Open AccessCloning and Expression of a Xylitol-4-Dehydrogenase Gene from Pantoea ananatis
Author(s) -
Johannes Aarnikunnas,
Anne Pihlajaniemi,
Airi Palva,
Matti Leisola,
Antti Nyyssölä
Publication year - 2006
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.72.1.368-377.2006
Subject(s) - operon , biology , open reading frame , biochemistry , nucleic acid sequence , microbiology and biotechnology , escherichia coli , dehydrogenase , reductase , gene , peptide sequence , sequence analysis , nitrite reductase , enzyme , nitrate reductase
ThePantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol tol -xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh ) from aP. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5′ region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated fromP. ananatis ATCC 43072 suggested thatxdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested thatxdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of theP. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. TheP. ananatis xdh gene was successfully overexpressed inEscherichia coli , XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD+ as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for thed -form of xylulose. Xylitol was converted tol -xylulose with a high yield (>80%) by the resting recombinant cells, and thel -xylulose was secreted into the medium. No evidence ofd -xylulose being synthesized by the recombinant cells was found.