.0% xylene, 0.4% PL, 0.5 U of nucleoside phosphorylase per ml, and 10% (wt/vol) washed cells of E. coli BL21/pTS17 (14). After a 4.5-h reaction at 30°C, the 2Ј-deoxyribonucleoside produced was analyzed by high-performance liquid chromatography as described below.…”

Section: Enzymes and Chemicalsmentioning

confidence: 99%

“…In a previous paper, DR5P production by a strain with an active DERA, Klebsiella pneumoniae B-4-4, was described (14). This strain produced DR5P from triose phosphates (G3P and dihydroxyacetone phosphate) and acetaldehyde.…”

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Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

Horinouchi

Ogawa

Sakai

et al. 2003

Appl Environ Microbiol

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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...

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Section: Enzymes and Chemicalsmentioning

confidence: 99%

Section: Enzymes and Chemicalsmentioning

confidence: 99%

Section: Enzymes and Chemicalsmentioning

confidence: 99%

mentioning

confidence: 99%

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Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

Horinouchi

Ogawa

Sakai

et al. 2003

Appl Environ Microbiol

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“…8) Triosephosphates are the intermediates of glycolysis, so if triosephosphates are supplied through glycolysis from cheap sugars, the process should become more practical. In a previous study, 9) we investigated the glycolytic function of DERAexpressing E. coli with several intermediates of glycolysis, such as glucose, fructose, fructose 6-phosphate, and fructose 1,6-diphosphate (FDP), and found that the phosphorylated intermediates, especially FDP, served as suitable precursors for G3P.…”

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Efficient Production of 2-Deoxyribose 5-Phosphate from Glucose and Acetaldehyde by Coupling of the Alcoholic Fermentation System of Baker’s Yeast and Deoxyriboaldolase-ExpressingEscherichia coli

Horinouchi

Ogawa

Kawano

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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Organic Synthesis with Isomerases

2022

Enzyme‐Based Organic Synthesis

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Microbial Production of 2-Deoxyribose 5-Phosphate from Acetaldehyde and Triosephosphate for the Synthesis of 2′-Deoxyribonucleosides

Cited by 32 publications

References 10 publications

Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

Efficient Production of 2-Deoxyribose 5-Phosphate from Glucose and Acetaldehyde by Coupling of the Alcoholic Fermentation System of Baker’s Yeast and Deoxyriboaldolase-ExpressingEscherichia coli

Organic Synthesis with Isomerases

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