Cloning of the xylitol dehydrogenase gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol.
Xylitol dehydrogenase (XDH) was purified from the cytoplasmic fraction of Gluconobacter oxydans ATCC 621. The purified enzyme reduced D-xylulose to xylitol in the presence of NADH with an optimum pH of around 5.0. Based on the determined NH2-terminal amino acid sequence, the gene encoding xdh was cloned, and its identity was confirmed by expression in Escherichia coli. The xdh gene encodes a polypeptide composed of 262 amino acid residues, with an estimated molecular mass of 27.8 kDa. The deduced amino acid sequence suggested that the enzyme belongs to the short-chain dehydrogenase/reductase family. Expression plasmids for the xdh gene were constructed and used to produce recombinant strains of G. oxydans that had up to 11-fold greater XDH activity than the wild-type strain. When used in the production of xylitol from D-arabitol under controlled aeration and pH conditions, the strain harboring the xdh expression plasmids produced 57 g/l xylitol from 225 g/l D-arabitol, whereas the control strain produced 27 g/l xylitol. These results demonstrated that increasing XDH activity in G. oxydans improved xylitol productivity.
Sugiyama
Masakazu
M
AminoScience Laboratories, Ajinomoto Co., Inc., Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan. masakazu_sugiyama@ajinomoto.com
Suzuki
Shun-ichi
S
Tonouchi
Naoto
N
Yokozeki
Kenzo
K
D016947:Acetobacteraceae / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D050542:D-Xylulose Reductase; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013402:Sugar Alcohols / Q000378:metabolism*; D014993:Xylitol / Q000096:biosynthesis*
D011994:Recombinant Proteins; D013402:Sugar Alcohols; D013401:Sugar Alcohol Dehydrogenases; D050542:D-Xylulose Reductase; D014993:Xylitol; C014999:arabitol