Allylic Hydroxylation Activity Is a Source of Saponin Chemodiversity in the Genus Glycyrrhiza.

Licorice (Glycyrrhiza) produces glycyrrhizin, a valuable triterpenoid saponin, which exhibits persistent sweetness and broad pharmacological activities. In the genus Glycyrrhiza, three species, Glycyrrhiza uralensis, Glycyrrhiza glabra and Glycyrrhiza inflata, produce glycyrrhizin as their main triterpenoid saponin, which has a ketone group at C-11. Other Glycyrrhiza species produce mainly oleanane-type saponins, which harbor homoannular or heteroannular diene structures that lack the C-11 ketone. Although the glycyrrhizin biosynthetic pathway has been fully elucidated, the pathway involving saponins with diene structures remains unclear. CYP88D6 from G. uralensis is a key enzyme in glycyrrhizin biosynthesis, catalyzing the sequential two-step oxidation of ß-amyrin at position C-11 to produce 11-oxo-ß-amyrin. In this study, we evaluated the functions of CYP88D6 homologs from the glycyrrhizin-producing species G. glabra and G. inflata and from the non-glycyrrhizin-producing species Glycyrrhiza pallidiflora and Glycyrrhiza macedonica, using yeast engineered to supply ß-amyrin as a substrate. Yeast expressing CYP88D6 homologs from glycyrrhizin-producing species produced 11-oxo-ß-amyrin. However, yeast expressing CYP88D6 homologs (such as CYP88D15) from the non-glycyrrhizin-producing Glycyrrhiza species accumulated oleana-9(11),12-dien-3ß-ol and oleana-11,13(18)-dien-3ß-ol; these diene compounds are non-enzymatic or yeast endogenous enzymatic dehydration derivatives of 11α-hydroxy-ß-amyrin, a direct reaction product of CYP88D15. These results suggest that the activities of CYP88D6 homologs, particularly their ability to catalyze the second oxidation, could influence glycyrrhizin productivity and diversify the chemical structures of saponins in Glycyrrhiza plants. A synthetic biological approach to engineer CYP88D15 could enable the production of pharmacologically active saponins with diene structures, such as saikosaponins, whose biosynthetic pathways have yet to be fully characterized.

Functional specialization of UDP-glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin.

Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side-effects. Here, we report that UDP-glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP-glucuronic acid to glycyrrhetinic acid 3-O-monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin-deficient (83-555) strain of G. uralensis. The natural variant showed loss of specificity for UDP-glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83-555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP-glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP-glucuronic acid. The functional arginine residue and resultant specificity for UDP-glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP-sugar selectivity for the rational engineering of sweet triterpenoid saponins.

Metabolomics with 15N Labeling for Characterizing Missing Monoterpene Indole Alkaloids in Plants.

Monoterpene indole alkaloids (MIAs) in medicinal plants remain uncharacterized owing to their complicated structure by metabolomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) despite their pharmaceutical importance. We demonstrate an untargeted metabolome analysis with 15nitrogen (N) labeling to characterize MIAs having an indolic skeleton in the flowers, leaves, petioles, stems, and roots of Catharanthus roseus. Principal component analysis using 15N- and nonlabeled metabolome data showed that N-containing metabolites (N-metabolites) are labeled with 15N. Paring of the 15N- and nonlabeled precursor ions were performed using the criteria of retention time, difference of m/z value, and a nonlabeled product ion at m/z 144.08 that indicates an indolic skeleton. The mass shift of the m/z value of the product and precursor ions to their 15N-labeled ions identified the number of N of their ions. Finally, molecular formula of 45 MIAs was unambiguously identified using the identified N number. The alkaloid network analysis using the MS/MS similarity showed the structural commonness and uniqueness among the MIAs. Of them, antirhine was identified using an authentic standard compound. Multimetabolomics using LC-MS/MS and imaging mass spectrometry showed that antirhine accumulates considerably in the epidermis and vascular cylinder of the roots. The developed approach showed the existence of the missing MIAs. The modification of this approach will identify other MIAs that contain a hydroxylated or methoxylated indolic skeleton.

Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin.

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 ß-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 ß-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-ß-amyrin (a possible biosynthetic intermediate between ß-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-ß-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 ß-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.

Licorice beta-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 beta-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 beta-amyrin at C-11 to produce 11-oxo-beta-amyrin, a possible biosynthetic intermediate between beta-amyrin and glycyrrhizin. CYP88D6 coexpressed with beta-amyrin synthase in yeast also catalyzed in vivo oxidation of beta-amyrin to 11-oxo-beta-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 beta-amyrin 11-oxidase in the glycyrrhizin pathway.

Spatial metabolomics using imaging mass spectrometry to identify the localization of asparaptine A in Asparagus officinalis.

Spatial metabolomics uses imaging mass spectrometry (IMS) to localize metabolites within tissue section. Here, we performed matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance-IMS (MALDI-FTICR-IMS) to identify the localization of asparaptine A, a naturally occurring inhibitor of angiotensin-converting enzyme, in green spears of asparagus (Asparagus officinalis). Spatial metabolome data were acquired in an untargeted manner. Segmentation analysis using the data characterized tissue-type-dependent and independent distribution patterns in cross-sections of asparagus spears. Moreover, asparaptine A accumulated at high levels in developing lateral shoot tissues. Quantification of asparaptine A in lateral shoots using liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated the IMS analysis. These results provide valuable information for understanding the function of asparaptine A in asparagus, and identify the lateral shoot as a potential region of interest for multiomics studies to examine gene-to-metabolite associations in the asparaptine A biosynthesis.

Camptothecin production by in vitro cultures and plant regeneration in Ophiorrhiza species.

Camptothecin derivatives are clinically used for the treatment of various human cancers. These derivatives are semi-synthesized from camptothecin which is isolated from the extracts of Camptotheca acuminata and Nothapodytes foetida. For the feasible production of camptothecin, the protocols for the tissue cultures of Ophiorrhiza species, O. pumila, O. liukiuensis and O. kuroiwai, have been established. The established aseptic plants and hairy roots produced camptothecin, and O. pumila hairy roots accumulated highest amount of camptothecin. Furthermore, we have established methods of plant regeneration from O. pumila hairy roots.

Producing the sulfur-containing metabolite asparaptine in Asparagus calluses and a suspension cell line.

Asparaptine, a conjugate of L-arginine and asparagusic acid, was found in green asparagus (Asparagus officinalis) using ultrahigh-resolution metabolomics for sulfur-containing metabolites (S-metabolites), called S-omics. Asparaptine has been shown to inhibit the activity of angiotensin-converting enzyme. Larger amounts of this S-metabolite are therefore required for further analysis; however, there are limitations that asparagus is a perennial plant and its spears, wherein asparaptine accumulates, can be mainly harvested at the spring to summer season. In order to overcome these, we prepared a callus and suspension cell line from green asparagus. Untargeted metabolome analysis using liquid chromatography-tandem mass spectrometry was performed in the materials as well as spears and three calluses derived from wild type Asparagus. The analysis demonstrated that the amount of asparaptine in the callus derived from the green asparagus was more than the others per mg dry weight. The suspension cell line treated with methyljasmonate showed the induction of asparaptine, suggesting that the asparaptine production is modifiable under appropriate culture conditions. The described materials can be utilized for the production of asparaptine and in integrated metabolomics to study the biosynthesis of this S-metabolite, which is currently unknown.

Molecular Basis of C-30 Product Regioselectivity of Legume Oxidases Involved in High-Value Triterpenoid Biosynthesis.

The triterpenes are structurally diverse group of specialized metabolites with important roles in plant defense and human health. Glycyrrhizin, with a carboxyl group at C-30 of its aglycone moiety, is a valuable triterpene glycoside, the production of which is restricted to legume medicinal plants belonging to the Glycyrrhiza species. Cytochrome P450 monooxygenases (P450s) are important for generating triterpene chemodiversity by catalyzing site-specific oxidation of the triterpene scaffold. CYP72A154 was previously identified from the glycyrrhizin-producing plant Glycyrrhiza uralensis as a C-30 oxidase in glycyrrhizin biosynthesis, but its regioselectivity is rather low. In contrast, CYP72A63 from Medicago truncatula showed superior regioselectivity in C-30 oxidation, improving the production of glycyrrhizin aglycone in engineered yeast. The underlying molecular basis of C-30 product regioselectivity is not well understood. Here, we identified two amino acid residues that control C-30 product regioselectivity and contribute to the chemodiversity of triterpenes accumulated in legumes. Amino acid sequence comparison combined with structural analysis of the protein model identified Leu149 and Leu398 as important amino acid residues for C-30 product regioselectivity. These results were further confirmed by mutagenesis of CYP72A154 homologs from glycyrrhizin-producing species, functional phylogenomics analyses, and comparison of corresponding residues of C-30 oxidase homologs in other legumes. These findings could be combined with metabolic engineering to further enhance the production of high-value triterpene compounds.

Transport of camptothecin in hairy roots of Ophiorrhiza pumila.

We have investigated the subcellular accumulation and transport of camptothecin (CPT), a monoterpene indole alkaloid, in hairy roots of Ophiorrhiza pumila. This hairy root produces high amounts of CPT and excretes it into the culture medium. When the hairy roots were exposed to UV radiation, autofluorescence emitted from CPT showed subcellular localization of CPT in the vacuole. Treatment with several inhibitors suggested that CPT excretion is a transporter-independent passive transport controlled by the concentration gradient of the compound. Interestingly, the hairy roots treated with brefeldin A, a vesicle transport inhibitor, showed increased CPT excretion. This could be explained by an increased transport rate of CPT from the endoplasmic reticulum (ER) to the cytoplasm when transport of CPT to the vacuole is blocked. The much higher concentration of CPT in the cytoplasm resulted in the increased excretion rate. This result indicates that CPT is biosynthesized at the ER and transported to accumulate in the vacuole by the same machinery that is used for vacuolar protein sorting. How O. pumila is insensitive to CPT is discussed.

Relationship between gastric mucosal IL-8 levels and histological gastritis in patients with Helicobacter pylori infection.

To determine the role of host immune responses in H. pylori infection, we examined the relationship between gastric mucosal IL-8 levels and histological gastritis in patients with H. pylori infection. Biopsy tissue obtained from 99 patients were homogenizedand mucosal IL-8 levels measured by ELISA. The gastric mucosal IL-8 levels in both the antrum and corpus were higher in patients with H. pylori than in H. pyloi negativepatients. IL-8 levels in the corpus but not the antrum correlated with the severity of the atrophy. The IL-1B polymorphism had no influence on the degree of IL-8 production. These findings indicate that IL-8 production is independent of IL-1B polymorphisms and IL-8 may play an important role in the development of atrophic gastritis.

Structural insight of DNA topoisomerases I from camptothecin-producing plants revealed by molecular dynamics simulations.

DNA topoisomerase I (Top1) catalyzes changes in DNA topology by cleaving and rejoining one strand of the double stranded (ds)DNA. Eukaryotic Top1s are the cellular target of the plant-derived anticancer indole alkaloid camptothecin (CPT), which reversibly stabilizes the Top1-dsDNA complex. However, CPT-producing plants, including Camptotheca acuminata, Ophiorrhiza pumila and Ophiorrhiza liukiuensis, are highly resistant to CPT because they possess point-mutated Top1. Here, the adaptive convergent evolution is reported between CPT production ability and mutations in their Top1, as a universal resistance mechanism found in all tested CPT-producing plants. This includes Nothapodytes nimmoniana, one of the major sources of CPT. To obtain a structural insight of the resistance mechanism, molecular dynamics simulations of CPT- resistant and -sensitive plant Top1s complexed with dsDNA and topotecan (a CPT derivative) were performed, these being compared to that for the CPT-sensitive human Top1. As a result, two mutations, Val617Gly and Asp710Gly, were identified in O. pumila Top1 and C. acuminata Top1, respectively. The substitutions at these two positions, surprisingly, are the same as those found in a CPT derivative-resistant human colon adenocarcinoma cell line. The results also demonstrated an increased linker flexibility of the CPT-resistant Top1, providing an additional explanation for the resistance mechanism found in CPT-producing plants. These mutations could reflect the long evolutionary adaptation of CPT-producing plant Top1s to confer a higher degree of resistance.

Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin producing plants.

Camptothecin derivatives are clinically used anti-neoplastic alkaloids that biogenetically belong to monoterpenoid indole alkaloids. Camptothecin-related alkaloids from the methanol extracts of Ophiorrhiza pumila, Camptotheca acuminata and Nothapodytes foetida plants were profiled and identified using a reverse-phase high performance liquid chromatography coupled with on-line photodiode array detection and electrospray-ionization ion-trap mass spectrometry. A natural 10-glycosyloxy camptothecin, chaboside, was accumulated in tissues of O. pumila but not in C. acuminata and N. foetida. Anthraquinones regarded as phytoalexins were present in the extracts of hairy roots and calli but not in the differentiated plants of O. pumila. These findings demonstrated a remarkable difference in the constituents between the differentiated plants and the hairy roots or calli tissues. The activity of strictosidine synthase, a key enzyme of camptothecin biosynthesis, was detected in the protein extracts of stems and roots of O. pumila, being correlated with the pattern of strictosidine synthase mRNA expression.

Rapid identification of a narcotic plant Papaver bracteatum using flow cytometry.

In May 2011, numerous poppy plants closely resembling Papaver bracteatum Lindl., a type of narcotic plant that is illegal in Japan, were distributed directly from several large flower shops or through online shopping throughout Japan, including the Tokyo Metropolitan area. In order to better identify the narcotic plants, the relative nuclear DNA content at the vegetative stage was measured by flow cytometric (FCM) analysis in 3 closely-related species of the genus Papaver section Oxytona, namely P. orientale, P. pseudo-orientale, and P. bracteatum, based on the difference between the chromosome numbers of these species. The results showed that the nuclear DNA content differed between these 3 species, and that most of the commercially distributed plants examined in this study could be identified as P. bracteatum. The remaining plants were P. pseudo-orientale, a non-narcotic plant. In addition, the FCM results for the identification of P. bracteatum completely agreed with the results obtained by the morphological analysis, the inter-genic spacer sequence of rpl16-rpl14 (PS-ID sequence) of chloroplast DNA, and the presence of thebaine. These results clearly indicate the usefulness of FCM analysis for the identification of P. bracteatum plants, including when they are in their vegetative stage.

Suppression of camptothecin biosynthetic genes results in metabolic modification of secondary products in hairy roots of Ophiorrhiza pumila.

Camptothecin is a monoterpenoid indole alkaloid that exhibits anti-tumor activity. In Ophiorrhiza pumila, production of camptothecin and its related alkaloids was high in the hairy roots, but not in the cell suspension culture derived from hairy roots. To identify the intermediates in camptothecin biosynthesis, expression of genes encoding tryptophan decarboxylase (TDC) and secologanin synthase (SLS), the two enzymes catalyzing the early steps in camptothecin biosynthesis, were suppressed in the hairy roots of O. pumila by RNA interference (RNAi), and metabolite changes were investigated. In most TDC- and SLS-suppressed lines, accumulation of camptothecin and related alkaloids, strictosidine, strictosamide, pumiloside, and deoxypumiloside was reduced. The accumulation levels of secologanin exhibited a strong negative correlation with the expression level of TDC, and that of loganin exhibited a negative correlation with the expression level of SLS. Some hairy root-specific chromatographic peaks detected by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) exhibited positive or negative correlation with TDC expression, suggesting their possible involvement in camptothecin biosynthesis.

Camptothecin: therapeutic potential and biotechnology.

Camptothecin (CPT) and its derivatives have been received considerable attention recently. Two semi-synthetic derivatives, topotecan and irinotecan, are currently prescribed as anticancer drugs. Several more are now in clinical trial. CPT is produced in many plants belonging to unrelated orders of angiosperms. At present, CPT supplied for pharmaceutical use is extracted from the plants, Camptotheca acuminata and Nothapodytes foetida. Several efforts have been made to sustain a stable production of CPT by in vitro cell cultures of C. acuminata, N. foetida and Ophiorrhiza pumila. Recent report showed that plants are not the only sources that produce CPT. CPT was reported to be produced from the endophytic fungus isolated from the inner bark of N. foetida. The hairy root cultures of C. acuminata and O. pumila produce and secrete CPT into the medium in large quantities. These reports suggest the possibility to develop large-scale production of CPT. In addition, recent advance in the cloning and characterization of biosynthetic enzymes involved in CPT biosynthetic pathway provides valuable information for developing genetically engineered CPT-producing plants.