SESQUITERPENE SYNTHASES FROM GRAND FIR (ABIES GRANDIS) - COMPARISON OF CONSTITUTIVE AND WOUND-INDUCED ACTIVITIES, AND CDNA ISOLATION, CHARACTERIZATION AND BACTERIAL EXPRESSION OF DELTA-SELINENE SYNTHASE AND GAMMA-HUMULENE SYNTHASE

Grand fir (Abies grandis) has been developed as a model system for til e study of oleoresin production in response to stem wounding and insec t attack. The turpentine fraction of the oleoresin was shown to contai n at least 38 sesquiterpenes that represent 12.5% of the turpentine, w ith the monoterpenes comprising the remainder. Assays of cell-free ext racts from grand fir stem with farnesyl diphosphate as substrate indic ated that the constitutive sesquiterpene synthases produced the same s esquiterpenes found in the oleoresin and that, in response to wounding , only two new products were synthesized, delta-cadinene and (E)-alpha -bisabolene. A similarity based cloning strategy yielded two new cDNA species from a stem cDNA library that, when expressed in Escherichia c oli and the gene products subsequently assayed, yielded a remarkable n umber of sesquiterpene products. The encoded enzymes have been named d elta-selinene synthase and gamma-humulene synthase based on the princi pal products formed; however, each enzyme synthesizes three major prod ucts and produces 34 and 52 total sesquiterpenes, respectively, thereb y accounting for many of the sesquiterpenes of the oleoresin, The dedu ced amino acid sequence of the delta-selinene synthase cDNA open readi ng frame encodes a protein of 581 residues (at 67.6 kDa), whereas that of the gamma-humulene synthase cDNA encodes a protein of 593 residues (at 67.9 kDa). The two amino acid sequences are 83% similar and 65% i dentical to each other and range in similarity from 65 to 67% and in i dentity from 43 to 46% when compared with the known sequences of monot erpene and diterpene synthases from grand fir. Although the two sesqui terpene synthases hom this gymnosperm do not very closely resemble ter pene synthases from angiosperm species (52-56% similarity and 26-30% i dentity), there are clustered regions of significant apparent homology between the enzymes of these two plant classes. The multi-step, multi -product reactions catalyzed by the sesquiterpene syntheses from grand fir are among the most complex of any terpenoid cyclase thus far desc ribed.