CDNA CLONING, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF 4S-(-)-LIMONENE SYNTHASE FROM PERILLA-FRUTESCENS

A molecular biological study on limonene synthase that catalyzes the c yclization of geranyldiphosphate to yield the olefin 4(S)-limonene, an intermediate in the biosynthesis of a monoterpenoid, perillaldehyde, in Perilla frutescens Britton has been carried out. We isolated and ch aracterized 10 cDNAs homologous to spearmint limonene synthase cDNA fr om an expression library constructed from cotyledons of a Perilla stra in homozygous for two pairs of dominant genes, G and H, which are resp onsible for the formation of 4(S)-limonene. Two of these cDNA clones w ere functionally expressed in Escherichia coli, yielding enzymes which were catalytically active in generating 4(S)-limonene from geranyldip hosphate. The longest open reading frame in the representative cDNA cl one PFLC1 consisted of 1812 nucleotides corresponding to 603 amino aci ds. 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, and gghh) of P. frutescens suggested that more than one copy of the PFLC1 DNA exists in strains having the HH g enotype. In contrast, no PPLC1 DNA sequences were found in the genomes of strains with the hh genotype that are incapable of producing cyclo hexanoid monoterpenes for lack of limonene synthase activity. Northern blot analyses, using a PFLC1 3'-flanking region as a hybridization pr obe, showed that PFLC1 mRNA accumulated in all the aerial parts of the GGHH plants, particularly in the leaves. In the ggHH plants, on the o ther hand, PFLC1 mRNA was detected only in minute amounts in the stem and calyx. (C) 1996 Academic Press, Inc.