Advanced metabolic engineering strategies for increasing artemisinin yield in Artemisia annua L.

Artemisinin, also known as 'Qinghaosu', is a chemically sesquiterpene lactone containing an endoperoxide bridge. Due to the high activity to kill Plasmodium parasites, artemisinin and its derivatives have continuously served as the foundation for antimalarial therapies. Natural artemisinin is unique to the traditional Chinese medicinal plant Artemisia annua L., and its content in this plant is low. This has motivated the synthesis of this bioactive compound using yeast, tobacco, and Physcomitrium patens systems. However, the artemisinin production in these heterologous hosts is low and cannot fulfil its increasing clinical demand. Therefore, A. annua plants remain the major source of this bioactive component. Recently, the transcriptional regulatory networks related to artemisinin biosynthesis and glandular trichome formation have been extensively studied in A. annua. Various strategies including (i) enhancing the metabolic flux in artemisinin biosynthetic pathway; (ii) blocking competition branch pathways; (iii) using transcription factors (TFs); (iv) increasing peltate glandular secretory trichome (GST) density; (v) applying exogenous factors; and (vi) phytohormones have been used to improve artemisinin yields. Here we summarize recent scientific advances and achievements in artemisinin metabolic engineering, and discuss prospects in the development of high-artemisinin yielding A. annua varieties. This review provides new insights into revealing the transcriptional regulatory networks of other high-value plant-derived natural compounds (e.g., taxol, vinblastine, and camptothecin), as well as glandular trichome formation. It is also helpful for the researchers who intend to promote natural compounds production in other plants species.

A chromosome-level genome assembly of anesthetic drug-producing Anisodus acutangulus provides insights into its evolution and the biosynthesis of tropane alkaloids.

Tropane alkaloids (TAs), which are anticholinergic agents, are an essential class of natural compounds, and there is a growing demand for TAs with anesthetic, analgesic, and spasmolytic effects. Anisodus acutangulus (Solanaceae) is a TA-producing plant that was used as an anesthetic in ancient China. In this study, we assembled a high-quality, chromosome-scale genome of A. acutangulus with a contig N50 of 7.4 Mb. A recent whole-genome duplication occurred in A. acutangulus after its divergence from other Solanaceae species, which resulted in the duplication of ADC1 and UGT genes involved in TA biosynthesis. The catalytic activities of H6H enzymes were determined for three Solanaceae plants. On the basis of evolution and co-expressed genes, AaWRKY11 was selected for further analyses, which revealed that its encoded transcription factor promotes TA biosynthesis by activating AaH6H1 expression. These findings provide useful insights into genome evolution related to TA biosynthesis and have potential implications for genetic manipulation of TA-producing plants.

Rigid flexible coupling contact mechanism for oral and maxillofacial skin and soft tissues.

BACKGROUND AND OBJECTIVES: The existing medical clinical treatment institutions mostly use rigid structures to come into contact with flexible skin. The rigid flexible coupled contact biomechanical model for the skin is the first step that urgently needs to be considered in the process of medical clinical operations. However, there has been currently no effective biomechanical contact model available. METHODS: Based on the principle of elastic interface deformation, the basic biomechanical characteristics of oral and maxillofacial skin and soft tissues were analyzed to address the unknown mechanism of rigid body and maxillofacial contact in oral imaging operations. A nonlinear characterization method for the mechanical properties of oral and maxillofacial skin soft tissues was proposed by deriving a general contact force model that takes into account energy dissipation. However, the problem of the inability to obtain analytical solutions for the parameters of the dynamic model exists. It is necessary to perform particle swarm parameter identification on different nonlinear contact models and verify the accuracy of the algorithm through numerical simulation. A maxillofacial contact experiment was conducted to verify the operation process of an oral imaging robot. RESULTS: After experimental analysis, it was found that the comprehensive average error between the model and the actual contact force was 0.13325 N. The absolute error of the maximum deformation displacement was below 0.18 N, which verified the effectiveness and safety of the contact model in the contact process of the oral imaging robot system. CONCLUSIONS: The results indicate that the output force of the model has been in good agreement with the actual contact force.

AaABI5 transcription factor mediates light and abscisic acid signaling to promote anti-malarial drug artemisinin biosynthesis in Artemisia annua.

Artemisia annua, a member of the Asteraceae family, remains the primary source of artemisinin. However, the artemisinin content in the existing varieties of this plant is very low. In this study, we found that the environmental factors light and phytohormone abscisic acid (ABA) could synergistically promote the expression of artemisinin biosynthetic genes. Notably, the increased expression levels of those genes regulated by ABA depended on light. Gene expression analysis found that AaABI5, a transcription factor belonging to the basic leucine zipper (bZIP) family, was inducible by the light and ABA treatment. Analysis of AaABI5-overexpressing and -suppressing lines suggested that AaABI5 could enhance artemisinin biosynthesis and activate the expression of four core biosynthetic genes. In addition, the key regulator of light-induced artemisinin biosynthesis, AaHY5, could bind to the promoter of AaABI5 and activate its expression. In conclusion, our results demonstrated that AaABI5 acts as an important molecular junction for the synergistic promotion of artemisinin biosynthesis by light and ABA signals, which provides a candidate gene for developing new germplasms of high-quality A. annua.

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.

AIE-YOLO: Auxiliary Information Enhanced YOLO for Small Object Detection.

Small object detection is one of the key challenges in the current computer vision field due to the low amount of information carried and the information loss caused by feature extraction. You Only Look Once v5 (YOLOv5) adopts the Path Aggregation Network to alleviate the problem of information loss, but it cannot restore the information that has been lost. To this end, an auxiliary information-enhanced YOLO is proposed to improve the sensitivity and detection performance of YOLOv5 to small objects. Firstly, a context enhancement module containing a receptive field size of 21×21 is proposed, which captures the global and local information of the image by fusing multi-scale receptive fields, and introduces an attention branch to enhance the expressive ability of key features and suppress background noise. To further enhance the feature expression ability of small objects, we introduce the high- and low-frequency information decomposed by wavelet transform into PANet to participate in multi-scale feature fusion, so as to solve the problem that the features of small objects gradually disappear after multiple downsampling and pooling operations. Experiments on the challenging dataset Tsinghua-Tencent 100 K show that the mean average precision of the proposed model is 9.5% higher than that of the original YOLOv5 while maintaining the real-time speed, which is better than the mainstream object detection models.

A Plasma Transmitting Source for Borehole Acoustic Reflection Imaging.

The detection depth of current borehole acoustic reflection imaging is only tens of meters without high resolution. This considerably limits its wide application in the identification and fine description of unconventional reservoirs and in the optimization of drilling trajectories. Increasing the directional energy from the transmitter to a geological structure is an excellent way to solve this issue. In this study, a plasma source with a parabolic reflector was introduced during borehole acoustic reflection imaging. First, an experimental system was built for testing the plasma source. Next, the acoustic-electrical characteristics and directional radiation of the source were studied using experiments and a numerical simulation. Finally, the advantages, disadvantages, and feasibility of the plasma-transmitting source were analyzed; some suggestions for further work on the source and its logging application were proposed. The experimental and simulation results show that the use of a plasma source with a parabolic reflector can increase the detection depth of borehole acoustic reflection imaging to hundreds of meters with high resolution. This is crucial in imaging the geological structures near boreholes and enhancing oil-gas exploration and development.

Mining of the CULLIN E3 ubiquitin ligase genes in the whole genome of Salvia miltiorrhiza.

CULLIN (CUL) proteins are E3 ubiquitin ligases that are involved in a wide variety of biological processes as well as in response to stress in plants. In Salvia miltiorrhiza, CUL genes have not been characterized and its role in plant development, stress response and secondary metabolite synthesis have not been studied. In this study, genome-wide analyses were performed to identify and to predict the structure and function of CUL of S. miltiorrhiza. Eight CUL genes were identified from the genome of S. miltiorrhiza. The CUL genes were clustered into four subgroups according to phylogenetic relationships. The CUL domain was highly conserved across the family of CUL genes. Analysis of cis-acting elements suggested that CUL genes might play important roles in a variety of biological processes, including abscission reaction acid (ABA) processing. To investigate this hypothesis, we treated hairy roots of S. miltiorrhiza with ABA. The expression of CUL genes varied obviously after ABA treatment. Co-expression network results indicated that three CUL genes might be involved in the biosynthesis of phenolic acid or tanshinone. In summary, the mining of the CUL genes in the whole genome of S. miltiorrhiza contribute novel information to the understanding of the CUL genes and its functional roles in plant secondary metabolites, growth and development.

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.

Basic Helix-Loop-Helix Transcription Factors AabHLH2 and AabHLH3 Function Antagonistically With AaMYC2 and Are Negative Regulators in Artemisinin Biosynthesis.

Plants have evolved sophisticated systems for regulating the biosynthesis of specialized phytochemicals. Artemisinin, which is a sesquiterpene lactone widely used in anti-malaria treatment, is produced by the Artemisia annua L. plant. However, the artemisinin content in A. annua is low and difficult to meet market demands. Studies have shown that artemisinin biosynthesis in A. annua has complex temporal and spatial specificity and is under tightly transcriptional regulation. However, the mechanism of transcriptional regulation of artemisinin biosynthesis remains unclear. In this study, we identified two MYC-type bHLH transcription factors (AabHLH2 and AabHLH3) as novel regulators of artemisinin biosynthesis. These bHLH TFs act as transcription repressors and function redundantly to negatively regulate artemisinin biosynthesis. Furthermore, AabHLH2 and AabHLH3 are nuclear proteins that bind to DNA elements with similar specificity to that of AaMYC2, but lack the conserved activation domain, suggesting that repression is achieved by competition for the same cis-regulatory elements. Together, our findings reveal a novel artemisinin biosynthesis regulatory network, provide new insight into how specialized metabolites are modulated in plants, and propose a model in which different bHLH TFs coordinated in regulating artemisinin production in the plant. Finally, this study provides some useful target genes for metabolic engineering of artemisinin production via CRISPR/Cas9 gene editing.

Water-Induced Self-Assembly and In Situ Mineralization within Plant Phenolic Glycol-Gel toward Ultrastrong and Multifunctional Thermal Insulating Aerogels.

Biopolymer/silica nanocomposite aerogels are highly attractive as thermally insulating materials for prevailing energy-saving engineering but are usually plagued by their lack of mechanical strength and environmental stability. Lignin is an appealing plant phenolic biopolymer due to its natural abundance, high stiffness, water repellency, and thermostability. However, integrating lignin and silica into high-performance 3D hybrid aerogels remains a substantial challenge due to the unstable co-sol process. In diatoms, the silicic acid stabilization prior to the condensation reaction is enhanced by the intervention of biomolecules in noncovalent interactions. Inspired by this mechanism, we herein rationally design an ultrastrong silica-mineralized lignin nanocomposite aerogel (LigSi) with an adjustable multilevel micro/nanostructure and arbitrary machinability through an unusual water-induced self-assembly and in situ mineralization based on ethylene glycol-stabilized lignin/siloxane colloid. The optimized LigSi exhibits an ultrahigh stiffness (a specific modulus of ∼376.3 kN m kg-1) and can support over 5000 times its own weight without obvious deformation. Moreover, the aerogel demonstrates a combination of outstanding properties, including superior and humidity-tolerant thermal insulation (maintained at ∼0.04 W m-1 K-1 under a relative humidity of 33-94%), excellent fire resistance withstanding an ∼1200 °C flame without disintegration, low near-infrared absorption (∼9%), and intrinsic self-cleaning/superhydrophobic performance (158° WCA). These advanced properties make it an ideal thermally insulating material for diversified applications in harsh environments. As a proof of concept, a dual-mode LigSi thermal device was designed to demonstrate the application prospect of combining passive heat-trapping and active heating in the building.

Genome-Wide Analysis of U-box E3 Ubiquitin Ligase Family in Response to ABA Treatment in Salvia miltiorrhiza.

Plant U-box (PUB) proteins are ubiquitin ligases (E3) involved in multiple biological processes and in response to plant stress. However, the various aspects of the genome and the differences in functions between the U-box E3 (UBE3) ubiquitin ligases remain quite obscure in Salvia miltiorrhiza. The 60 UBE3 genes in the S. miltiorrhiza genome were recognized in the present study. The phylogenetic analysis, gene structure, motifs, promoters, and physical and chemical properties of the genes were also examined. Based on the phylogenetic relationship, the 60 UBE3 genes were categorized under six different groups. The U-box domain was highly conserved across the family of UBE3 genes. Analysis of the cis-acting element revealed that the UBE3 genes might play an important role in a variety of biological processes, including a reaction to the abscisic acid (ABA) treatment. To investigate this hypothesis, an ABA treatment was developed for the hairy roots of S. miltiorrhiza. Thirteen out of the UBE3 genes significantly increased after the ABA treatment. The co-expression network revealed that nine UBE3 genes might be associated with phenolic acids or tanshinone biosynthesis. The findings of the present study brought fresh and new understanding to the participation of the UBE3 gene family in plants, specifically in their biological responses mediated by the ABA. In S. miltiorrhiza, this gene family may be crucial during the ABA treatment. Significantly, the results of this study contribute novel information to the understanding of the ubiquitin ligase gene and its role in plant growth.

Dihydrotanshinone I inhibits ovarian tumor growth by activating oxidative stress through Keap1-mediated Nrf2 ubiquitination degradation.

Dihydrotanshinone I (DHT), a bioactive compound in Salvia miltiorrhiza, was reported to exhibit cytotoxicity against various malignancies. However, the underlying mechanism on ovarian cancer remains unclear. Here, DHT inhibited cell viability of ovarian cancer HO8910PM, SKOV3, A2780 and ES2 cells. It showed moderate inhibitory effect on ovarian epithelial IOSE80 cells and lower toxicity than chemotherapy drugs. DHT induced apoptosis and G2 cell cycle arrest accompanied by reduced expression of Bcl-2, Caspase-3, and increased Bax. Meanwhile, DHT increased ROS accumulation, decreased mitochondrial membrane potential and activated oxidative stress in HO8910PM and ES2 cells. Mechanistically, DHT inhibited Nrf2 and p62 expression, Nrf2 target genes and enzymes, and Nrf2 nuclear translocation, while increased the expression of Nrf2 inhibitor Keap1. NAC, a ROS scavenger, rescued DHT-induced proliferation inhibition, ROS generation and Nrf2 inhibition. DHT alleviated tBHQ-induced Nrf2 expression and increased its mRNA level. However, the proteasome inhibitor MG132 blocked DHT-induced Nrf2 inhibition, suggesting a post-translational regulation manner. DHT enhanced Nrf2 binding with Keap1, leading to potentiated Nrf2 ubiquitination degradation. Furthermore, Nrf2 and p62 overexpression blocked DHT-induced Nrf2 and p62 inhibition. Consistent with the in vitro results, DHT significantly delayed tumor growth in HO8910PM and ES2 xenograft nude mice, decreased tumor marker HE4 and CA125 levels, reversed the abnormally expressed proteins including Ki67, Nrf2, p62, Keap1, Bcl-2, CyclinB1, Cdc-2, and antioxidant enzymes SOD, CAT in vivo. Serum from DHT-treated mice also inhibited cell growth in vitro. Taken together, DHT exhibits anti-ovarian tumor effect by activating oxidative stress through ubiquitination-mediated Nrf2 degradation. Our findings implicate a potential application of DHT for ovarian cancer therapy.

AaWRKY17, a positive regulator of artemisinin biosynthesis, is involved in resistance to Pseudomonas syringae in Artemisia annua.

Artemisia annua, a traditional Chinese medicinal plant, remains the only plant source for artemisinin production, yet few genes have been identified to be involved in both the response to biotic stresses, such as pathogens, and artemisinin biosynthesis. Here, we isolated and identified the WRKY transcription factor (TF) AaWRKY17, which could significantly increase the artemisinin content and resistance to Pseudomonas syringae in A. annua. Yeast one-hybrid (Y1H), dual-luciferase (dual-LUC), and electrophoretic mobility shift assay (EMSA) results showed that AaWRKY17 directly bound to the W-box motifs in the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase (ADS) and promoted its expression. Real-time quantitative PCR (RT-qPCR) analysis revealed that the transcript levels of two defense marker genes, Pathogenesis-Related 5 (PR5) and NDR1/HIN1-LIKE 10 (NHL10), were greatly increased in AaWRKY17-overexpressing transgenic A. annua plants. Additionally, overexpression of AaWRKY17 in A. annua resulted in decreased susceptibility to P. syringae. These results indicated that AaWRKY17 acted as a positive regulator in response to P. syringae infection. Together, our findings demonstrated that the novel WRKY transcription factor AaWRKY17 could potentially be used in transgenic breeding to improve the content of artemisinin and pathogen tolerance in A. annua.

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.

Optimal Trajectory Planning of the Variable-Stiffness Flexible Manipulator Based on CADE Algorithm for Vibration Reduction Control.

Robotic manipulators are widely used for precise operation in the medical field. Vibration suppression control of robotic manipulators has become a key issue affecting work stability and safety. In this paper an optimal trajectory planning control method to suppress the vibration of a variable-stiffness flexible manipulator considering the rigid-flexible coupling is proposed. Through analyzing the elastic deformation of the variable-stiffness flexible manipulator, a distributed dynamic physical model of the flexible manipulator is constructed based on the Hamilton theory. Based on the mathematical model of the system, the design of the vibration damping controller of the flexible manipulator is proposed, and the control system with nonlinear input is considered for numerical analysis. According to the boundary conditions, the vibration suppression effect of the conventional and the variable-stiffness flexible manipulator is compared. The motion trajectory of the variable-stiffness flexible manipulator and compare the vibration response from different trajectories. Then, with minimum vibration displacement, minimum energy consumption and minimum trajectory tracking deviation as performance goals, the trajectory planning of the variable-stiffness flexible manipulator movement is carried out based on the cloud adaptive differential evolution (CADE) optimization algorithm. The validity of the proposed trajectory planning method is verified by numerical simulation.

Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila.

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.

AaMYB15, an R2R3-MYB TF in Artemisia annua, acts as a negative regulator of artemisinin biosynthesis.

Artemisinin is a secondary metabolite extracted from Artemisia annua. As an effective antimalarial component certified by WHO, artemisinin has extensive economical values. Numerous studies about transcription factors positively regulating artemisinin biosynthesis have been published while negative regulators are rarely reported. In the present study, we identified AaMYB15 as the first R2R3-MYB that negatively regulates artemisinin biosynthesis in A. annua. Experimental evidences showed that AaMYB15 is a transcription factor within nucleus and predominantly expressed in glandular secretory trichomes (GSTs) in A. annua where artemisinin is synthesized and accumulated. The expression of AaMYB15 was induced by dark and JA treatment. Overexpression of AaMYB15 led to a significant decline in the expression levels of key enzyme genes ADS, CYP, DBR2, and ALDH1 and a significant decrease in the artemisinin contents of transgenic A. annua. AaMYB15 directly bound to the promoter of AaORA, a reported positive regulator of artemisinin biosynthesis in JA signaling pathway, to repress its transcriptional activity, thus downregulating the expression levels of downstream key enzyme genes and negatively regulating the artemisinin biosynthesis. Our study provides candidate gene for improvement of A. annua germplasm and new insights into the artemisinin biosynthesis regulation network mediated by light and JA.