Sunday, July 29, 2007
P41

Microbial production of 2'-deoxyribonucleoside from glucose, acetaldehyde and nucleobase through multi-step enzymatic reactions

Jun Ogawa, Nobuyuki Horinouchi, and Sakayu Shimizu. Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502, Japan

With the spread of PCR techniques and use of new antiviral nucleosides, and the advent of antisense drugs for cancer therapy, there will be a need for DNA building block, 2’-deoxyribonucleoside (dNS).  Here, we report a microbial process for dNS production from easily available materials, glucose, acetaldehyde and nucleobase.  In this process, microorganisms possessing glycolytic enzymes, deoxyriboaldolase (DERA), phosphopentomutase (PPMase) and nucleoside phosphorylase (NPase), were used as catalysts.  Metabolic investigation of Escherichia coli transformant expressing acetaldehyde-tolerant DERA from Klebsiella pneumoniae showed that fructose 1,6-diphosphate (FDP) is a potential substrate for 2-deoxyribose 5-phosphate (DR5P), a key intermediate for dNS synthesis.  Based on these results, coupling of glycolytic pathway and DERA reaction was investigated.  The glycolytic enzymes in baker’s yeast gave FDP from glucose driven by the energy of ATP generated from adenosine by alcoholic fermentation of the yeast.  FDP was further transformed to DR5P in the presence of acetaldehyde by DERA-expressing E. coli via D-glyceraldehyde 3-phosphate.  The DR5P produced was further transformed to dNS via 2-deoxyriobse 1-phosphate in the presence of a nucleobase by E. coli transformant expressing PPMase and commercial NPase. By operating all above reactions in a one-pot, 75 mM 2’-deoxyinosine was produced from 600 mM glucose, 430 mM acetaldehyde, 70 mM adenine, and 20 mM adenosine as an energy carrier. 

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