cDNA cloning, characterization, and functional expression of 4S-(-)- limonene synthase from Perilia frutescens

Akiko Kubo, Kazufumi Yazaki, Mamoru Tabata, Gisho Honda, Rodney Croteau

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

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, in Perilla frutescens Britton has been carried out. We isolated and characterized 10 cDNAs homologous to spearmint limonene synthase cDNA from an expression library constructed from cotyledons of a Perilla strain homozygous for two pairs of dominant genes, G and H, which are responsible for the formation of 4(S)-limonene. Two of these cDNA clones were functionally expressed in Escherichia 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, and gghh) of P. frutescens suggested that more than one copy of the PFLC1 DNA exists in strains having the HH genotype. In contrast, no PFLC1 DNA sequences were found in the genomes of strains with the hh genotype that are incapable of producing cyclohexanoid monoterpenes for lack of limonene synthase activity. Northern blot analyses, using a PFLC13'-flanking region as a hybridization probe, showed that PFLC1 mRNA accumulated in all the aerial parts of the GGHH plants, particularly in the leaves. In the ggHH plants, on the other hand, PFLC1 mRNA was detected only in minute amounts in the stem and calyx.

Original languageEnglish
Pages (from-to)280-287
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume332
Issue number2
DOIs
Publication statusPublished - 1996 Aug 15
Externally publishedYes

Fingerprint

Cloning
Organism Cloning
Perilla frutescens
Mentha spicata
Complementary DNA
Monoterpenes
Genotype
Open Reading Frames
Perilla
Clone Cells
Genes
Aerial Plant Components
Castor Bean
Dominant Genes
Messenger RNA
Tobacco
Cotyledon
DNA sequences
Cyclization
Biosynthesis

Keywords

  • genetic analysis
  • l-limonene
  • limonene synthase
  • Perilla frutescens

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

cDNA cloning, characterization, and functional expression of 4S-(-)- limonene synthase from Perilia frutescens. / Kubo, Akiko; Yazaki, Kazufumi; Tabata, Mamoru; Honda, Gisho; Croteau, Rodney.

In: Archives of Biochemistry and Biophysics, Vol. 332, No. 2, 15.08.1996, p. 280-287.

Research output: Contribution to journalArticle

Kubo, Akiko ; Yazaki, Kazufumi ; Tabata, Mamoru ; Honda, Gisho ; Croteau, Rodney. / cDNA cloning, characterization, and functional expression of 4S-(-)- limonene synthase from Perilia frutescens. In: Archives of Biochemistry and Biophysics. 1996 ; Vol. 332, No. 2. pp. 280-287.
@article{90170ba0cab945a6a5244b7f9d90a2da,
title = "cDNA cloning, characterization, and functional expression of 4S-(-)- limonene synthase from Perilia frutescens",
abstract = "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, in Perilla frutescens Britton has been carried out. We isolated and characterized 10 cDNAs homologous to spearmint limonene synthase cDNA from an expression library constructed from cotyledons of a Perilla strain homozygous for two pairs of dominant genes, G and H, which are responsible for the formation of 4(S)-limonene. Two of these cDNA clones were functionally expressed in Escherichia 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, and gghh) of P. frutescens suggested that more than one copy of the PFLC1 DNA exists in strains having the HH genotype. In contrast, no PFLC1 DNA sequences were found in the genomes of strains with the hh genotype that are incapable of producing cyclohexanoid monoterpenes for lack of limonene synthase activity. Northern blot analyses, using a PFLC13'-flanking region as a hybridization probe, showed that PFLC1 mRNA accumulated in all the aerial parts of the GGHH plants, particularly in the leaves. In the ggHH plants, on the other hand, PFLC1 mRNA was detected only in minute amounts in the stem and calyx.",
keywords = "genetic analysis, l-limonene, limonene synthase, Perilla frutescens",
author = "Akiko Kubo and Kazufumi Yazaki and Mamoru Tabata and Gisho Honda and Rodney Croteau",
year = "1996",
month = "8",
day = "15",
doi = "10.1006/abbi.1996.0343",
language = "English",
volume = "332",
pages = "280--287",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - cDNA cloning, characterization, and functional expression of 4S-(-)- limonene synthase from Perilia frutescens

AU - Kubo, Akiko

AU - Yazaki, Kazufumi

AU - Tabata, Mamoru

AU - Honda, Gisho

AU - Croteau, Rodney

PY - 1996/8/15

Y1 - 1996/8/15

N2 - 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, in Perilla frutescens Britton has been carried out. We isolated and characterized 10 cDNAs homologous to spearmint limonene synthase cDNA from an expression library constructed from cotyledons of a Perilla strain homozygous for two pairs of dominant genes, G and H, which are responsible for the formation of 4(S)-limonene. Two of these cDNA clones were functionally expressed in Escherichia 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, and gghh) of P. frutescens suggested that more than one copy of the PFLC1 DNA exists in strains having the HH genotype. In contrast, no PFLC1 DNA sequences were found in the genomes of strains with the hh genotype that are incapable of producing cyclohexanoid monoterpenes for lack of limonene synthase activity. Northern blot analyses, using a PFLC13'-flanking region as a hybridization probe, showed that PFLC1 mRNA accumulated in all the aerial parts of the GGHH plants, particularly in the leaves. In the ggHH plants, on the other hand, PFLC1 mRNA was detected only in minute amounts in the stem and calyx.

AB - 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, in Perilla frutescens Britton has been carried out. We isolated and characterized 10 cDNAs homologous to spearmint limonene synthase cDNA from an expression library constructed from cotyledons of a Perilla strain homozygous for two pairs of dominant genes, G and H, which are responsible for the formation of 4(S)-limonene. Two of these cDNA clones were functionally expressed in Escherichia 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, and gghh) of P. frutescens suggested that more than one copy of the PFLC1 DNA exists in strains having the HH genotype. In contrast, no PFLC1 DNA sequences were found in the genomes of strains with the hh genotype that are incapable of producing cyclohexanoid monoterpenes for lack of limonene synthase activity. Northern blot analyses, using a PFLC13'-flanking region as a hybridization probe, showed that PFLC1 mRNA accumulated in all the aerial parts of the GGHH plants, particularly in the leaves. In the ggHH plants, on the other hand, PFLC1 mRNA was detected only in minute amounts in the stem and calyx.

KW - genetic analysis

KW - l-limonene

KW - limonene synthase

KW - Perilla frutescens

UR - http://www.scopus.com/inward/record.url?scp=0030586724&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030586724&partnerID=8YFLogxK

U2 - 10.1006/abbi.1996.0343

DO - 10.1006/abbi.1996.0343

M3 - Article

VL - 332

SP - 280

EP - 287

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 2

ER -