Molecular cloning and functional expression of a multifunctional triterpene synthase cDNA from a mangrove species Kandelia candel (L.) Druce

• Published in: Phytochemistry (Oxford) Vol. 67; no. 23; pp. 2517 - 2524
• Main Authors: Basyuni, Mohammad, Oku, Hirosuke, Inafuku, Masashi, Baba, Shigeyuki, Iwasaki, Hironori, Oshiro, Keichiro, Okabe, Takafumi, Shibuya, Masaaki, Ebizuka, Yutaka
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 01-12-2006; Elsevier
• Subjects: Amino Acid Sequence; Biological and medical sciences; Cloning, Molecular; complementary DNA; DNA, Complementary - genetics; enzyme activity; Enzymes; Fundamental and applied biological sciences. Psychology; Gene Expression; isomerases; Kandelia candel; Kandelia candel (L.) druce; Lupeol; lupeol synthase; Mangrove; Metabolism; Molecular Sequence Data; Molecular Structure; Multifunctional triterpene synthase; Oxidosqualene cyclase; Phylogeny; Plant physiology and development; plant proteins; Plant Proteins - genetics; Plant Proteins - metabolism; recombinant fusion proteins; Rhizophoraceae; Rhizophoraceae - enzymology; Rhizophoraceae - genetics; sequence analysis; triterpene synthase; Triterpenes - chemistry; Triterpenes - metabolism; triterpenoids; α-Amyrin; β-Amyrin; Japan; α-Amyrin; Mangrove; β-Amyrin; Rhizophoraceae; Oxidosqualene cyclase; Multifunctional triterpene synthase; Kandelia candel (L.) druce; Lupeol; Root; Enzyme; Homology; Synthase; Characterization; Conserved sequence; Complementary DNA; Dicotyledones; Angiospermae; Spermatophyta; Aminoacid sequence
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2006.09.016

An oxidosqualene cyclase homologue was cloned from
Kandelia candel (L.) Druce, and identified as a multifunctional triterpene synthase able to produce lupeol, β-amyrin and α-amyrin in a 2:1:1 ratio by expression in the mutant yeast strain GIL77.
Homology based PCRs with degenerate primers designed from the conserved sequences among the known oxidosqualene cylases (OSCs) have resulted in cloning of a triterpene synthase (
KcMS) from the young roots of
Kandelia candel (L.) Druce (Rhizophoraceae).
KcMS consists of a 2286 bp open reading frame, which codes for 761 amino acids. The deduced amino acid sequence showed 79% homology to a lupeol synthase from
Ricinus communis suggesting it to be a lupeol synthase of
K. candel.
KcMS was expressed in a lanosterol synthase deficient yeast with the expression vector pYES2 under the control of
GAL1 promoter. GC–MS analysis showed that the transformant accumulated a mixture of lupeol, β-amyrin and α-amyrin in a 2:1:1 ratio, indicating that
KcMS encodes a multifunctional triterpene synthase, although it showed high sequence homology to a
R. communis lupeol synthase. This is the first OSC cloning from mangrove tree species.