MONOTERPENE SYNTHASES FROM COMMON SAGE (SALVIA-OFFICINALIS) - CDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF (-SABINENE SYNTHASE, 1,8-CINEOLE SYNTHASE, AND (+)-BORNYL DIPHOSPHATE SYNTHASE())

Common sage (Salvia officinalis) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including member s of the p-menthane (1,8-cineole), pinane (alpha- and beta-pinene), th ujane (isothujone), camphane (camphene), and bornane (camphor) familie s. An homology-based polymerase chain reaction cloning strategy was de veloped and used to isolate the cDNAs encoding three multiproduct mono terpene synthases from this species that were functionally expressed i n Escherichia coli. The heterologously expressed synthases produce (+) -bornyl diphosphate, 1,8-cineole, and (+)-sabinene, respectively, as t heir major products from geranyl diphosphate. The bornyl diphosphate s ynthase also produces significant amounts of (+)-alpha-pinene, (+)-cam phene, and (+/-)-limonene. The 1,8-cineole synthase produces significa nt amounts of (+)- and (-)-alpha-pinene, (+)- and (-)-beta-pinene, myr cene and (+)-sabinene, and the (+)-sabinene synthase produces signific ant quantities of gamma-terpinene and terpinolene. All three enzymes a ppear to be translated as preproteins bearing an amino-terminal plasti d targeting sequence, consistent with the plastidial origin of monoter penes in plants. Deduced sequence analysis and size exclusion chromato graphy indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, co nsistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products ge nerated by the recombinant (+)-bornyl diphosphate synthase suggest tha t this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzy mes.