Takeyoshi Takahashi

Biosynthesis of triterpenoids in cultured cells, and regenerated and wild plant organs of Taraxacum officinale

Abstract Undifferentiated cultured cells of Taraxacum officinale produced oleanolic and ursolic acids as major triterpenoids in addition to triterpenols composed mainly of α- and β-amyrins. Regenerated and wild plants contained additional triterpenols, i.e. taraxasterol and lupeol, but negligible quantities of triterpene acids. [2- 14 C]Mevalonic acid was fed to callus cells, wild plant organs, and shoot and root segments of plants redifferentiated from callus. After 48 hr incubation, triterpenols, phytosterols and triterpene acids were equally labelled in callus cultures; in contrast, triterpene acids were strongly labelled in the differentiated tissues. The incorporation of radioactivity into α- and β-amyrins was highest in the callus where taraxasterol and lupeol were not labelled substantially; the latter were labelled in differentiated organs, agreeing with the accumulation patterns. A time course experiment with shoot segments showed a different production pattern of cycloartane triterpenes from other classes of triterpenoids. The results are discussed in relation to organ-specific biosynthesis of specific triterpenoids.

Licodione Synthase, a Cytochrome P450 Monooxygenase Catalyzing 2-Hydroxylation of 5-Deoxyflavanone, in Cultured Glycyrrhiza echinata L. Cells

Cultured Glycyrrhiza echinata L. (Leguminosae) cells produce a retrochalcone echinatin (4,4[prime]-dihydroxy-2-methoxychalcone) and its biosynthetic intermediate licodione [1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione, a dibenzoylmethane (keto form) or its enol tautomer ([beta]-hydroxychalcone)], when treated with elicitor-active substances, e.g. yeast extract. A microsomal fraction (160,000g pellet) prepared from yeast extract-induced suspension cultures of G. echinata catalyzed the formation of licodione from (2S)-liquiritigenin (7,4[prime]-dihydroxyflavanone) in the presence of NADPH and air. This licodione synthase activity was shown to be dependent on cytochrome P450 by its microsomal localization, requirement of NAD(P)H and O2 for activity, and inhibition by typical cytochrome P450 inhibitors. Licodione synthase activity transiently increased in the cells after treatment with yeast extract. When (2S)-naringenin (5,7,4[prime]-trihydroxyflavanone) and NADPH were incubated with the same microsomal preparation, a polar compound, which further converted into apigenin (5,7,4[prime]-trihydroxyflavone) when treated with acid, was produced. The reaction mechanism of licodione synthase is likely to be 2-hydroxylation of the flavanone molecule and subsequent hemiacetal opening and is possibly the same as the previously suggested mechanism of flavone synthase II from soybean and, furthermore, closely related to isoflavone synthase from Pueraria lobata.

Triterpenoid biosynthesis in tissue cultures of Glycyrrhiza glabra var. glandulifera.

The incorporation of [1-14C]acetate and [214 C]mevalonate into free and esterified triterpen-3-ols was examined in original plant organs and tissue cultures of Glycyrrhiza glabra var. glandulifera. Both substrates labeled β-amyrin, an oleanane-type triterpene, and cycloartenol and 24-methylenecycloartanol, both of which are intermediates of phytosterol biosynthesis. The label in esterified triterpenes was distributed mainly in phytosterol intermediates, but not in β-amyrin. The ratio of βamyrin formation among the three triterpenes from [2-14C]mevalonate was relatively high in stolon segments and in root cultures, but negligible in callus cultures. Administration of a specific inhibitor of squalene-2, 3-epoxide:cycloartenol (lanosterol) cyclase caused a marked increase of β-amyrin synthesis in root suspension cultures, and of 24-methylenecycloartanol synthesis in cell suspension cultures, from [2-14C]mevalonate.

Cloning of cytochrome P450 cDNAs from cultured Glycyrrhiza echinata L. cells and their transcriptional activation by elicitor-treatment

From a cDNA library of cultured Glycyrrhiza echinata L. (Fabaceae) cells treated with yeast extract (YE) elicitor, eight cytochrome P450 (P450) fragments (Ge-1 to 8) were isolated using the PCR method based on conserved sequences of P450s. Comparison of amino acid sequences revealed that two of the fragments (Ge-1 and 2) belong to the CYP73 family encoding trans-cinnamic acid 4-hydroxylase. Ge-4 had 63% identity with the sequence included in CYP93, which has recently been reported to be induced by methyl jasmonate in soybean cells (Suzuki et al., FEBS Lett., 383 (1996) 83–86), and Ge-6 and 7 were highly homologous to the partial sequence of CYP84 encoding ferulic acid 5-hydroxylase of Arabidopsis thaliana (Meyer et al., Proc. Natl. Acad. Sci. USA, 93 (1996) 6869–6874). Others (Ge-3, 5 and 8) displayed homology less than 43% with known plant P450s. A full-length cDNA (CYP73A14) containing Ge-1 sequence was isolated from the cDNA library, and its amino acid sequence showed 91–93% identity with CYP73 of other leguminous plant species. Northern blot analysis of mRNAs of G. echinata cells indicated that CYP73 (Ge-1 and 2) genes were transcribed at the early stages post-elicitation (<10 h). The transcription level of the Ge-1 gene was much higher than that of Ge-2. Transcription of Ge-3, 5, 6 and 7 was also activated by elicitation, but no clear signal from Ge-4 and 8 was observed. When total RNAs of cultured cells of Medicago sativa were analyzed by Northern blotting, transcripts hybridized with G. echinata cDNA clones containing Ge-1, 3, 5 and 6 sequences could be detected. Possible involvement of cloned P450s in elicitor-induced phenylpropanoid/flavonoid biosynthesis is discussed.

Enzymatic synthesis of 6′-deoxychalcone in cultured Glycyrrhiza echinata cells

Abstract5-Deoxy-(iso)flavonoids are biosynthesized from 6′-deoxychalcone (isoliquiritigenin). The coaction of a reductase with chalcone synthase (CHS) has been established in soybean cells to be responsible for the synthesis of 6′-deoxychalcone. Western blot analysis of crude extracts from cultured cells of Glycyrrhiza echinata, another member of the Leguminosae, revealed proteins which cross-react with an antiserum raised against the soybean reductase. DEAE-Cellulose chromatography of the extract yielded fractions which showed CHS activity but not deoxychalcone synthase activity, and these fractions were also negative in Western blot analysis. In contrast, fractions displaying positive signals with the antiserum were also able to synthesize 6′-deoxychalcone/5-deoxyflavanone. These results indicate that in G. echinata, too, synthesis of 6′-deoxychalcone is likely to be performed by the coaction of the reductase and CHS. Induction of the reductase and CHS by yeast extract treatment of the cells was demonstrated.

Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus

Shionone is the major triterpenoid component of Aster tataricus possessing a unique all six‐membered tetracyclic skeleton and 3‐oxo‐4‐monomethyl structure. To clarify its biosynthetic process, an oxidosqualene cyclase cDNA was isolated from A. tataricus, and the function of the enzyme was determined in lanosterol synthase‐deficient yeast. The cyclase yielded ca. 90% shionone and small amounts of β‐amyrin, friedelin, dammara‐20,24‐dienol, and 4‐epishionone and was designated as a shionone synthase (SHS). Transcripts of SHS were detected in A. tataricus organs, confirming its involvement in shionone biosynthesis. SHS was shown to have evolved in the Asteraceae from β‐amyrin synthase lineages and acquired characteristic species‐ and product‐specificities.

Properties and partial purification of squalene synthase from cultured cells of dandelion

Squalene synthase (SQS) activity was detected in the microsomal fractions of suspension-cultured cells of dandelion (Taraxacum officinale), which produce cycloartane (involved in phytosterol biosynthesis) and other classes (e.g. oleanane and ursane) of triterpenoids. High SQS activity was detected at the late logarithmic growth stage of the culture. The activity was solubilized from the microsomes by the use of detergents (glycodeoxycholate and octylthioglucopyranoside), and chromatographed on triple-joint-columns composed of cation-exchange, anion-exchange and hydroxylapatite resins. Active fractions were further subjected to chromatography on hydroxylapatite and anion-exchange reins, followed by fast protein liquid chromatography (FPLC) on a gel-filtration column. About a 50-fold purification compared with the microsomal activity was achieved at the hydroxylapatite stage, and, although the recovery was poor, the activity was retained in the final FPLC fractions.