Construction of deoxyriboaldolase-overexpressing Escherichia coli and its application to 2-deoxyribose 5-phosphate synthesis from glucose and acetaldehyde for 2'-deoxyribonucleoside production.
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.
Horinouchi
Nobuyuki
N
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Ogawa
Jun
J
Sakai
Takafumi
T
Kawano
Takako
T
Matsumoto
Seiichiro
S
Sasaki
Mie
M
Mikami
Yoichi
Y
Shimizu
Sakayu
S
D000079:Acetaldehyde / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003853:Deoxyribonucleosides / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005947:Glucose / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012267:Ribosemonophosphates / Q000096:biosynthesis*; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
D003853:Deoxyribonucleosides; D011994:Recombinant Proteins; D012267:Ribosemonophosphates; C058245:2-deoxyribose 5-phosphate; D000446:Aldehyde-Lyases; C020019:deoxyribose-phosphate aldolase; D000079:Acetaldehyde; D005947:Glucose