Abstract
Common sage (Salvia officinalis) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of the p-menthane (1,8-cineole), pinane (alpha- and beta-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families. An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli. The heterologously expressed synthases produce (+)-bornyl diphosphate, 1, 8-cineole, and (+)-sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces significant amounts of (+)-alpha-pinene, (+)-camphene, and (+/-)-limonene. The 1,8-cineole synthase produces significant amounts of (+)- and (-)-alpha-pinene, (+)- and (-)-beta-pinene, myrcene and (+)-sabinene, and the (+)-sabinene synthase produces significant quantities of gamma-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes.
Highlights
The nucleotide sequences reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data banks under accession numbers AF051899 (1,8-cineole synthase), AF051900, and AF051901
We describe the homology-based cloning, and subsequent sequencing and heterologous expression, of three monoterpene synthase cDNA genes from sage, the recombinant enzymes from which produce three different major types of cyclic monoterpene products, (ϩ)-sabinene, 1,8-cineole, and (ϩ)-bornyl diphosphate, respectively (Scheme 1)
As a possible alternative to protein-based cloning of terpene synthases, a homology-based PCR strategy was recently proposed [22] that was developed by comparison of the deduced amino acid sequences of cDNAs encoding a monoterpene synthase [23], a sesquiterpene synthase [24], and a diterpene synthase [25] of phylogenetically distant angiosperm species
Summary
The nucleotide sequences reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data banks under accession numbers AF051899 (1,8-cineole synthase), AF051900 (bornyl diphosphate synthase), and AF051901 (sabinene synthase). We describe the homology-based cloning, and subsequent sequencing and heterologous expression, of three monoterpene synthase cDNA genes from sage, the recombinant enzymes from which produce three different major types of cyclic monoterpene products, (ϩ)-sabinene (a bicyclic olefin), 1,8-cineole (a bicyclic ether), and (ϩ)-bornyl diphosphate (a bicyclic diphosphate ester), respectively (Scheme 1).
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