Yoichi Mikami

Construction of deoxyriboaldolase-overexpressing Escherichia coli and its application to 2-deoxyribose 5-phosphate synthesis from glucose and acetaldehyde for 2'-deoxyribonucleoside production.

ABSTRACT 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.

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

2-Deoxyribose 5-phosphate was produced from acetaldehyde and dihydroxyacetone phosphate via D-glyceraldehyde 3-phosphate by Klebsiella pneumoniae B-4-4 through deoxyriboaldolase- and triosephosphate isomerase-catalyzing reactions. Under the optimum conditions, 98.7 mM 2-deoxyribose 5-phosphate was produced from 200 mM acetaldehyde and 117 mM dihydroxyacetone phosphate in 2 h with a molar yield of 84%. The 2-deoxyriobse 5-phosphate produced was directly transformed to 2′-deoxyribonucleoside by phosphopentomutase- and nucleoside phosphorylase-catalyzing reactions.

Antiviral substances with systemic effects produced by basidiomycetes such as Fomes fomentarius

Inhibitors of plant virus infection with systemic effects were found in the culture filtrates of Basidiomycetes such as Fomes fomentarius and Schizophyllum commune. These inhibitors were widely distributed in Agaricales and Polyporales. The inhibitors designated as BAS (Basidiomycete Antiviral Substance) were highly active against the mechanical transmission of tobacco mosaic virus (TMV). No toxic effect was observed on the host plants. BAS-F, a polysaccharide produced by F. fomentarius, almost completely inhibited infection, when BAS-F at 2 μg/ml was applied to the same surface of leaves of Xanthi-nc tobacco 24 h before TMV inoculation to the upper surface of the leaves, and 500/0 inhibition was shown when BAS-F at 10 μg/ml was applied to the under surface of leaves. BAS-F also induced systemic resistance to the non-treated leaves when it was applied to only one leaf of the plant. BAS-F also had similar effects against the infection of TMV on bell pepper and tomato plants.

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli.

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker’s yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker’s yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.

Enzymatic Synthesis of 5-Methyluridine from Adenosine and Thymine with High Efficiency.

5-Methyluridine (5MU) was synthesized efficiently from adenosine, thymine, and phosphate by a combination of adenosine deaminase (ADA), purine nucleoside phosphorylase (PUNP), pyrimidine nucleoside phosphorylase (PYNP), and xanthine oxidase (XOD). Adenosine was converted into inosine first by ADA. 5MU and hypoxanthine were synthesized from inosine and thymine by PUNP and PYNP. The hypoxanthine formed was converted into urate via xanthine by XOD. After inosine was completely consumed, an equilibrium state, in which 5MU, thymine, ribose-1-phosphate, and phosphate were involved, was achieved. At the equilibrium state, the maximum yield of 5MU was obtained. The yield of 5MU was 74%, when the initial concentrations of adenosine, thymine, and phosphate were 5 mM each. On the other hand, in the absence of ADA or XOD the yield of 5MU was 1.8%. Several kinds of nucleosides were also synthesized with high yield by the same method.