Tomoyoshi Akashi

Licorice β-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin

Glycyrrhizin, a major bioactive compound derived from the underground parts of Glycyrrhiza (licorice) plants, is a triterpene saponin that possesses a wide range of pharmacological properties and is used worldwide as a natural sweetener. Because of its economic value, the biosynthesis of glycyrrhizin has received considerable attention. Glycyrrhizin is most likely derived from the triterpene β-amyrin, an initial product of the cyclization of 2,3-oxidosqualene. The subsequent steps in glycyrrhizin biosynthesis are believed to involve a series of oxidative reactions at the C-11 and C-30 positions, followed by glycosyl transfers to the C-3 hydroxyl group; however, no genes encoding relevant oxidases or glycosyltransferases have been identified. Here we report the successful identification of CYP88D6, a cytochrome P450 monooxygenase (P450) gene, as a glycyrrhizin-biosynthetic gene, by transcript profiling-based selection from a collection of licorice expressed sequence tags (ESTs). CYP88D6 was characterized by in vitro enzymatic activity assays and shown to catalyze the sequential two-step oxidation of β-amyrin at C-11 to produce 11-oxo-β-amyrin, a possible biosynthetic intermediate between β-amyrin and glycyrrhizin. CYP88D6 coexpressed with β-amyrin synthase in yeast also catalyzed in vivo oxidation of β-amyrin to 11-oxo-β-amyrin. CYP88D6 expression was detected in the roots and stolons by RT-PCR; however, no amplification was observed in the leaves or stems, which is consistent with the accumulation pattern of glycyrrhizin in planta. These results suggest a role for CYP88D6 as a β-amyrin 11-oxidase in the glycyrrhizin pathway.

Flavonoids of leguminous plants: structure, biological activity, and biosynthesis.

Flavonoids are common to most vascular plants and are postulated to be involved in a number of plant functions. Leguminous plants are intriguing materials for studies on flavonoids for several reasons. First, legumes produce flavonoids with specific chemical structures i.e. isoflavonoids and 5- deoxy( iso)f lavonoids. Second, leg um i nous flavonoids are postulated to play important roles both as defensive compounds against pathogenic microorganisms and as chemical signals in symbiotic nitrogen fixation. Third, as constituents of food and forage, leguminous flavonoids have striking biological activity in humans and animals. This article reviews the chemical structure, biological activity, and biosynthesis of leguminous flavonoids. Recent achievements in the identification and characterization of cytochrome P450 monooxygenases, which play important roles in (iso) flavonoid biosynthesis, are also introduced. Perspectives of molecular genetic studies on flavonoids with the model legume are discussed.

Triterpene Functional Genomics in Licorice for Identification of CYP72A154 Involved in the Biosynthesis of Glycyrrhizin

This work reports the identification of a cytochrome P450 monooxygenase that is responsible for the biosynthesis of glycyrrhizin, a triterpenoid saponin found in licorice. The results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide proof of concept for engineering the production of high-value triterpenoid products in yeasts. Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton β-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding β-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-β-amyrin (a possible biosynthetic intermediate between β-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-β-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of β-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.

Identification of a cytochrome P450 cDNA encoding (2S)-flavanone 2-hydroxylase of licorice (Glycyrrhiza echinata L.; Fabaceae) which represents licodione synthase and flavone synthase II1

The microsome of insect cells expressing CYP Ge‐5 (CYP93B1), a cytochrome P450 cDNA of licorice (Glycyrrhiza echinata L.), catalyzed the formation of [14C]licodione and [14C]‐2‐hydroxynaringenin from (2S)‐[14C]liquiritigenin and (2S)‐[14C]naringenin, respectively. On acid treatment, the products were converted to 14C‐labeled 7,4′‐dihydroxyflavone and apigenin. Eriodictyol was also converted to luteolin by the reaction with the microsome of yeast expressing CYP93B1 and subsequent acid treatment. CYP93B1 was thus shown to encode (2S)‐flavanone 2‐hydroxylase, which has previously been designated to licodione synthase and flavone synthase II depending on the substrates employed.

Cytochrome P450s in flavonoid metabolism

In this review, cytochrome P450s characterized at the molecular level catalyzing aromatic hydroxylations, aliphatic hydroxylations and skeleton formation in the flavonoid metabolism are surveyed. They are involved in the biosynthesis of anthocyanin pigments and condensed tannin (CYP75, flavonoid 3′,5′-hydroxylase and 3′-hydroxylase), flavones [CYP93B, (2S)-flavanone 2-hydroxylase and flavone synthase II], and leguminous isoflavonoid phytoalexins [CYP71D9, flavonoid 6-hydroxylase; CYP81E, isoflavone 2′-hydroxylase and 3′-hydroxylase; CYP93A, 3,9-dihydroxypterocarpan 6a-hydroxylase; CYP93C, 2-hydroxyisoflavanone synthase (IFS)]. Other P450s of the flavonoid metabolism include methylenedioxy bridge forming enzyme, cyclases producing glyceollins, flavonol 6-hydroxylase and 8-dimethylallylnaringenin 2′-hydroxylase. Mechanistic studies on the unusual aryl migration by CYP93C, regulation of IFS expression in plant organs and its biotechnological applications are introduced, and flavonoid metabolisms by non-plant P450s are also briefly discussed.

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.

Identification of cDNAs encoding pterocarpan reductase involved in isoflavan phytoalexin biosynthesis in Lotus japonicus by EST mining

Isoflavans and pterocarpans are the major biosynthetically connected phytoalexins in legumes. A search of the expressed sequence tag library of a model legume Lotus japonicus, which produces an (−)‐isoflavan, for homologs of phenylcoumaran benzylic ether reductase catalyzing the reductive cleavage of dihydrofurans, yielded seven full‐length cDNAs, and the encoded proteins were analyzed in vitro. Four of them cleaved the dihydrofuran of a pterocarpan medicarpin to yield an isoflavan (−)‐vestitol and were designated pterocarpan reductase (PTR). Two PTRs displayed enantiospecificity to (−)‐medicarpin, representing genuine L. japonicus PTRs, while the other two lacked enantiospecificity and were presumed to be evolutionarily primitive types.

Genome-wide Analyses of the Structural Gene Families Involved in the Legume-specific 5-Deoxyisoflavonoid Biosynthesis of Lotus japonicus

Abstract A model legume Lotus japonicus (Regel) K. Larsen is one of the subjects of genome sequencing and functional genomics programs. In the course of targeted approaches to the legume genomics, we analyzed the genes encoding enzymes involved in the biosynthesis of the legume-specific 5-deoxyisoflavonoid of L. japonicus, which produces isoflavan phytoalexins on elicitor treatment. The paralogous biosynthetic genes were assigned as comprehensively as possible by biochemical experiments, similarity searches, comparison of the gene structures, and phylogenetic analyses. Among the 10 biosynthetic genes investigated, six comprise multigene families, and in many cases they form gene clusters in the chromosomes. Semi-quantitative reverse transcriptase–PCR analyses showed coordinate up-regulation of most of the genes during phytoalexin induction and complex accumulation patterns of the transcripts in different organs. Some paralogous genes exhibited similar expression specificities, suggesting their genetic redundancy. The molecular evolution of the biosynthetic genes is discussed. The results presented here provide reliable annotations of the genes and genetic markers for comparative and functional genomics of leguminous plants.

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.

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.