[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

IMP: bridging the gap for medicinal plant genomics.

Medicinal plants have garnered significant attention in ethnomedicine and traditional medicine due to their potential antitumor, anti-inflammatory and antioxidant properties. Recent advancements in genome sequencing and synthetic biology have revitalized interest in natural products. Despite the availability of sequenced genomes and transcriptomes of these plants, the absence of publicly accessible gene annotations and tabular formatted gene expression data has hindered their effective utilization. To address this pressing issue, we have developed IMP (Integrated Medicinal Plantomics), a freely accessible platform at https://www.bic.ac.cn/IMP. IMP curated a total of 8 565 672 genes for 84 high-quality genome assemblies, and 2156 transcriptome sequencing samples encompassing various organs, tissues, developmental stages and stimulations. With the integrated 10 analysis modules, users could simply examine gene annotations, sequences, functions, distributions and expressions in IMP in a one-stop mode. We firmly believe that IMP will play a vital role in enhancing the understanding of molecular metabolic pathways in medicinal plants or plants with medicinal benefits, thereby driving advancements in synthetic biology, and facilitating the exploration of natural sources for valuable chemical constituents like drug discovery and drug production.

Functional characterization of CYP81C16 involved in the tanshinone biosynthetic pathway in Salvia miltiorrhiza.

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.

[Dead heart of pith-decayed Scutellariae Radix: a study based on multi-omics].

Dead heart is an important trait of pith-decayed Scutellariae Radix. The purpose of this study was to clarify the scientific connotation of the dead heart using multi-omics. Metabolomics and transcriptomics combined with multivariate statistical analysis such as principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA) were used to systematically compare the differences in chemical composition and gene expression among phloem, outer xylem and near-dead xylem of pith-decayed Scutella-riae Radix. The results revealed significant differences in the contents of flavonoid glycosides and aglycones among the three parts. Compared with phloem and outer xylem, near-dead xylem had markedly lowered content of flavonoid glycosides(including baicalin, norwogonin-7-O-ß-D-glucuronide, oroxylin A-7-O-ß-D-glucuronide, and wogonoside) while markedly increased content of aglycones(including 3,5,7,2',6'-pentahydroxy dihydroflavone, baicalin, wogonin, and oroxylin A). The differentially expressed genes were mainly concentrated in KEGG pathways such as phenylpropanoid metabolism, flavonoid biosynthesis, ABC transporter, and plant MAPK signal transduction pathway. This study systematically elucidated the material basis of the dead heart of pith-decayed Scutellariae Radix with multiple growing years. Specifically, the content of flavonoid aglycones was significantly increased in the near-dead xylem, and the gene expression of metabolic pathways such as flavonoid glycoside hydrolysis, interxylary cork development and programmed apoptosis was significantly up-regulated. This study provided a theoretical basis for guiding the high-quality production of pith-decayed Scutellariae Radix.

Functional diversity of diterpene synthases in Aconitum plants.

Members of the Aconitum genus within the Ranunculaceae family are known to accumulate a broad array of medicinal and toxic diterpenoid alkaloids (DAs). Historically, ent-copalyl diphosphate (ent-CPP) was considered the sole precursor in DAs biosynthesis. However, the recent discovery of ent-8,13-CPP synthase in A. gymnandrum Maxim., which participates in ent-atiserene biosynthesis, raises the question of whether this gene is conserved throughout the Aconitum genus. In this study, RNA sequencing and PacBio Iso-sequencing were employed to identify diterpene synthases (diTPSs) in four additional Aconitum species with distinct DA compositions. In vitro and in vivo analyses functionally characterized a diverse array of 10 class II and 9 class I diTPSs. In addition to the identification of seven class II diTPSs as ent-CPP synthases, three other synthases generating ent-8,13-CPP, 8,13-CPP, and 8α-hydroxy-CPP were also discovered. Four class I kaurene synthases-like (KSLs) were observed to react with ent-CPP to yield ent-kaurene. Three KSLs not only reacted with ent-CPP but also ent-8,13-CPP to produce ent-atiserene. AsiKSL2-1 was found to react with 8α-hydroxy-CPP to produce Z-abienol and AsiKSL2-2 exhibited no activity with any of the four intermediates. This research delineates the known diterpene biosynthesis pathways in six Aconitum species and explores the highly divergent diterpene synthases within the genus, which are consistent with their phylogeny and may be responsible for the differential distribution of diterpenoid alkaloids in root and aerial parts. These findings contribute valuable insights into the diversification of diterpene biosynthesis and establish a solid foundation for future investigation into DA biosynthetic pathways in Aconitum.

Phylogenetic analysis and functional characterization of norcoclaurine synthase involved in benzylisoquinoline alkaloids biosynthesis in Stephania tetrandra.

Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites that possess diverse pharmaceutical properties and are exclusively accumulated in specific plant genera. The Pictet-Spengler condensation, catalyzed by norcoclaurine synthase (NCS), represents a key enzymatic reaction in the biosynthetic pathway of BIAs. While NCS genes have been identified in several plant families such as Papaveraceae, Berberidaceae, and Ranunculaceae, no NCS genes have been reported in Menispermaceae, which is another genus known to accumulate BIAs. Here, NCSs were isolated and functionally characterized from the Menispermaceae family plant Stephania tetrandra. In vitro enzyme assay identified two functional StNCSs which could catalyze the formation of (S)-norcoclaurine. These functionally characterized genes were then integrated into engineered yeast to enable the production of norcoclaurine. Phylogenetic analysis of the NCS enzymes revealed that the StNCSs predominantly clustered into two clades. The functional StNCSs clustered with known NCSs, highlighting the presence of a specific NCS catalytic domain. This study not only provides additional genetic components for the synthetic biology-based production of BIAs in yeast but also contributes to the understanding of the phylogenetic relationships and structure-function relationship of NCS genes involved in the origin and production of BIAs.

[Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro].

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production.

Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32 mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.

Integrating Metabolomics and Transcriptomics to Unveil Atisine Biosynthesis in Aconitum gymnandrum Maxim.

Diterpene alkaloids (DAs) are characteristic compounds in Aconitum, which are classified into four skeletal types: C18, C19, C20, and bisditerpenoid alkaloids. C20-DAs are thought to be the precursor of the other types. Their biosynthetic pathway, however, is largely unclear. Herein, we combine metabolomics and transcriptomics to unveil the methyl jasmonate (MJ) inducible biosynthesis of DAs in the sterile seedling of A. gymnandrum, the only species in the Subgenus Gymnaconitum (Stapf) Rapaics. Target metabolomics based on root and aerial portions identified 51 C19-DAs and 15 C20-DAs, with 40 inducible compounds. The highest content of C20-DA atisine was selected for further network analysis. PacBio Isoform sequencing integrated with RNA sequencing not only provided the full-length transcriptome but also their response to induction, revealing 1994 genes that exhibited up-regulated expression. Further, 38 genes involved in terpenoid biosynthesis were identified, including 7 diterpene synthases. In addition to the expected function of the four diterpene synthases, AgCPS5 was identified to be a new ent-8,13-CPP synthase in Aconitum and could also combine with AgKSL1 to form the C20-DAs precursor ent-atiserene. Combined with multiple network analyses, six CYP450 and seven 2-ODD genes predicted to be involved in the biosynthesis of atisine were also identified. This study not only sheds light on diterpene synthase evolution in Aconitum but also provides a rich dataset of full-length transcriptomes, systemic metabolomes, and gene expression profiles, setting the groundwork for further investigation of the C20-DAs biosynthesis pathway.

Catalytic promiscuity of O-methyltransferases from Corydalis yanhusuo leading to the structural diversity of benzylisoquinoline alkaloids.

O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, Corydalis yanhusuo, a plant used in traditional Chinese medicine due to the analgesic effects of its BIA-active compounds, was employed to analyze the catalytic characteristics of O-methyltransferases in the formation of BIA diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an O-methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4'- and 6-O-methylations of 1-BIAs was found. CyOMT6 was found to perform two sequential methylations at both 9- and 2-positions of the essential intermediate of tetrahydroprotoberberines, (S)-scoulerine. Two O-methyltransferases (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with the 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2-O-methyltransferase) and the 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, results of integrated phylogenetic molecular docking analysis and site-directed mutation suggested that residues at sites 172, 306, 313, and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at the 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of the 6-position and to expand substrate scopes. This work provides insight into O-methyltransferases in producing BIA diversity in C. yanhusuo and genetic elements for producing BIAs by metabolic engineering and synthetic biology.

Identification and characterization of two Isatis indigotica O-methyltransferases methylating C-glycosylflavonoids.

Isatis indigotica accumulates several active substances, including C-glycosylflavonoids, which have important pharmacological activities and health benefits. However, enzymes catalyzing the methylation step of C-glycosylflavonoids in I. indigotica remain unknown. In this study, three O-methyltransferases (OMTs) were identified from I. indigotica that have the capacity for O-methylation of the C-glycosylflavonoid isoorientin. The Type II OMTs IiOMT1 and IiOMT2 efficiently catalyze isoorientin to form isoscoparin, and decorate one of the aromatic vicinal hydroxyl groups on flavones and methylate the C6, C8, and 3'-hydroxyl positions to form oroxylin A, wogonin, and chrysoeriol, respectively. However, the Type I OMT IiOMT3 exhibited broader substrate promiscuity and methylated the C7 and 3'-hydroxyl positions of flavonoids. Further site-directed mutagenesis studies demonstrated that five amino acids of IiOMT1/IiOMT2 (D121/D100, D173/D149, A174/A150R, N200/N176, and D248/D233) were critical residues for their catalytic activity. Additionally, only transient overexpression of Type II OMTs IiOMT1 and IiOMT2 in Nicotiana benthamiana significantly increased isoscoparin accumulation, indicating that the Type II OMTs IiOMT1 and IiOMT2 could catalyze the methylation step of C-glycosylflavonoid, isoorientin at the 3'-hydroxyl position. This study provides insights into the biosynthesis of methylated C-glycosylflavonoids, and IiOMTs could be promising catalysts in the synthesis of bioactive compounds.

[Enzymatic characterization of lignan glucosyltransferase of Isatis indigotica].

The lignan glycosyltransferase UGT236(belonging to the UGT71 B family) from Isatis indigotica can catalyze the production of phloridzin from phloretin in vitro. UGT236 shares high identity with P2'GT from apple. In this study, the recombinant plasmid pET28 a-MBP-UGT236 was transferred into Escherichia coli Rosetta(DE3) cells and induced by isopropyl-ß-D-thiogalactoside(IPTG). The purified UGT236 protein was used for enzymatic characterization with phloretin as substrate. The results showed that UGT236 had the optimal reaction temperature of 40 ℃ and the optimal pH 8(Na_2HPO_4-NaH_2PO_4 system). The UGT236 activity was inhibited by Ni~(2+) and Al~(3+), enhanced by Fe~(2+), Co~(2+), and Mn~(2+), and did not affected by Mg~(2+), Ca~(2+), Li~+, Na~+, or K~+. The K_m, K_(cat), and K_(cat)/K_m of phloretin were 61.03 µmol·L~(-1), 0.01 s~(-1), and 157.11 mol~(-1)·s~(-1)·L, and those of UDPG were 183.6 µmol·L~(-1), 0.01 s~(-1), and 51.91 mol~(-1)·s~(-1)·L, respectively. The possible active sites were predicted by homologous modeling and molecular docking. By mutagenisis and catalytic activity detection, three key active sites, Glu391, His15, and Thr141, were identified, while Phe146 was related to product diversity. In summary, we found that the lignan glycosyltransferase UGT236 from I.indigotica could catalyze the reaction of phloretin into phloridzin. Several key amino acid residues were identified by structure prediction, molecular docking, and site-mutagenesis, which provided a basis for studying the specificity and diversity of phloretin glycoside products. This study can provide a reference for artificially producing glycosyltransferase elements with high efficiency and specific catalysis.

Network Pharmacology Research and Dual-omic Analyses Reveal the Molecular Mechanism of Natural Product Nodosin Inhibiting Muscle-Invasive Bladder Cancer in Vitro and in Vivo.

Bladder cancer, specifically, muscle-invasive bladder cancer (MIBC), is among the most common malignant tumors. Patients with MIBC who cannot tolerate standard drugs require novel treatments. Targeting apoptosis may help treat cancer, which may be achieved with the use of some natural products. Nodosin, found in Isodon serra (Maxim.) Kudo (known as Xihuangcao), may inhibit bladder cancer cells. Transcriptomics and proteomics dual-omic analyses revealed the network pharmacological mechanism: (1) blocking the S phase by up-regulating RPA2, CLSPN, MDC1, PDCD2L, and E2F6 gene expressions, suppressing cancer cell proliferation; (2) inducing apoptosis and autophagy and restraining ferroptosis by up-regulating HMOX1, G0S2, SQSTM1, FTL, SLC7A11, and AIFM2 gene expressions; (3) preventing cancer cell migration by down-regulating NEXN, LIMA1, CFL2, PALLD, and ITGA3 gene expressions. In vivo, nodosin inhibited bladder cancer cell growth in a model of xenograft tumor in nude mice. This study is the first to report basic research findings on the network pharmacological mechanism of cytotoxicity of bladder cancer cells by nodosin, providing novel evidence for the application of nodosin in the field of oncology; however, other mechanisms may be involved in the effects of nodosin for further research. These findings provide a foundation for the development of novel MIBC drugs.

Functional Characterization of a 2OGD Involved in Abietane-Type Diterpenoids Biosynthetic Pathway in Salvia miltiorrhiza.

Salvia miltiorrhiza is one of the most commonly used Chinese medicinal herbs. Tanshinones, the most abundant lipid-soluble bioactive constituents of S. miltiorrhiza, are a class of structural highly oxidized abietane-type diterpenoids with multiple pharmacological activities. Although several enzymes, including diterpene synthase, cytochrome P450, and Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD), have been functionally characterized in biosynthesis of abietane-type diterpenoids, the highly oxidized structure and complex secondary metabolic network of tanshinones imply that more oxidases should be characterized. Here, we identified a new 2OGD (Sm2OGD25) from S. miltiorrhiza. Molecular cloning and functional studies in vitro showed that Sm2OGD25 could catalyze the hydroxylation of sugiol at C-15 and C-16 positions to produce hypargenin B and crossogumerin C, respectively. The phylogenetic analysis of the DOXC family demonstrated that Sm2OGD25 belongs to the DOXC54 clade. Furthermore, structural modeling and site-directed mutagenesis characterization revealed the importance of the hydrogen-bonding residue Y339 and the hydrophobic residues (V122, F129, A144, A208, F303, and L344) in substrate binding and enzyme activity. This study will promote further studies on the catalytic characterization of plant 2OGDs and the secondary metabolic biosynthesis network of diterpenoids.

Functional Characterization of UDP-Glycosyltransferases Involved in Anti-viral Lignan Glycosides Biosynthesis in Isatis indigotica.

Isatis indigotica is a popular herbal medicine with its noticeable antiviral properties, which are primarily due to its lignan glycosides such as lariciresinol-4-O-ß-D-glucoside and lariciresinol-4,4'-bis-O-ß-D-glucosides (also called clemastanin B). UDP-glucose-dependent glycosyltransferases are the key enzymes involved in the biosynthesis of these antiviral metabolites. In this study, we systematically characterized the UGT72 family gene IiUGT1 and two UGT71B family genes, IiUGT4 and IiUGT71B5a, with similar enzymatic functions. Kinetic analysis showed that IiUGT4 was more efficient than IiUGT1 or IiUGT71B5a for the glycosylation of lariciresinol. Further knock-down and overexpression of these IiUGTs in I. indigotica's hairy roots indicates that they play different roles in planta: IiUGT71B5a primarily participates in the biosynthesis of coniferin not pinoresinol diglucoside, and IiUGT1 primarily participates in the biosynthesis of pinoresinol diglucoside, while IiUGT4 is responsible for the glycosylation of lariciresinol and plays a dominant role in the biosynthesis of lariciresinol glycosides in I. indigotica. Analysis of the molecular docking and site-mutagenesis of IiUGT4 have found that key residues for its catalytic activity are H373, W376, E397, and that F151 could be associated with substrate preference. This study elucidates the biosynthetic route of anti-viral lignan glycosides in I. indigotica, and provides the foundation for the production of anti-viral lignan glycosides via synthetic biology under the heterologous model.

The chromosome-scale assembly of endive (Cichorium endivia) genome provides insights into the sesquiterpenoid biosynthesis.

Endive (Cichorium endivia L.) is a leafy vegetable in the Asteraceae family. Sesquiterpene lactones (STLs) in endive leaves bring a bitter taste that varies between varieties. Despite their importance in breeding varieties with unique flavours, sesquiterpenoid biosynthesis pathways in endive are poorly understood. We assembled a chromosome-scale endive genome of 641 Mb with a contig N50 of 5.16 Mb and annotated 46,711 protein-coding genes. Several gene families, especially terpene synthases (TPS) genes, expanded significantly in the C. endivia genome. STLs biosynthesis-related genes and TPS genes in more bitter varieties have shown a higher level of expression, which could be attributed to genomic variations. Our results penetrate the origin and diversity of bitter taste and facilitate the molecular breeding of endive varieties with unique bitter tastes. The high-quality endive assembly would provide a reference genome for studying the evolution and diversity of Asteraceae.

TMT-Based Quantitative Proteomics Analysis Reveals the Panoramic Pharmacological Molecular Mechanism of ß-Elemonic Acid Inhibition of Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide but has limited available therapeutic methods; therefore, there is a need to develop highly efficient prevention and treatment strategies. Here, we investigated the anti-cancer activity of ß-elemonic acid (EA) in CRC in vitro and in vivo. Our results showed that EA inhibited cell proliferation and migration in the CRC cell lines SW480 and HCT116. Moreover, EA significantly suppressed the growth of transplanted colorectal tumors in nude mice. Interestingly, high-throughput tandem mass tag (TMT)-based quantitative proteomics indicated that EA mainly targets tumor mitochondria and attenuates the translation of 54 mitochondrial ribosome proteins, many of which are discovered significantly upregulated in clinical CRC patients. More interestingly, EA at a low concentration (lower than 15 µg/ml) repressed the cell cycle by downregulating CDK1, CDK6, and CDC20, whereas at a high concentration (higher than 15 µg/ml), caused a non-apoptotic cell death-ferroptosis via downregulating ferritin (FTL) and upregulating transferrin (TF), ferroxidase (CP), and acyl-CoA synthetase long-chain family member 4 (ACSL4). This is the first report on the panoramic molecular mechanism of EA against CRC, which would make great contributions to developing a novel drug for colorectal cancer therapy.

Diterpene synthases from Leonurus japonicus elucidate epoxy-bridge formation of spiro-labdane diterpenoids.

Spiro-9,13-epoxy-labdane diterpenoids are commonly found in Leonurus species, particularly in Leonurus japonicus Houtt., which is a medicinal herb of long-standing use in Asia and in which such spiro-heterocycles are present in at least 38 diterpenoids. Here, through generation of a transcriptome and functional characterization of six diterpene synthases (diTPSs) from L. japonicus, including three class II diTPSs (LjTPS1, LjTPS3, and LjTPS4) and three class I diTPSs (LjTPS5, LjTPS6, and LjTPS7), formation of the spiro-9,13-epoxy-labdane backbone was elucidated, along with identification of the relevant diTPSs for production of other labdane-related diterpenes. Similar to what has been found with diTPSs from other plant species, while LjTPS3 specifically produces the carbon-9 (C9) hydroxylated bicycle peregrinol diphosphate (PPP), the subsequently acting LjTPS6 yields a mixture of four products, largely labda-13(16),14-dien-9-ol, but with substantial amounts of viteagnusin D and the C13-S/R epimers of 9,13-epoxy-labda-14-ene. Notably, structure-function analysis identified a critical residue in LjTPS6 (I420) in which single site mutations enable specific production of the 13S epimer. Indeed, extensive mutagenesis demonstrated that LjTPS6:I420G reacts with PPP to both specifically and efficiently produce 9,13S-epoxy-labda-14-ene, providing a specialized synthase for further investigation of derived diterpenoid biosynthesis. The results reported here provide a strong foundation for future studies of the intriguing spiro-9,13-epoxy-labdane diterpenoid metabolism found in L. japonicus.

Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases.

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2-7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.

Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae.

Borneol is a precious monoterpenoid with two chiral structures, (-)-borneol and (+)-borneol. Bornyl diphosphate synthase is the key enzyme in the borneol biosynthesis pathway. Many (+)-bornyl diphosphate synthases have been reported, but no (-)-bornyl diphosphate synthases have been identified. Blumea balsamifera leaves are rich in borneol, almost all of which is (-)-borneol. In this study, we identified a high-efficiency (-)-bornyl diphosphate synthase (BbTPS3) from B. balsamifera that converts geranyl diphosphate (GPP) to (-)-bornyl diphosphate, which is then converted to (-)-borneol after dephosphorylation in vitro. BbTPS3 exhibited a K m value of 4.93 ± 1.38 µM for GPP, and the corresponding k cat value was 1.49 s-1. Multiple strategies were applied to obtain a high-yielding (-)-borneol producing yeast strain. A codon-optimized BbTPS3 protein was introduced into the GPP high-yield strain MD, and the resulting MD-B1 strain produced 1.24 mg·L-1 (-)-borneol. After truncating the N-terminus of BbTPS3 and adding a Kozak sequence, the (-)-borneol yield was further improved by 4-fold to 4.87 mg·L-1. Moreover, the (-)-borneol yield was improved by expressing the fusion protein module of ERG20F96W-N127W-YRSQI-t14-BbTPS3K2, resulting in a final yield of 12.68 mg·L-1 in shake flasks and 148.59 mg·L-1 in a 5-L bioreactor. This work is the first reported attempt to produce (-)-borneol by microbial fermentation.