Biosci. Biotechnol. Biochem., 70 (6), 1371–1378, 2006 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 y Nobuyuki HORINOUCHI,1 Jun OGAWA,1; Takako KAWANO,1 Takafumi SAKAI,1 Kyota SAITO,1 Seiichiro MATSUMOTO,2 Mie SASAKI,2 Yoichi MIKAMI,2 and Sakayu SHIMIZU1 1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan 2Tokyo Laboratory, Yuki Gosei Kogyo Co., Ltd., 3-37-1 Sakashita, Itabashi-ku, Tokyo 174-0043, Japan Received December 5, 2005; Accepted February 19, 2006; Online Publication, June 23, 2006 [doi:10.1271/bbb.50648] 2-Deoxyribose 5-phosphate production through cou- of Klebsiella pneumoniae B-4-4 with triosephosphates pling of the alcoholic fermentation system of baker’s such as D-glyceraldehyde 3-phosphate (G3P) and dihy- yeast and deoxyriboaldolase-expressing Escherichia coli droxyacetone phosphate (DHAP) and acetaldehyde as was investigated. In this process, baker’s yeast generates starting materials.8) Triosephosphates are the intermedi- fructose 1,6-diphosphate from glucose and inorganic ates of glycolysis, so if triosephosphates are supplied phosphate, and then the E. coli convert the fructose 1,6- through glycolysis from cheap sugars, the process diphosphate into 2-deoxyribose 5-phosphate via D-glyc- should become more practical. In a previous study,9) eraldehyde 3-phosphate. Under the optimized condi- we investigated the glycolytic function of DERA- tions with toluene-treated yeast cells, 356 mM (121 g/l) expressing E. coli with several intermediates of glycol- fructose 1,6-diphosphate was produced from 1,111 mM ysis, such as glucose, fructose, fructose 6-phosphate, and glucose and 750 mM potassium phosphate buffer (pH fructose 1,6-diphosphate (FDP), and found that the 6.4) with a catalytic amount of AMP, and the reaction phosphorylated intermediates, especially FDP, served as supernatant containing the fructose 1,6-diphosphate was suitable precursors for G3P. used directly as substrate for 2-deoxyribose 5-phosphate Processes for the production of various substances production with the E. coli cells. With 178 mM enzy- through coupling with the powerful fermentative ability matically prepared fructose 1,6-diphosphate and 400 of baker’s yeast as the energy source were established by mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2- Tochikura and colleagues.10–12) The sugar-fermentative deoxyribose 5-phosphate was produced. The molar yield system of baker’s yeast was used as the ATP donor in of 2-deoxyribose 5-phosphate as to glucose through the these processes. Temporary accumulation of FDP in the total two step reaction was 22.1%. The 2-deoxyribose course of ATP regeneration has been reported.13–15) 5-phosphate produced was converted to 2-deoxyribose Based on these reports, we designed a novel meta- with a molar yield of 85% through endogenous or bolic and enzymatic DR5P production system consisting exogenous phosphatase activity. of glucose fermentation and DERA-catalyzed aldol condensation with glucose and acetaldehyde as starting Key words: 2-deoxyribose 5-phosphate; 2-deoxyribose; materials. In this study, we tried to optimize FDP deoxyriboaldolase; alcoholic fermentation; production from glucose and inorganic phosphate with 20-deoxyribonucleoside toluene-treated baker’s yeast (toluene-treated yeast), and DR5P production from enzymatically prepared FDP and The synthesis of antiviral 20-deoxyribonucleosides acetaldehyde by DERA-expressing E. coli (Fig. 1). such as AZT requires a deoxyribose component as a Further convenient conversion of DR5P to 2-deoxyri- starting material, but chemical synthesis of the deoxy- bose (DR) was also examined. ribose frame is a tedious process with many protection and deprotection steps.1–4) Materials and Methods We performed 2-deoxyribose 5-phosphate (DR5P) production by means of deoxyriboaldolase 5–7) (DERA) Preparation of a toluene-treated yeast. Pressed y To whom correspondence should be addressed. Tel: +81-75-753-6122; Fax: +81-75-753-6128; E-mail: [email protected] Abbreviations: DR5P, 2-deoxyribose 5-phosphate; DERA, deoxyriboaldolase; FDP, fructose 1,6-diphosphate; G3P, D-glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; LB, Luria-Bertani; DR, 2-deoxyribose 1372 N. HORINOUCHI et al. ATP ATP ADP ADP AMP AMP Glucose FDP ADP ATP CH OH 2 O O P O HO O P OH OH Ethanol, CO2 HO OH OH HO Pi Pi Baker's yeast FDP O OH DERA CHO CH OH P O 2 CHOH C=O HO CH2O P CH2O P DR5P G3P DHAP DERA-expressing CH3CHO E. coli Fig. 1. DR5P Synthesis from Glucose and Acetaldehyde through the Fermentation Energy of Baker’s Yeast and DERA-Expressing E. coli. baker’s yeast (Oriental Yeast, Tokyo) was incubated resulting supernatants were measured as described with 16.6% (v/v) toluene in 176 mM potassium phos- below after checking the accumulation of DR by TLC. phate buffer (pH 7.0) at 37 C for 1 h with standing. The The averages of three separate experiments, which were suspension was centrifuged (3;500 Â g, 20 min), and the reproducible within Æ 10%, are presented in the text resulting pellet was used as the source of glycolytic and figures. enzymes (toluene-treated yeast). DR production: DR production was carried out by means of temperature shift or phosphatase addition. As Culture conditions for DERA-expressing E. coli. for temperature shift, the complete reaction mixture for DERA-expressing E. coli 10B5/pTS8 was cultivated DR5P production was further incubated at 47 C for at 37 C in Luria-Bertani (LB) medium (1% peptone, 22 h and centrifuged (15;000 Â g, 15 min), and then the 0.5% yeast extract, 1% NaCl, pH 7.2) for 12 h. Cells amount of DR in the resulting supernatant was assayed. were harvested by centrifugation (8;000 Â g, 10 min). As for phosphatase addition, the reaction mixture for After washing with a 0.85% NaCl solution, the cells DR5P production was centrifuged (15;000 Â g, 15 min), were used for DR5P synthesis. and the resulting supernatant was incubated at 80 C for 1 h, and then 2% (w/v) of Sumizyme PM (Shin Nihon Reaction conditions. FDP production: The standard Chemical, Anjo, Japan) was added. After 4 h incubation reaction mixture comprised, in 10 ml, 1,111 mM glucose, at 37 C, the reaction mixture was centrifuged (15;000 Â 500 mM K2HPO4,30mM MgSO4.7H2O, 15 mM AMP. g, 15 min) and the amount of DR in the supernatant was 2Na and 60% (w/v) toluene-treated yeast. The reactions determined. The averages of three separate experiments, were carried out at 37 C for 3–7 h with standing. The which were reproducible within Æ 10%, are presented supernatants obtained on centrifugation (15;000 Â g, in the text. 15 min) were subjected to FDP analysis and then used for DR5P production as an enzymatically prepared FDP Analytical methods. FDP analysis: FDP was measured solution. The averages of three separate experiments, enzymatically by monitoring a decrease in absorbance at which were reproducible within Æ 10%, are presented 340 nm of NADH through coupled reactions catalyzed in the text and figures. by FDP aldolase, triosephosphate isomerase (TPI), and DR5P production: The standard reaction mixture a-glycerophosphate dehydrogenase (GDH). Twenty ml comprised, in 0.5–60 ml, 50% (v/v) enzymatically of FDP-containing sample solution was added to 140 ml prepared FDP solution, 200 mM acetaldehyde, 200 mM of a reaction mixture comprising 150 mM Tris/HCl potassium phosphate buffer (pH 7.0), 15 mM MgSO4. (pH 7.4) and 0.5 mM NADH. Then 20 ml of a TPI/GDH 7H2O, 0.4% (v/v) polyoxyethylenelaurylamine, 1.0% solution (Sigma, St. Louis, MO) containing 50 U of TPI (v/v) xylene, and 12.5% (w/v) wet cells of E. coli and 5 U of GDH was added to decompose contaminating 10B5/pTS8. The reactions were carried out at 28 C for triosephosphates. After 10 min incubation at 30 C, 20 ml 2–4 h with shaking (120 rpm), followed by centrifuga- of FDP aldolase solution (Sigma) containing 10 U of tion (15;000 Â g, 15 min). The amounts of DR5P in the FDP aldolase from rabbit muscle was added, and then Enzymatic 2-Deoxyribose 5-Phosphate Production 1373 ) A M 250 mM Pi 500 mM Pi 750 mM Pi1000 mM Pi 1250 mM Pi 450 300 150 FDP production (m 0 357 7 357 357 35 357 Reaction time (h) ) M B 250 mM Pi 500 mM Pi 750 mM Pi1000 mM Pi 1250 mM Pi 160 120 80 40 0 DR5P production (m 3 h 5 h 7 h 3 h 5 h 7 h 3 h 5 h 7 h 3 h 5 h 7 h 3 h 5 h 7 h Reaction time for FDP production Fig. 2. Effects of the Inorganic Phosphate Concentration on FDP (A) and DR5P (B) Production. A, The reactions were carried out in 10 ml under the standard conditions, except for the inorganic phosphate concentration, for 7 h. B, The reaction mixtures, comprising in 300 ml 25% (v/v) enzymatically prepared FDP solution (with various initial inorganic phosphate concentrations and reaction times), 200 mM KH2PO4,15mM MgSO4.7H2O, 0.4% (v/v) polyoxyethylenelaurylamine, and 1% (v/v) xylene, were incubated at 28 C for 2 h with shaking (120 rpm). Pi, inorganic phosphate. the decrease in NADH was monitored at a wavelength of Result 340 nm with a Spectra Max 190 (Molecular Devices, Sunnyvale, CA) after 20 min incubation at 30 C. The Optimization of FDP production with baker’s yeast FDP concentration was calculated based on the decrease Reaction pH and temperature: The effect of the in NADH using a calibration curve obtained with reaction pH (in a range of 6.0–9.0) and temperature (in a authentic FDP solutions of known concentrations.