O-020. Isolation and Characterization of Isoeugenol-Degrading Gene from Pseudomonas nitroreducens Jin1

J-Y. Ryu1, K. W. Widmer1, J-H. Ahn2, M. J. Sadowsky3, H-G. Hur1;
1Gwangju Inst. of Sci. and Technology, Gwangju, REPUBLIC OF KOREA, 2Konkuk Univ., Seoul, REPUBLIC OF KOREA, 3Univ. of Minnesota, St. Paul, MN.

Vanillin has been widely used as a flavoring agent in foods, beverages, and pharmaceuticals. Eugenol and isoeugenol, plant-originated phenylpropanoid compounds, are abundant and used in the production of vanillin. Even though short metabolic pathways are known to be involved in the biotransformation of isoeugenol to vanillin, only one research group has published the isoeugenol degrading gene. In this study, we constructed the genomic library of Pseudomonas nitroreducens Jin1 which is able to utilize eugenol and isoeugenol as carbon sources. The library consisted of approximately 1,100 independent recombinant clones with an insert size of 50 - 100 kb. One clone exhibited a decrease of isoeuegenol in the culture medium. About 140 kb inserted DNA was sequenced and possible open reading frames (ORFs) were annotated. The deduced amino acid sequence of ORF18 of 1437 bp, exhibited 81.4% amino acid identity to isoeugenol monooxygenase from Pseudomonas putida IE27. The ORF18 was cloned and the expressed protein metabolized isoeugenol to vanillin in recombinant E. coli. This work is only the second reported attempt to clone the vanillin-producing gene from isoeugenol