Cloning of the xylitol dehydrogenase gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2003-03, Vol.67 (3), p.584-591
• Main Authors: Sugiyama, M. (Ajinomoto Co. Inc., Kawasaki, Kanagawa (Japan). Central Research Labs.), Suzuki, S, Tonouchi, N, Yokozeki, K
• Format: Article
• Language: English
• Subjects: Acetobacteraceae - enzymology; Acetobacteraceae - genetics; Amino Acid Sequence; BACTERIA; Base Sequence; Biological and medical sciences; Cloning, Molecular; D-Xylulose Reductase; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Gluconobacter; Hydrogen-Ion Concentration; MOLECULAR CLONING; Molecular Sequence Data; OXIDOREDUCTASES; Plasmids - genetics; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; short-chain dehydrogenase/reductase family; Substrate Specificity; Sugar Alcohol Dehydrogenases - genetics; Sugar Alcohol Dehydrogenases - isolation & purification; Sugar Alcohol Dehydrogenases - metabolism; Sugar Alcohols - metabolism; XYLITOL; Xylitol - biosynthesis; xylitol dehydrogenase
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.67.584

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 NH 2 -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.