cDNA Cloning, Characterization, and Functional Expression of 4S-(−)-Limonene Synthase fromPerilla frutescens

A molecular biological study on limonene synthase that catalyzes the cyclization of geranyldiphosphate to yield the olefin 4(S)-limonene, an intermediate in the biosynthesis of a monoterpenoid, perillaldehyde, inPerilla frutescensBritton has been carried out. We isolated and characterized 10 cDNAs homologous to spearmint limonene synthase cDNA from an expression library constructed from cotyledons of aPerillastrain homozygous for two pairs of dominant genes,GandH,which are responsible for the formation of 4(S)-limonene. Two of these cDNA clones were functionally expressed inEscherichia coli,yielding enzymes which were catalytically active in generating 4(S)-limonene from geranyldiphosphate. The longest open reading frame in the representative cDNA clone PFLC1 consisted of 1812 nucleotides corresponding to 603 amino acids. Its identity to the ORFs of spearmint limonene synthase, tobacco 5-epi-aristolochene synthase, and castor bean casbene synthase were 65, 35, and 30%, respectively. Genomic Southern blot analyses of various genotypes (GGHH, GGhh, ggHH,andgghh) ofP. frutescenssuggested that more than one copy of the PFLC1 DNA exists in strains having theHHgenotype. In contrast, no PFLC1 DNA sequences were found in the genomes of strains with thehhgenotype that are incapable of producing cyclohexanoid monoterpenes for lack of limonene synthase activity. Northern blot analyses, using a PFLC1 3′-flanking region as a hybridization probe, showed that PFLC1 mRNA accumulated in all the aerial parts of theGGHHplants, particularly in the leaves. In theggHHplants, on the other hand, PFLC1 mRNA was detected only in minute amounts in the stem and calyx.