GERMACRENE-C SYNTHASE FROM LYCOPERSICON-ESCULENTUM CV VFNT CHERRY TOMATO - CDNA ISOLATION, CHARACTERIZATION, AND BACTERIAL EXPRESSION OF THE MULTIPLE PRODUCT SESQUITERPENE CYCLASE

Germacrene C was found by GC-MS and NMR analysis to be the most abunda nt sesquiterpene in the leaf oil of Lycopersicon esculentum cv, VFNT C herry, with lesser amounts of germacrene A, guaia-6,9-diene, germacren e B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enz yme preparations from leaves catalyzed the divalent metal ion-dependen t cyclization of [1-H-3]farnesyl diphosphate to these same sesquiterpe ne olefins, as determined by radio-CC, To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases, With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp f ragment that was used as a hybridization probe to screen the cDNA libr ary, Thirty-one clones were evaluated for functional expression of ter penoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA iso lates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacre ne A B, and D, None of the expressed proteins was active with geranylg eranyl diphosphate; however, one truncated protein converted geranyl d iphosphate to the moneterpene limonene, The cDNA inserts specify a ded uced polypeptide of 548 amino acids (M-r = 64,114), and sequence compa rison with other plant sesquiterpene cyclases indicates that germacren e C synthase most closely resembles cotton delta-cadinene synthase (50 % identity).