Changfu Li

Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus

Catharanthus roseus is an important medicinal plant and the sole commercial source of monoterpenoid indole alkaloids (MIA), anticancer compounds. Recently, triterpenoids like ursolic acid and oleanolic acid have also been found in considerable amounts in C. roseus leaf cuticular wax layer. These simple pentacyclic triterpenoids exhibit various pharmacological activities such as anti-inflammatory, anti-tumor and anti-microbial properties. Using the EST collection from C. roseus leaf epidermome (http://www.ncbi.nlm.nih.gov/dbEST), we have successfully isolated a cDNA (CrAS) encoding 2,3-oxidosqualene cyclase (OSC) and a cDNA (CrAO) encoding amyrin C-28 oxidase from the leaves of C. roseus. The functions of CrAS and CrAO were analyzed in yeast (Saccharomyces cerevisiae) systems. CrAS was characterized as a novel multifunctional OSC producing α- and β-amyrin in a ratio of 2.5:1, whereas CrAO was a multifunctional C-28 oxidase converting α-amyrin, β-amyrin and lupeol to ursolic-, oleanolic- and betulinic acids, respectively, via a successive oxidation at the C-28 position of the substrates. In yeast co-expressing CrAO and CrAS, ursolic- and oleanolic acids were detected in the yeast cell extracts, while the yeast cells co-expressing CrAO and AtLUP1 from Arabidopsis thaliana produced betulinic acid. Both CrAS and CrAO genes show a high expression level in the leaf, which was consistent with the accumulation patterns of ursolic- and oleanolic acids in C. roseus. These results suggest that CrAS and CrAO are involved in the pentacyclic triterpene biosynthesis in C. roseus.Catharanthus roseus is an important medicinal plant and the sole commercial source of monoterpenoid indole alkaloids (MIA), anticancer compounds. Recently, triterpenoids like ursolic acid and oleanolic acid have also been found in considerable amounts in C. roseus leaf cuticular wax layer. These simple pentacyclic triterpenoids exhibit various pharmacological activities such as anti-inflammatory, anti-tumor and anti-microbial properties. Using the EST collection from C. roseus leaf epidermome ( http://www.ncbi.nlm.nih.gov/dbEST ), we have successfully isolated a cDNA (CrAS) encoding 2,3-oxidosqualene cyclase (OSC) and a cDNA (CrAO) encoding amyrin C-28 oxidase from the leaves of C. roseus. The functions of CrAS and CrAO were analyzed in yeast (Saccharomyces cerevisiae) systems. CrAS was characterized as a novel multifunctional OSC producing α- and β-amyrin in a ratio of 2.5:1, whereas CrAO was a multifunctional C-28 oxidase converting α-amyrin, β-amyrin and lupeol to ursolic-, oleanolic- and betulinic acids, respectively, via a successive oxidation at the C-28 position of the substrates. In yeast co-expressing CrAO and CrAS, ursolic- and oleanolic acids were detected in the yeast cell extracts, while the yeast cells co-expressing CrAO and AtLUP1 from Arabidopsis thaliana produced betulinic acid. Both CrAS and CrAO genes show a high expression level in the leaf, which was consistent with the accumulation patterns of ursolic- and oleanolic acids in C. roseus. These results suggest that CrAS and CrAO are involved in the pentacyclic triterpene biosynthesis in C. roseus.

Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis

The medicinal plant Xanthium strumarium L. (X. strumarium) is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs) are a class of 21–24 nucleotide (nt) non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

Identification and Functional Characterization of Sesquiterpene Synthases from Xanthium strumarium

Xanthium strumarium synthesizes various pharmacologically active sesquiterpenes. The molecular characterization of sesquiterpene biosynthesis in X. strumarium has not been reported so far. In this study, the cDNAs coding for three sesquiterpene synthases (designated as XsTPS1, XsTPS2 and XsTPS3) were isolated using the X. strumarium transcriptome that we recently constructed. XsTPS1, XsTPS2 and XsTPS3 were revealed to have primary activities forming germacrene D, guaia-4,6-diene and germacrene A, respectively, by either ectopic expression in yeast cells or purified recombinant protein-based in vitro assays. Quantitative real-time PCRs and metabolite analysis for the different plant parts showed that the transcript abundance of XsTPS1-XsTPS3 is consistent with the accumulation pattern of their enzymatic products, supporting their biochemical functions in vivo. In particular, we discovered that none of the XsTPS2 product, guaia-4,6-diene, can be detected in one of the X. strumarium cultivars used in this study (it was named the Hubei-cultivar), in which a natural deletion of two A bases in the XsTPS2 cDNA disrupts its activity, which further confirmed the proposed biochemical role of XsTPS2 in X. strumarium in vivo.

Identifying three ecological chemotypes of Xanthium strumarium glandular trichomes using a combined NMR and LC-MS method.

Xanthanolides, as the sesquiterpene lactones, are reportedly the major components for the pharmacological properties of X. strumarium L. species. Phytochemical studies indicated that the glandular structures on the surface of plant tissues would form the primary sites for the accumulation of this class of the compounds. As the interface between plants and their natural enemies, glandular trichomes may vary with respect to which of their chemicals are sequestered against different herbivores in different ecologies. However, to date, no data are available on the chemical characterisation of X. strumarium glandular cells. In this study, the trichome secretions of the X. strumarium species originating from nineteen unique areas across eleven provinces in China, were analysed by HPLC, LC-ESI-MS and NMR. For the first time three distinct chemotypes of X. strumarium glandular trichomes were discovered along with the qualitative and quantitative evaluations of their presence of xanthanolides; these were designated glandular cell Types I, II, and III, respectively. The main xanthanolides in Type I cells were 8-epi-xanthatin and xanthumin while no xanthatin was detected. Xanthatin, 8-epi-xanthatin, and xanthumin dominated in Type II cells with comparable levels of each being present. For Type III cells, significantly higher concentrations of 8-epi-xanthatin or xanthinosin (relative to xanthatin) were detected with xanthinosin only being observed in this type. Further research will focus on understanding the ecological and molecular mechanism causing these chemotype differences in X. strumarium glandular structures.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides.

An Alternative Pathway for Formononetin Biosynthesis in Pueraria lobata

The O-methylation is an important tailing process in Pueraria lobata isoflavone metabolism, but the molecular mechanism governing it remains not elucidated. This manuscript describes the mining of key O-methyltransferases (OMTs) involved in the process. Using our previously constructed P. lobata transcriptome, the OMT candidates were searched, extensively analyzed, and their functions were investigated by expression in yeast, Escherichia coli, or Glycine max hairy roots. Here, we report the identification of the key OMT gene responsible for formononetin production in P. lobata (designated as PlOMT9). PlOMT9 primarily functions as an isoflavone-specific 4′-O-methyltransferase, although it shows high sequence identities with isoflavone 7-O-methyltransferases. Moreover, unlike the previously reported OMTs that catalyze the 4′-O-methylation for formononetin biosynthesis at the isoflavanone stage, PlOMT9 performs this modifying step at the isoflavone level, using daidzein rather than 2,7,4′-trihydroxy-isoflavanone as the substrate. Gene expression analyses and metabolite profiling supported its proposed roles in P. lobata. Using the system of transgenic G. max hairy roots, the role of PlOMT9 in the biosynthesis of formononetin was further demonstrated in vivo.

Molecular Cloning and Functional Characterization of a Novel (Iso)flavone 4′,7-O-diglucoside Glucosyltransferase from Pueraria lobata

Pueraria lobata roots accumulate a rich source of isoflavonoid glycosides, including 7-O- and 4′-O-mono-glucosides, and 4′,7-O-diglucosides, which have numerous human health benefits. Although, isoflavonoid 7-O-glucosyltranferases (7-O-UGTs) have been well-characterized at molecular levels in legume plants, genes, or enzymes that are required for isoflavonoid 4′-O- and 4′,7-O-glucosylation have not been identified in P. lobata to date. Especially for the 4′,7-O-di-glucosylations, the genetic control for this tailing process has never been elucidated from any plant species. Through transcriptome mining, we describe here the identification and characterization of a novel UGT (designated PlUGT2) governing the isoflavonoid 4′,7-O-di-glucosylations in P. lobata. Biochemical roles of PlUGT2 were assessed by in vitro assays with PlUGT2 protein produced in Escherichia coli and analyzed for its qualitative substrate specificity. PlUGT2 was active with various (iso)flavonoid acceptors, catalyzing consecutive glucosylation activities at their O-4′ and O-7 positions. PlUGT2 was most active with genistein, a general isoflavone in legume plants. Real-time PCR analysis showed that PlUGT2 is preferentially transcribed in roots relative to other organs of P. lobata, which is coincident with the accumulation pattern of 4′-O-glucosides and 4′,7-O-diglucosides in P. lobata. The identification of PlUGT2 would help to decipher the P. lobata isoflavonoid glucosylations in vivo and may provide a useful enzyme catalyst for an efficient biotransformation of isoflavones or other natural products for food or pharmacological purposes.

Discovery of Several Novel Targets that Enhance β-Carotene Production in Saccharomyces cerevisiae

β-Carotene is the precursor of vitamin A, and also exhibits multiple pharmaceutical functions by itself. In comparison to chemical synthesis, the production of β-carotene in microbes by metabolic engineering strategy is relatively inexpensive. Identifying genes enhancing β-carotene production in microbes is important for engineering a strain of producing higher yields of β-carotene. Most of previous efforts in identifying the gene targets have focused on the isoprenoid pathway where the β-carotene biosynthesis belongs. However, due to the complex interactions between metabolic fluxes, seemingly irrelevant genes that are outside the isoprenoid pathway might also affect β-carotene biosynthesis. To this end, here we provided an example that several novel gene targets, which are outside the isoprenoid pathway, have improving effects on β-carotene synthesis in yeast cells, when they were over-expressed. Among these targets, the class E protein of the vacuolar protein-sorting pathway (Did2) led to the highest improvement in β-carotene yields, which was 2.1-fold to that of the corresponding control. This improvement was further explained by the observation that the overexpression of the DID2 gene generally boosted the transcriptions of β-carotene pathway genes. The mechanism by which the other targets improve the production of β-carotene is discussed.

Molecular Cloning and Functional Characterization of a Novel Isoflavone 3′-O-methyltransferase from Pueraria lobata

Pueraria lobata roots accumulate 3′-, 4′- and 7-O-methylated isoflavones and many of these methylated compounds exhibit various pharmacological activities. Either the 4′- or 7-O-methylation activity has been investigated at molecular levels in several legume species. However, the gene encoding the isoflavone 3′-O-methyltransferase (OMT) has not yet been isolated from any plant species. In this study, we reported the first cDNA encoding the isoflavone 3′-OMT from P. lobata (designated PlOMT4). Heterologous expressions in yeast and Escherichia coli cells showed that the gene product exhibits an enzyme activity to methylate the 3′-hydroxy group of the isoflavone substrate. The transcript abundance of PlOMT4 matches well with its enzymatic product in different organs of P. lobata and in the plant roots in response to methyl jasmonate elicitation. Integration of the biochemical with metabolic and transcript data supported the proposed function of PlOMT4. The identification of PlOMT4 would not only help to understand the isoflavonoid metabolism in P. lobata but also potentially provide an enzyme catalyst for methylating existing drug candidates to improve their hydrophobicity.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Dioscin Biosynthesis in Dioscorea zingiberensis

Dioscorea zingiberensis is a perennial herb native to China. The rhizome of D. zingiberensis has long been used as a traditional Chinese medicine to treat rheumatic arthritis. Dioscin is the major bioactive ingredient conferring the medicinal property described in Chinese pharmacopoeia. Several previous studies have suggested cholesterol as the intermediate to the biosynthesis of dioscin, however, the biosynthetic steps to dioscin after cholesterol remain unknown. In this study, a comprehensive D. zingiberensis transcriptome derived from its leaf and rhizome was constructed. Based on the annotation using various public databases, all possible enzymes in the biosynthetic steps to cholesterol were identified. In the late steps beyond cholesterol, cholesterol undergoes site-specific oxidation by cytochrome P450s (CYPs) and glycosylation by UDP-glycosyltransferases (UGTs) to yield dioscin. From the D. zingiberensis transcriptome, a total of 485 unigenes were annotated as CYPs and 195 unigenes with a sequence length above 1000 bp were annotated as UGTs. Transcriptomic comparison revealed 165 CYP annotated unigenes correlating to dioscin biosynthesis in the plant. Further phylogenetic analysis suggested that among those CYP candidates four of them would be the most likely candidates involved in the biosynthetic steps from cholesterol to dioscin. Additionally, from the UGT annotated unigenes, six of them were annotated as 3-O-UGTs and two of them were annotated as rhamnosyltransferases, which consisted of potential UGT candidates involved in dioscin biosynthesis. To further explore the function of the UGT candidates, two 3-O-UGT candidates, named Dz3GT1 and Dz3GT2, were cloned and functionally characterized. Both Dz3GT1 and Dz3GT2 were able to catalyze a C3-glucosylation activity on diosgenin. In conclusion, this study will facilitate our understanding of dioscin biosynthesis pathway and provides a basis for further mining the genes involved in dioscin biosynthesis.