The Experts below are selected from a list of 129 Experts worldwide ranked by ideXlab platform
Guoyin Kai - One of the best experts on this subject based on the ideXlab platform.
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divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila
BMC Biology, 2021Co-Authors: Mengquan Yang, Qiang Wang, Yining Liu, Xiaolong Hao, Can Wang, Yuchen Liang, Jianbo Chen, Youli Xiao, Guoyin KaiAbstract:The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches.
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characterization and phylogenetic analysis of the complete chloroplast genome of Ophiorrhiza pumila rubiaceae
Mitochondrial DNA Part B, 2021Co-Authors: Qikai Huang, Guoyin KaiAbstract:Ophiorrhiza pumila (Rubiaceae) is an herbaceous plant that grows streamside in forest gullies or wetlands in the shade. Complete chloroplast genome of O. pumila was obtained and analyzed its phylogeny relationship within Rubiaceae plants. The results showed that the genome had a typical quadripartite structure of 154,385 bp, and contained a total of 112 unique genes, including 79 protein-coding genes, 29 tRNA genes, and 4 rRNA genes. Phylogenetic analysis suggested that O. pumila is sister to a highly supported clade composed of 10 species including Morinda officinalis, Gynochthodes cochinchinensis, Saprosma merrillii, Hedyotis ovata, Foonchewia guangdongensis, Dunnia sinensis, Paederia scandens, Leptodermis scabrida, Rubia cordifolia, and Galium mollugo. The complete chloroplast genome provides valuable information for the phylogenetic analysis of O. pumila.
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cloning characterization and enzymatic identification of a new tryptophan decarboxylase from Ophiorrhiza pumila
Biotechnology and Applied Biochemistry, 2021Co-Authors: Can Wang, Dawei You, Yue Feng, Chengtao Sun, Yao Wang, Degang Zhao, Guoyin KaiAbstract:Tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyzes tryptophan decarboxylation to form tryptamine through the cofactor pyridoxal-5'-phosphate (PLP), a crucial stage in the production of the terpenoid indole alkaloids like camptothecin (CPT). A new gene encoding TDC was identified from the CPT-producing plant Ophiorrhiza pumila by transcriptome analysis, termed OpTDC2. It contained a 1,536 bp open reading frame that encodes a 511 amino acid protein with a molecular mass of 57.01 kDa and an isoelectric point of 6.39. Multiple sequence alignment and phylogenetic tree analysis showed the closest similarity (85%) with the TDC from Mitragyna speciosa. Moreover, the highest expression of OpTDC2 was observed in the O. pumila root. To achieve high-efficiency expression of OpTDC2 in Escherichia coli, we fused the TF tag onto the N-terminal of the OpTDC2. Optimum enzymatic activity was observed at 45 °C, pH 8 and cofactor concentration of 0.1 mM. The catalytic reaction was strongly inhibited by metal ions of Cu2+ , Zn2+ , and Fe2+ . The l-tryptophan was particularly catalyzed compared with d-tryptophan. Besides, the Km and kcat of the OpTDC2 were 1.08 mM and 0.78 Sec-1 , respectively. The results provided information on new functional OpTDC2 that might be used in synthetic biology for the enhanced biosynthesis of CPT in O. pumila.
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Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila
'Springer Science and Business Media LLC', 2021Co-Authors: Mengquan Yang, Qiang Wang, Yining Liu, Xiaolong Hao, Can Wang, Yuchen Liang, Jianbo Chen, Youli Xiao, Guoyin KaiAbstract:Abstract Background The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. Results In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. Conclusions In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches
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the transcription factor opwrky2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila
Horticulture research, 2021Co-Authors: Xiaolong Hao, Chenhong Xie, Qingyan Ruan, Xichen Zhang, Bing Han, Jun Qian, Wei Zhou, Hanswilhelm Nutzmann, Guoyin KaiAbstract:The limited bioavailability of plant-derived natural products with anticancer activity poses major challenges to the pharmaceutical industry. An example of this is camptothecin, a monoterpene indole alkaloid with potent anticancer activity that is extracted at very low concentrations from woody plants. Recently, camptothecin biosynthesis has been shown to become biotechnologically amenable in hairy-root systems of the natural producer Ophiorrhiza pumila. Here, time-course expression and metabolite analyses were performed to identify novel transcriptional regulators of camptothecin biosynthesis in O. pumila. It is shown here that camptothecin production increased over cultivation time and that the expression pattern of the WRKY transcription factor encoding gene OpWRKY2 is closely correlated with camptothecin accumulation. Overexpression of OpWRKY2 led to a more than three-fold increase in camptothecin levels. Accordingly, silencing of OpWRKY2 correlated with decreased camptothecin levels in the plant. Further detailed molecular characterization by electrophoretic mobility shift, yeast one-hybrid and dual-luciferase assays showed that OpWRKY2 directly binds and activates the central camptothecin pathway gene OpTDC. Taken together, the results of this study demonstrate that OpWRKY2 acts as a direct positive regulator of camptothecin biosynthesis. As such, a feasible strategy for the over-accumulation of camptothecin in a biotechnologically amenable system is presented.
Kazuki Saito - One of the best experts on this subject based on the ideXlab platform.
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function of ap2 erf transcription factors involved in the regulation of specialized metabolism in Ophiorrhiza pumila revealed by transcriptomics and metabolomics
Frontiers in Plant Science, 2016Co-Authors: Nirin Udomsom, Kazuki Saito, Ryo Nakabayashi, Amit Rai, Hideyuki Suzuki, Jun Okuyama, Ryosuke Imai, Tetsuya Mori, Mami YamazakiAbstract:The hairy roots (HR) of Ophiorrhiza pumila produce camptothecin (CPT), a monoterpenoid indole alkaloid used as a precursor in the synthesis of chemotherapeutic drugs. O. pumila HR culture is considered as a promising alternative source of CPT, however, the knowledge about the biosynthetic pathway and regulatory mechanism is still limited. In this study, five genes that encode AP2/ERF transcription factors, namely OpERF1 to OpERF5, were isolated from HR of O. pumila. Phylogenetic analysis of AP2/ERF protein sequences suggested the close evolutionary relationship of OpERF1 with stress-responsive ERF factors in Arabidopsis and of OpERF2 with ERF factors reported to regulate alkaloid production, such as ORCA3 in Catharanthus roseus, NIC2 locus ERFs in tobacco, and JRE4 in tomato. We generated the transgenic HR lines of O. pumila, ERF1i and ERF2i, in which the expression of OpERF1 and OpERF2, respectively, was suppressed using RNA interference technique. The transcriptome and metabolome of these suppressed HR were analyzed for functional characterization of OpERF1 and OpERF2. Although significant changes were not observed in the metabolome, including CPT and related compounds, the suppression of OpERF2 resulted in reduced expression of genes in the 2-C-methyl- D -erythritol 4-phosphate and secologanin-strictosidine pathways, which supply a precursor, strictosidine, for CPT biosynthesis. Furthermore, while it was not conclusive for OpERF1, enrichment analysis of differentially expressed genes in the suppressed HR showed that the gene ontology terms for oxidation-reduction, presumably involved in secondary metabolite pathways, were enriched in the ERF2i downregulated gene set. These results suggest a positive role of OpERF2 in regulating specialized metabolism in O. pumila.
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Function of AP2/ERF transcription factors involved in the regulation of specialized metabolism in <i>Ophiorrhiza pumila</i> revealed by transcriptomics and metabolomics
Frontiers Media S.A., 2016Co-Authors: Nirin Udomsom, Kazuki Saito, Ryo Nakabayashi, Amit Rai, Hideyuki Suzuki, Jun Okuyama, Ryosuke Imai, Tetsuya Mori, Mami YamazakiAbstract:The hairy roots (HR) of Ophiorrhiza pumila produce camptothecin (CPT), a monoterpenoid indole alkaloid used as a precursor in the synthesis of chemotherapeutic drugs. O. pumila HR culture is considered as a promising alternative source of CPT, however, the knowledge about the biosynthetic pathway and regulatory mechanism is still limited. In this study, five genes that encode AP2/ERF transcription factors, namely OpERF1 to OpERF5, were isolated from HR of O. pumila. Phylogenetic analysis of AP2/ERF protein sequences suggested the close evolutionary relationship of OpERF1 with stress-responsive ERF factors in Arabidopsis and of OpERF2 with ERF factors reported to regulate alkaloid production, such as ORCA3 in Catharanthus roseus, NIC2 locus ERFs in tobacco, and JRE4 in tomato. We generated the transgenic HR lines of O. pumila, ERF1i and ERF2i, in which the expression of OpERF1 and OpERF2, respectively, was suppressed using RNA interference technique. The transcriptome and metabolome of these suppressed HR were analyzed for functional characterization of OpERF1 and OpERF2. Although significant changes were not observed in the metabolome, including CPT and related compounds, the suppression of OpERF2 resulted in reduced expression of genes in the 2-C-methyl- D -erythritol 4-phosphate and secologanin-strictosidine pathways, which supply a precursor, strictosidine, for CPT biosynthesis. Furthermore, while it was not conclusive for OpERF1, enrichment analysis of differentially expressed genes in the suppressed HR showed that the gene ontology terms for oxidation-reduction, presumably involved in secondary metabolite pathways, were enriched in the ERF2i downregulated gene set. These results suggest a positive role of OpERF2 in regulating specialized metabolism in O. pumila
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suppression of camptothecin biosynthetic genes results in metabolic modification of secondary products in hairy roots of Ophiorrhiza pumila
Phytochemistry, 2013Co-Authors: Takashi Asano, Hiroshi Sudo, Kazuki Saito, Kanae Kobayashi, Emi Kashihara, Ryosuke Sasaki, Yoko Iijima, Koh Aoki, Daisuke Shibata, Mami YamazakiAbstract: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.
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transport of camptothecin in hairy roots of Ophiorrhiza pumila
Phytochemistry, 2007Co-Authors: Supaart Sirikantaramas, Hiroshi Sudo, Mami Yamazaki, Takashi Asano, Kazuki SaitoAbstract: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.
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regeneration of transformed Ophiorrhiza pumila plants producing camptothecin
Plant Biotechnology, 2004Co-Authors: Ikumi Watase, Hiroshi Sudo, Mami Yamazaki, Kazuki SaitoAbstract:Camptothecin derivatives are clinically used as anti-tumor alkaloids that are currently obtained by extraction from intact plants. Seeking for the alternative sources for commercial production and for fundamental study, cell and tissue cultures have been investigated. In the present study, we developed a method for regeneration of Ophiorrhiza pumila plant from hairy roots transformed with Agrobacterium rhizogenes. The regeneration frequency was over 83%. Integration of a rol B gene from T-DNA of A. rhizogenes was confirmed by polymerase chain reaction in both of the hairy roots and the regenerated plants. The transformed plants accumulated camptothecin in amounts of 66-111% compared with that in the wild-type plants.
Mami Yamazaki - One of the best experts on this subject based on the ideXlab platform.
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function of ap2 erf transcription factors involved in the regulation of specialized metabolism in Ophiorrhiza pumila revealed by transcriptomics and metabolomics
Frontiers in Plant Science, 2016Co-Authors: Nirin Udomsom, Kazuki Saito, Ryo Nakabayashi, Amit Rai, Hideyuki Suzuki, Jun Okuyama, Ryosuke Imai, Tetsuya Mori, Mami YamazakiAbstract:The hairy roots (HR) of Ophiorrhiza pumila produce camptothecin (CPT), a monoterpenoid indole alkaloid used as a precursor in the synthesis of chemotherapeutic drugs. O. pumila HR culture is considered as a promising alternative source of CPT, however, the knowledge about the biosynthetic pathway and regulatory mechanism is still limited. In this study, five genes that encode AP2/ERF transcription factors, namely OpERF1 to OpERF5, were isolated from HR of O. pumila. Phylogenetic analysis of AP2/ERF protein sequences suggested the close evolutionary relationship of OpERF1 with stress-responsive ERF factors in Arabidopsis and of OpERF2 with ERF factors reported to regulate alkaloid production, such as ORCA3 in Catharanthus roseus, NIC2 locus ERFs in tobacco, and JRE4 in tomato. We generated the transgenic HR lines of O. pumila, ERF1i and ERF2i, in which the expression of OpERF1 and OpERF2, respectively, was suppressed using RNA interference technique. The transcriptome and metabolome of these suppressed HR were analyzed for functional characterization of OpERF1 and OpERF2. Although significant changes were not observed in the metabolome, including CPT and related compounds, the suppression of OpERF2 resulted in reduced expression of genes in the 2-C-methyl- D -erythritol 4-phosphate and secologanin-strictosidine pathways, which supply a precursor, strictosidine, for CPT biosynthesis. Furthermore, while it was not conclusive for OpERF1, enrichment analysis of differentially expressed genes in the suppressed HR showed that the gene ontology terms for oxidation-reduction, presumably involved in secondary metabolite pathways, were enriched in the ERF2i downregulated gene set. These results suggest a positive role of OpERF2 in regulating specialized metabolism in O. pumila.
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Function of AP2/ERF transcription factors involved in the regulation of specialized metabolism in <i>Ophiorrhiza pumila</i> revealed by transcriptomics and metabolomics
Frontiers Media S.A., 2016Co-Authors: Nirin Udomsom, Kazuki Saito, Ryo Nakabayashi, Amit Rai, Hideyuki Suzuki, Jun Okuyama, Ryosuke Imai, Tetsuya Mori, Mami YamazakiAbstract:The hairy roots (HR) of Ophiorrhiza pumila produce camptothecin (CPT), a monoterpenoid indole alkaloid used as a precursor in the synthesis of chemotherapeutic drugs. O. pumila HR culture is considered as a promising alternative source of CPT, however, the knowledge about the biosynthetic pathway and regulatory mechanism is still limited. In this study, five genes that encode AP2/ERF transcription factors, namely OpERF1 to OpERF5, were isolated from HR of O. pumila. Phylogenetic analysis of AP2/ERF protein sequences suggested the close evolutionary relationship of OpERF1 with stress-responsive ERF factors in Arabidopsis and of OpERF2 with ERF factors reported to regulate alkaloid production, such as ORCA3 in Catharanthus roseus, NIC2 locus ERFs in tobacco, and JRE4 in tomato. We generated the transgenic HR lines of O. pumila, ERF1i and ERF2i, in which the expression of OpERF1 and OpERF2, respectively, was suppressed using RNA interference technique. The transcriptome and metabolome of these suppressed HR were analyzed for functional characterization of OpERF1 and OpERF2. Although significant changes were not observed in the metabolome, including CPT and related compounds, the suppression of OpERF2 resulted in reduced expression of genes in the 2-C-methyl- D -erythritol 4-phosphate and secologanin-strictosidine pathways, which supply a precursor, strictosidine, for CPT biosynthesis. Furthermore, while it was not conclusive for OpERF1, enrichment analysis of differentially expressed genes in the suppressed HR showed that the gene ontology terms for oxidation-reduction, presumably involved in secondary metabolite pathways, were enriched in the ERF2i downregulated gene set. These results suggest a positive role of OpERF2 in regulating specialized metabolism in O. pumila
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suppression of camptothecin biosynthetic genes results in metabolic modification of secondary products in hairy roots of Ophiorrhiza pumila
Phytochemistry, 2013Co-Authors: Takashi Asano, Hiroshi Sudo, Kazuki Saito, Kanae Kobayashi, Emi Kashihara, Ryosuke Sasaki, Yoko Iijima, Koh Aoki, Daisuke Shibata, Mami YamazakiAbstract: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.
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coupling deep transcriptome analysis with untargeted metabolic profiling in Ophiorrhiza pumila to further the understanding of the biosynthesis of the anti cancer alkaloid camptothecin and anthraquinones
Plant and Cell Physiology, 2013Co-Authors: Mami Yamazaki, Yasuyo Yamazaki, Keiichi Mochida, Takashi Asano, Ryo Nakabayashi, Motoaki Chiba, Nirin Udomson, Dayan B Goodenowe, Ushio SankawaAbstract:The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila.
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transport of camptothecin in hairy roots of Ophiorrhiza pumila
Phytochemistry, 2007Co-Authors: Supaart Sirikantaramas, Hiroshi Sudo, Mami Yamazaki, Takashi Asano, Kazuki SaitoAbstract: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.
Can Wang - One of the best experts on this subject based on the ideXlab platform.
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divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila
BMC Biology, 2021Co-Authors: Mengquan Yang, Qiang Wang, Yining Liu, Xiaolong Hao, Can Wang, Yuchen Liang, Jianbo Chen, Youli Xiao, Guoyin KaiAbstract:The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches.
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cloning characterization and enzymatic identification of a new tryptophan decarboxylase from Ophiorrhiza pumila
Biotechnology and Applied Biochemistry, 2021Co-Authors: Can Wang, Dawei You, Yue Feng, Chengtao Sun, Yao Wang, Degang Zhao, Guoyin KaiAbstract:Tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyzes tryptophan decarboxylation to form tryptamine through the cofactor pyridoxal-5'-phosphate (PLP), a crucial stage in the production of the terpenoid indole alkaloids like camptothecin (CPT). A new gene encoding TDC was identified from the CPT-producing plant Ophiorrhiza pumila by transcriptome analysis, termed OpTDC2. It contained a 1,536 bp open reading frame that encodes a 511 amino acid protein with a molecular mass of 57.01 kDa and an isoelectric point of 6.39. Multiple sequence alignment and phylogenetic tree analysis showed the closest similarity (85%) with the TDC from Mitragyna speciosa. Moreover, the highest expression of OpTDC2 was observed in the O. pumila root. To achieve high-efficiency expression of OpTDC2 in Escherichia coli, we fused the TF tag onto the N-terminal of the OpTDC2. Optimum enzymatic activity was observed at 45 °C, pH 8 and cofactor concentration of 0.1 mM. The catalytic reaction was strongly inhibited by metal ions of Cu2+ , Zn2+ , and Fe2+ . The l-tryptophan was particularly catalyzed compared with d-tryptophan. Besides, the Km and kcat of the OpTDC2 were 1.08 mM and 0.78 Sec-1 , respectively. The results provided information on new functional OpTDC2 that might be used in synthetic biology for the enhanced biosynthesis of CPT in O. pumila.
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Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila
'Springer Science and Business Media LLC', 2021Co-Authors: Mengquan Yang, Qiang Wang, Yining Liu, Xiaolong Hao, Can Wang, Yuchen Liang, Jianbo Chen, Youli Xiao, Guoyin KaiAbstract:Abstract Background The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. Results In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)-strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21-(S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. Conclusions In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches
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genome wide identification and comparative analysis of the teosinte branched 1 cycloidea proliferating cell factors 1 2 transcription factors related to anti cancer drug camptothecin biosynthesis in Ophiorrhiza pumila
Frontiers in Plant Science, 2021Co-Authors: Xiaolong Hao, Can Wang, Yao Wang, Min Shi, Zhigang Zhou, Guoyin KaiAbstract:Ophiorrhiza pumila (O. pumila; Op) is a medicinal herbaceous plant, which can accumulate camptothecin (CPT). CPT and its derivatives are widely used as chemotherapeutic drugs for treating malignant tumors. Its biosynthesis pathway has been attracted significant attention. Teosinte branched 1/cycloidea/proliferating cell factors 1/2 (TCP) transcription factors (TFs) regulate a variety of physiological processes, while TCP TFs are involved in the regulation of CPT biosynthesis remain unclear. In this study, a systematic analysis of the TCP TFs family in O. pumila was performed. A total of 16 O. pumila TCP (OpTCP) genes were identified and categorized into two subgroups based on their phylogenetic relationships with those in Arabidopsis thaliana. Tissue-specific expression patterns revealed that nine OpTCP genes showed the highest expression levels in leaves, while the other seven OpTCPs showed a higher expression level in the stems. Co-expression, phylogeny analysis, and dual-luciferase (Dual-LUC) assay revealed that OpTCP15 potentially plays important role in CPT and its precursor biosynthesis. In addition, the subcellular localization experiment of candidate OpTCP genes showed that they are all localized in the nucleus. Our study lays a foundation for further functional characterization of the candidate OpTCP genes involved in CPT biosynthesis regulation and provides new strategies for increasing CPT production.
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wrky transcription factor opwrky1 acts as a negative regulator of camptothecin biosynthesis in Ophiorrhiza pumila hairy roots
Plant Cell Tissue and Organ Culture, 2020Co-Authors: Limei Zhao, Guoyin Kai, Can Wang, Yao Wang, Wei Zhou, Yanlin MingAbstract:Camptothecin (CPT) has unique anti-tumor activities, and its analogs such as irinotecan and topotecan are clinically used as anti-cancer drugs. Recently, CPT biosynthesis pathway is gradually being resolved. However, few studies have been conducted on transcription factors (TFs), which regulate CPT biosynthesis in Ophiorrhiza pumila. In this study, we cloned and functionally identified a new OpWRKY1 TF from O. pumila that was preferentially expressed in stems and responded to exogenous elicitors such as gibberellin, acetylsalicylic acid, salicylic acid, abscisic acid and 1-aminocyclopropane-carboxylic acid. Subcellular localization assay showed that OpWRKY1 was specifically expressed in the nucleus. Transgenic results revealed that over-expression of OpWRKY1 significantly repressed CPT accumulation in the hairy roots by inhibiting the expression of several CPT biosynthesis genes, especially OpCPR. Furthermore, Dual-Luciferase assay and yeast-one-hybrid assay showed that OpCPR was a target of OpWRKY1. The above results elucidated how OpWRKY1 directly down-regulated the transcription of OpCPR and inhibited CPT biosynthesis in O. pumila. Our findings indicate that OpWRKY1 acts as a negative regulator of CPT biosynthesis in O. pumila and provide new insights on metabolic engineering of CPT metabolism in the future. Overexpression of OpWRKY1 inhibits CPT accumulation in transgenic O. pumila hairy roots by reducing the expression of CPT biosynthetic gene OpCPR.
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biosynthesis of camptothecin in silico and in vivo tracer study from 1 13c glucose
Plant Physiology, 2004Co-Authors: Yasuyo Yamazaki, Hiroshi Sudo, Mariko Kitajima, Hiromitsu Takayama, Norio Aimi, Mami Yamazaki, Masanori Arita, Kazuki SaitoAbstract:Camptothecin derivatives are clinically used antitumor alkaloids that belong to monoterpenoid indole alkaloids. In this study, we investigated the biosynthetic pathway of camptothecin from [1-13C]glucose (Glc) by in silico and in vivo studies. The in silico study measured the incorporation of Glc into alkaloids using the Atomic Reconstruction of Metabolism software and predicted the labeling patterns of successive metabolites from [1-13C]Glc. The in vivo study followed incorporation of [1-13C]Glc into camptothecin with hairy roots of Ophiorrhiza pumila by 13C nuclear magnetic resonance spectroscopy. The 13C-labeling pattern of camptothecin isolated from the hairy roots clearly showed that the monoterpene-secologanin moiety was synthesized via the 2C-methyl-d-erythritol 4-phosphate pathway, not via the mevalonate pathway. This conclusion was supported by differential inhibition of camptothecin accumulation by the pathway-specific inhibitors (fosmidomycin and lovastatin). The quinoline moiety from tryptophan was also labeled as predicted by the Atomic Reconstruction of Metabolism program via the shikimate pathway. These results indicate that camptothecin is formed by the combination of the 2C-methyl-d-erythritol 4-phosphate pathway and the shikimate pathway. This study provides the innovative example for how a computer-aided comprehensive metabolic analysis will refine the experimental design to obtain more precise biological information.
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camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila cloning characterization and differential expression in tissues and by stress compounds
Plant and Cell Physiology, 2003Co-Authors: Yasuyo Yamazaki, Hiroshi Sudo, Norio Aimi, Mami Yamazaki, Kazuki SaitoAbstract:Camptothecin derivatives are clinically used anti-tumor compounds that biogenetically belong to a group of monoterpenoid indole alkaloids (TIA). We have already established a hairy root culture of Ophiorrhiza pumila (Rubiaceae) that produces camptothecin. The present study describes the cloning and characterization of cDNAs encoding strictosidine synthase (OpSTR; EC 4.3.3.2) and tryptophan decarboxylase (OpTDC; EC 4.1.1.28), two key enzymes in the biosynthesis of TIA from hairy roots of O. pumila. We also isolated the cDNA coding for NADPH:cytochrome P450 reductase (OpCPR; EC 1.6.2.4) that is presumed to be indirectly involved in camptothecin synthesis. The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli. The tissue-specific and stress-inducible expression patterns of OpSTR and OpTDC were quite similar, unlike those of OpCPR. The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis. Plant stress compounds like salicylic acid repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis.
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camptothecin related alkaloids from hairy roots of Ophiorrhiza pumila
Tetrahedron, 2002Co-Authors: Mariko Kitajima, Hiromitsu Takayama, Kazuki Saito, Satoshi Yoshida, Kyoko Yamagata, Mio Nakamura, Hiroko Seki, Norio AimiAbstract:Hairy roots of Ophiorrhiza pumila, a rubiaceous camptothecin-producing plant, were obtained. From an investigation of the secondary metabolites, it was found that the hairy roots produced camptothecin and its related alkaloids, i.e. (3S)-pumiloside, (3S)- and (3R)-deoxypumilosides and strictosamide. We also isolated two new camptothecinoids, OPHR-23 and OPHR-17, the structures of which including the absolute configurations, were determined by spectroscopic analyses and chemical conversion from tryptamine and secologanin.
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Bioreactor production of camptothecin by hairy root cultures of Ophiorrhiza pumila
Biotechnology Letters, 2002Co-Authors: Hiroshi Sudo, Norio Aimi, Mami Yamazaki, Takashi Yamakawa, Kazuki SaitoAbstract:A large-scale culture of hairy root of Ophiorrhiza pumila using a modified 3 l bioreactor was established. The hairy roots, incited by infection of Agrobacterium rhizogenes were grown in the bioreactor equipped with a stainless net. The final concentration of camptothecin was 0.0085% fresh wt of tissue, and the total production of camptothecin, an anti-neoplastic quinoline alkaloid, reached 22 mg over 8 weeks' culture in the reactor. Approx. 17% (3.6 mg) of the total camptothecin produced was excreted into the culture medium.
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feasible production of camptothecin by hairy root culture of Ophiorrhiza pumila
Plant Cell Reports, 2001Co-Authors: Kazuki Saito, Mariko Kitajima, Hiromitsu Takayama, Mami Yamazaki, H Sudo, M Kosekinakamura, Norio AimiAbstract:Camptothecin derivatives are used clinically as anti-tumor alkaloids. Camptothecin and its related compounds are at present obtained by extraction from intact plants, but transformed plant cell cultures may be an alternative source of production. We have established a hairy root culture of Ophiorriza pumila (Rubiaceae) transformed by Agrobacterium rhizogenes strain 15834. This hairy root culture grew well, increasing by 16-fold during 5 weeks in liquid culture, and it produced camptothecin as a main alkaloid up to 0.1% per dry weight of the cells. Interestingly, not only the hairy root cells contained camptothecin, but the culture medium also accumulated substantial amounts. Camptothecin content in the medium was increased by the presence of a polystyrene resin (Diaion HP-20) that absorbed camptothecin. Camptothecin was easily recovered from the resin. Our method is the most feasible and commercially applicable way to produce camptothecin by in vitro cell culture.