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Business Unit Cell Cybernetics, Plant Research International, Wageningen, the Netherlands; 2University of Wu¨rzburg, Chair of Food Chemistry, Germany; 3Laboratory of Plant Physiology, Wageningen University, the Netherlands Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. | Eur. J. Biochem. 269 3160-3171 2002 FEBS 2002 doi 10.1046 j.1432-1033.2002.02985.x Monoterpene biosynthesis in lemon Citruslimorì cDNA isolation and functional analysis of four monoterpene synthases Joost Liicker1 Mazen K. El Tamer1 Wilfried Schwab2 Francel W. A. Verstappen1 Linus H. W. van der Plas3 Harro J. Bouwmeester1 and Harrie A. Verhoeven1 1 Business Unit Cell Cybernetics Plant Research International Wageningen the Netherlands 2 University of Wurzburg Chair of Food Chemistry Germany 3Laboratory of Plant Physiology Wageningen University the Netherlands Citrus limon possesses a high content and large variety of monoterpenoids especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were -limonene two cDNAs - -b-pinene and y-ter-pinene. All enzymes exhibited a pH optimum around 7 addition of Mn2 as bivalent metal ion cofactor resulted in higher activity than Mg2 with an optimum concentration of 0.6 mM. Km values ranged from 0.7 to 3.1 IM. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil corresponding to more than 90 of the main components present. Keywords Citrus limon functional expression -limon-ene synthase - -b-pinene synthase y-terpinene synthase. Lemon Citrus limon L. Burm. f. is a member of the large Rutaceae family containing 130 genera in seven subfamilies with many important .
