Ophiorrhiza Pumila Research Articles

Proxy-approach in understanding the bisubstrate activity of strictosidine synthases

Besides tryptamine (1) and secologanin (2), non-cognate substrates also undergo a Pictet-Spengler reaction (PSR) catalyzed by strictosidine synthases (STR) with differing catalytic properties. We characterized the bisubstrate binding aspect of catalysis – order, affinity, and cooperativity – with STR orthologs from Rauvolfia serpentina (RsSTR) and Ophiorrhiza pumila (OpSTR) by an isothermal titration calorimetry (ITC) based ‘proxy approach’ that employed a non-reactive tryptamine analog (m1) to capture its inert ternary complexes with STRs and (2). ITC studies with OpSTR and (2) revealed ‘tryptamine-first’ cooperative binding with (1) and a simultaneous cooperative binding with (m1). Binding cooperativity among (m1) and (2) towards OpSTR was higher than RsSTR. Crystallographic study of RsSTR-(m1) complex helped to understand the unreactive binding of (m1) in terms of orientation and interactions in the RsSTR pocket. PSR with (m1) was revealed to be energetically unfeasible by the density functional theory (DFT) scans of the first hydrogen abstraction by RsSTR. The effect of pH on the bisubstrate binding to OpSTR was deciphered by molecular dynamics simulations (MDS), which also provided a molecular basis for the stability of complex of OpSTR with (m1) and (2). Therefore, we investigated STRs from a substrate binding perspective to inform drug-design and rational enzyme engineering efforts.

CaERF1- mediated ABA signal positively regulates camptothecin biosynthesis by activating the iridoid pathway in Camptotheca acuminata

Camptotheca acuminata is one of the primary sources of camptothecin (CPT), which is widely used in the treatment of human malignancies because of its inhibitory activity against DNA topoisomerase I. Although several transcription factors have been identified for regulating CPT biosynthesis in other species, such as Ophiorrhiza pumila, the specific regulatory components controlling CPT biosynthesis in C. acuminata have yet to be definitively determined. In this study, CaERF1, an DREB subfamily of the APETALA2/ethylene response factors (AP2ERFs), was identified in C. acuminata. The transient overexpression and silencing of CaERF1 in C. acuminata leaves confirmed that it positively regulates the accumulation of CPT by inducing the expression of CaCYC1 and CaG8O in the iridoid pathway. Results of transient transcriptional activity assay and yeast one-hybrid assays have showed that CaERF1 transcriptionally activates the expression of CaCYC1 and CaG8O by binding to RAA and CEI elements in the promoter regions of these two genes. Furthermore, the expression of CaCYC1 and CaG8O in CaERF1-silenced leaves was less sensitive to ABA treatment, indicating that CaERF1 is a crucial component involved in ABA-regulated CPT biosynthesis in C. acuminata.

Comprehensive analysis of OpHD-ZIP transcription factors related to the regulation of camptothecin biosynthesis in Ophiorrhiza pumila

Ophiorrhiza pumila, as a folk herb belonging to the Rubiaceae family, has become a potential source of camptothecin (CPT), which is a monoterpenoid indole alkaloid with good antitumor property. However, the camptothecin content in this herb is low, and is far from meeting the increasing clinical demand. Understanding the transcriptional regulation of camptothecin biosynthesis provides an effective strategy for improvement of camptothecin yield. Previous studies have demonstrated several transcription factors that are related to camptothecin biosynthesis, while the functions of HD-ZIP members in O. pumila have not been investigated yet. In this study, 32 OpHD-ZIP transcription factor members were genome-wide identified. Phylogenetic tree showed that these OpHD-ZIP proteins are divided into four subfamilies. Based on the transcriptome data, nine OpHD-ZIP genes were shown to be predominantly expressed in O. pumila roots, which were in line with the camptothecin biosynthetic genes. Co-expression analysis showed that OpHD-ZIP7 and OpHD-ZIP20 were potentially related to the modulation of camptothecin biosynthesis. Dual-luciferase reporter assays (Dual-LUC) showed that both OpHD-ZIP7 and OpHD-ZIP20 could activate the expression of camptothecin biosynthetic genes OpIO and OpTDC. In conclusion, this study offered the promising data for exploring the roles of OpHD-ZIP transcription factors in regulating camptothecin biosynthesis.

OpNAC1 transcription factor regulates the biosynthesis of the anticancer drug camptothecin by targeting loganic acid O-methyltransferase in Ophiorrhiza pumila.

Camptothecin (CPT) is an anticancer pentacyclic quinoline alkaloid widely used to treat cancer patients worldwide. However, the biosynthetic pathway and transcriptional regulation of camptothecin are largely unknown. Ophiorrhiza pumila, the herbaceous plant from the Rubiaceae family, has emerged as a model plant for studying camptothecin biosynthesis and regulation. In this study, a high-quality reference genome of O. pumila with estimated size of ~456.90 Mb was reported, and the accumulation level of camptothecin in roots was higher than that in stems and leaves. Based on its spatial distribution in the plant, we examined gene functions and expression by combining genomics with transcriptomic analysis. Two loganic acid O-methyltransferase (OpLAMTs) were identified in strictosidine-producing plant O. pumila, and enzyme catalysis assays showed that OpLAMT1 and not OpLAMT2 could convert loganic acid into loganin. Further knock-out of OpLAMT1 expression led to the elimination of loganin and camptothecin accumulation in O. pumila hairy roots. Four key residues were identified in OpLAMT1 protein crucial for the catalytic activity of loganic acid to loganin. By co-expression network, we identified a NAC transcription factor, OpNAC1, as a candidate gene for regulating camptothecin biosynthesis. Transgenic hairy roots and biochemical assays demonstrated that OpNAC1 suppressed OpLAMT1 expression. Here, we reported on two camptothecin metabolic engineering strategies paving the road for industrial-scale production of camptothecin in CPT-producing plants.

In vitro and in vivo anti-lymphoma effects of Ophiorrhiza pumila extract.

Background: Current therapeutic strategies on patients with lymphomas remains limited. Previously we found the suppressive effect of Ophiorrhiza pumila (OPE) on hepatocarcinoma. In present study, the effect of OPE on lymphoma in vitro and in vivo were investigated.Methods: CCK-8 assay was applied to detect the effect of OPE on cell proliferation. Flow cytometry was used to analyze the effect of OPE on cell cycle distribution and apoptosis. Xenograft mouse model was conducted to determine the anti-tumor activity of OPE. TNUEL assay was performed to detect the apoptosis in tumor tissues. Western blot and immuno-histochemistry were used to determine protein expression.Results: In vitro tests indicate that OPE suppressed A20 cell proliferation in a dose- and time-dependent manner. OPE treatment induced cell cycle arrest at S phase and elevated apoptosis in A20 cells. OPE displayed a significant inhibition in tumor growth in a mouse xenograft model. OPE promoted apoptosis of tumor cell in the mouse model Cleaved caspase 3 expression and Bax/Bcl2 ratio were also enhanced. In addition, OPE suppressed A20 cell viability partially by reducing phosphorylation of EGFR.Conclusions: Our data showed that OPE suppressed the proliferation of lymphoma cells and promoted apoptosis in vitro and in vivo, which might be partially mediated by inactivating EGFR signaling.

Genome-wide survey of the GATA gene family in camptothecin-producing plant Ophiorrhiza pumila

BackgroundOphiorrhiza pumila (Rubiaceae) is capable of producing camptothecin (CPT), one monoterpene indole alkaloid extensively employed in the treatment of multiple cancers. Transcription factors (TFs) GATA are a group of transcription regulators involved in plant development and metabolism, and show the feature of binding to the GATA motif within the promoters of target genes. However, GATA TFs have not been characterized in O. pumila.ResultIn this study, a total of 18 GATA genes classified into four subfamilies were identified, which randomly distributed on 11 chromosomes of O. pumila. Synteny analysis of GATA genes between O. pumila and other plant species such as Arabidopsis thaliana, Oryza sativa, Glycine max, Solanum lycopersicum, Vitis vinifera, and Catharanthus roseus genomes were analyzed. Tissue expression pattern revealed that OpGATA1 and OpGATA18 were found to be correlated with ASA, MK, CPR and GPPS, which were highly expressed in leaves. OpGATA7, showed high expression in roots as most of the CPT biosynthetic pathway genes did, suggesting that these OpGATAs may be potential candidates regulating CPT biosynthesis in O. pumila.ConclusionsIn this study, we systematically analyzed the OpGATA TFs, and provided insights into the involvement of OpGATA TFs from O. pumila in CPT biosynthesis.

Identification of WRKY transcription factors involved in regulating the biosynthesis of the anti-cancer drug camptothecin in Ophiorrhiza pumila.

Camptothecin is a chemotherapeutic drug widely used to treat various cancers. Ophiorrhiza pumila is an ideal plant model for the study of camptothecin production, with various advantages for studying camptothecin biosynthesis and regulation. The DNA-binding WRKY transcription factors have a key regulatory role in secondary metabolite biosynthesis in plants. However, little is currently known about their involvement in camptothecin biosynthesis in O. pumila. We identified 46 OpWRKY genes unevenly distributed on the 11 chromosomes of O. pumila. Phylogenetic and multiple sequence alignment analyses divided the OpWRKY proteins into three subfamilies. Based on spatial expression and co-expression, we targeted the candidate gene OpWRKY6. Overexpression of OpWRKY6 significantly reduced the accumulation of camptothecin compared with the control. Conversely, camptothecin accumulation increased in OpWRKY6 knockout lines. Further biochemical assays showed that OpWRKY6 negatively regulates camptothecin biosynthesis from both the iridoid and shikimate pathways by directly downregulating the gene expression of OpGES, Op10HGO, Op7DLH, and OpTDC. Our data provide direct evidence for the involvement of WRKYs in the regulation of camptothecin biosynthesis and offer valuable information for enriching the production of camptothecin in plant systems.

A High-Density Genetic Map Enables Genome Synteny and QTL Mapping of Vegetative Growth and Leaf Traits in Gardenia.

The gardenia is a traditional medicinal horticultural plant in China, but its molecular genetic research has been largely hysteretic. Here, we constructed an F1 population with 200 true hybrid individuals. Using the genotyping-by-sequencing method, a high-density sex-average genetic map was generated that contained 4,249 SNPs with a total length of 1956.28 cM and an average genetic distance of 0.46 cM. We developed 17 SNP-based Kompetitive Allele-Specific PCR markers and found that 15 SNPs were successfully genotyped, of which 13 single-nucleotide polymorphism genotypings of 96 F1 individuals showed genotypes consistent with GBS-mined genotypes. A genomic collinearity analysis between gardenia and the Rubiaceae species Coffea arabica, Coffea canephora and Ophiorrhiza pumila showed the relativity strong conservation of LG11 with NC_039,919.1, HG974438.1 and Bliw01000011.1, respectively. Lastly, a quantitative trait loci analysis at three phenotyping time points (2019, 2020, and 2021) yielded 18 QTLs for growth-related traits and 31 QTLs for leaf-related traits, of which qBSBN7-1, qCD8 and qLNP2-1 could be repeatably detected. Five QTL regions (qCD8 and qSBD8, qBSBN7 and qSI7, qCD4-1 and qLLLS4, qLNP10 and qSLWS10-2, qSBD10 and qLLLS10) with potential pleiotropic effects were also observed. This study provides novel insight into molecular genetic research and could be helpful for further gene cloning and marker-assisted selection for early growth and development traits in the gardenia.

Chromosome-level assembly of the Neolamarckia cadamba genome provides insights into the evolution of cadambine biosynthesis.

Neolamarckia cadamba (Roxb.), a close relative of Coffea canephora and Ophiorrhiza pumila, is an important traditional medicine in Southeast Asia. Three major glycosidic monoterpenoid indole alkaloids (MIAs), cadambine and its derivatives 3β-isodihydrocadambine and 3β-dihydrocadambine, accumulate in the bark and leaves, and exhibit antimalarial, antiproliferative, antioxidant, anticancer and anti-inflammatory activities. Here, we report a chromosome-scale N. cadamba genome, with 744.5 Mb assembled into 22 pseudochromosomes with contig N50 and scaffold N50 of 824.14 Kb and 29.20 Mb, respectively. Comparative genomic analysis of N. cadamba with Co. canephora revealed that N. cadamba underwent a relatively recent whole-genome duplication (WGD) event after diverging from Co. canephora, which contributed to the evolution of the MIA biosynthetic pathway. We determined the key intermediates of the cadambine biosynthetic pathway and further showed that NcSTR1 catalyzed the synthesis of strictosidine in N. cadamba. A new component, epoxystrictosidine (C27H34N2O10, m/z 547.2285), was identified in the cadambine biosynthetic pathway. Combining genome-wide association study (GWAS), population analysis, multi-omics analysis and metabolic gene cluster prediction, this study will shed light on the evolution of MIA biosynthetic pathway genes. This N. cadamba reference sequence will accelerate the understanding of the evolutionary history of specific metabolic pathways and facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants.

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.

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.

Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila

BackgroundThe 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.ResultsIn 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.ConclusionsIn 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.

Characterization and phylogenetic analysis of the complete chloroplast genome of Ophiorrhiza pumila (Rubiaceae)

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.

Chromosome-level genome assembly of Ophiorrhiza pumila reveals the evolution of camptothecin biosynthesis

Plant genomes remain highly fragmented and are often characterized by hundreds to thousands of assembly gaps. Here, we report chromosome-level reference and phased genome assembly of Ophiorrhiza pumila, a camptothecin-producing medicinal plant, through an ordered multi-scaffolding and experimental validation approach. With 21 assembly gaps and a contig N50 of 18.49 Mb, Ophiorrhiza genome is one of the most complete plant genomes assembled to date. We also report 273 nitrogen-containing metabolites, including diverse monoterpene indole alkaloids (MIAs). A comparative genomics approach identifies strictosidine biogenesis as the origin of MIA evolution. The emergence of strictosidine biosynthesis-catalyzing enzymes precede downstream enzymes’ evolution post γ whole-genome triplication, which occurred approximately 110 Mya in O. pumila, and before the whole-genome duplication in Camptotheca acuminata identified here. Combining comparative genome analysis, multi-omics analysis, and metabolic gene-cluster analysis, we propose a working model for MIA evolution, and a pangenome for MIA biosynthesis, which will help in establishing a sustainable supply of camptothecin.

The transcription factor OpWRKY2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila

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.

Computational biotechnology guides elucidation of the biosynthesis of the plant anticancer drug camptothecin

Camptothecin is a clinically important monoterpene indole alkaloid (MIAs) used for treating various cancers. Currently, the production of this biopharmaceutical hinges on its extraction from camptothecin-producing plants, leading to high market prices and supply bottlenecks. While synthetic biology combined with metabolic approaches could represent an attractive alternative approach to manufacturing, it requires firstly a complete biosynthetic pathway elucidation, which is, unfortunately, severely missing in species naturally accumulating camptothecin. This knowledge gap can be attributed to the lack of high-quality genomic resources of medicinal plant species. In such a perspective, Yamazaki and colleagues produced the first described and experimentally validated chromosome-level plant genome assembly of Ophiorrhiza pumila, a prominent source plant of camptothecin for the pharmaceutical industry. More specifically, they have developed a method incorporating Illumina reads, PacBio single-molecule reads, optical mapping and Hi-C sequencing, followed by the experimental validation of contig orientation within scaffolds, using fluorescence in situ hybridization (FISH) analysis. This relevant strategy resulted in the most contiguous and complete de novo plant reference genome described to date, which can streamline the sequencing of new plant genomes. Further mining approaches, including integrative omics analysis, phylogenetics, gene cluster evaluation and comparative genomics were successfully used to puzzle out the evolutionary origins of MIA metabolism and revealed a short-list of high confidence MIA biosynthetic genes for functional validation.

Transcriptome-wide identification, characterization, and phylogenomic analysis of cytochrome P450s from Nothapodytes nimmoniana reveal candidate genes involved in the camptothecin biosynthetic pathway.

The plant Nothapodytes nimmoniana is an important source of camptothecin (CPT), an anticancer compound widely used in the treatment of colorectal, lung, and ovarian cancers. CPT is biosynthesized by the combination of the seco-iridoid and indole pathways in plants. The majority of the biosynthetic steps and associated genes still remain unknown. Certain reactions in the seco-iridoid pathway are catalyzed by cytochrome P450 enzymes. Hence, identifying transcriptionally active cytochrome P450 genes becomes essential in the elucidation of the CPT biosynthetic pathway. Here, we report the identification of 94 cytochrome P450s from the assembled transcriptomic data from leaf and root tissues of N. nimmoniana. The identified cytochrome P450 genes were full length and possessed all four conserved characteristic signature motifs of cytochrome P450 genes. Phylogenetic analysis of the protein sequences revealed their evolution and diversification and further categorized them into A-type (52.12%) and non-A-type (47.87%) cytochrome P450s. These 94 sequences represent 38 families and 63 subfamilies of cytochrome P450s. We also compared the transcriptional activity of identified cytochrome P450s with the expression of their homologs in the CPT-producing plant Ophiorrhiza pumila. Based on expression profiles and quantitative PCR validation, we propose NnCYP81CB1 and NnCYP89R1 as candidate cytochrome P450 genes involved in camptothecin biosynthesis in N. nimmoniana.

Effects of Concentration and Temperature of Nutrient Solution on Growth and Camptothecin Accumulation of Ophiorrhiza pumila.

The medicinal plant, Ophiorrhiza pumila, naturally grows on the floors of humid inland forests in subtropical areas. It accumulates camptothecin (CPT), which is used as an anti-tumor agent, in all organs. We investigated the optimal hydroponic root-zone environments for growth and CPT accumulation in O. pumila in a plant factory. In experiment 1, to determine the appropriate nutrient solution concentration (NSC), O. pumila was cultivated using four concentrations (0.125, 0.25, 0.5, and 1.0 times) of a commercial solution for 63 days after the start of treatment (DAT). The electrical conductivity of these NSCs was 0.6, 0.9, 1.5, and 2.7 dS m−1, respectively. The total dry weights at 0.25 and 0.5 NSCs were higher than those at the other two NSCs. CPT content at 0.25 NSC was significantly higher than those at other NSCs. In experiment 2, to investigate an appropriate nutrient solution temperature (NST), O. pumila was cultivated at four NSTs (10, 20, 26, and 35 °C, named as T10, T20, T26, and T36, respectively) for 35 DAT. The growth and CPT content at T20 was the highest among the treatments. Therefore, root-zone environments of 0.25 NSC and 20 °C of NST produced the best growth and CPT accumulation in O. pumila.

Cloning, characterization, and enzymatic identification of a new tryptophan decarboxylase from Ophiorrhiza pumila

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 1,536bp open reading frame that encodes a 511 amino acid protein with a molecular mass of 57.01kDa 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 0.1mM. 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 1.08mM and 0.78Sec-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.

WRKY transcription factor OpWRKY1 acts as a negative regulator of camptothecin biosynthesis in Ophiorrhiza pumila hairy roots

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.

Targeted metabolic engineering of committed steps improves anti-cancer drug camptothecin production in Ophiorrhiza pumila hairy roots

Camptothecin (CPT) is a natural compound with remarkable antitumor activities due to its special mechanism of inhibiting DNA topoisomerase I. Ophiorrhiza pumila was found to produce camptothecin. By comparative transcriptome profile analysis of O.pumila plantlet roots and hairy roots with suspension cell culture (CSC), two key CPT biosynthetic genes including OpG10H and OpSLS with significantly higher expression level were selected for metabolic engineering. Camptothecin content in hairy root lines mediated by CRISPR-Cas9 system (OpG10H or OpSLS knock-out lines) was obviously reduced, suggesting that both OpG10H and OpSLS play pivotal roles in CPT biosynthesis. Meanwhile individual overexpression of OpG10H (G line) or OpSLS (S line) in O.pumila hairy roots significantly elevated the production of CPT, the highest content was 2.40 mg/g in G line and 3.28 mg/g in S line. Subsequently co-introduction of OpG10H and OpSLS (SG line) resulted in higher concentration of CPT, reaching 3.5 mg/g in SG-17 line, showing cooperative effect than single gene. Besides, camptothecin crude extract from transgenic hairy roots exhibited excellent anti-tumor activity. In summary, the present study shows genetic manipulation of OpG10H and OpSLS revealed by comparative transcriptome analysis is an effective strategy for production of CPT in O.pumila, which sheds new insights for enhancement of other valuable bioactive products.