The Zinc-Finger Protein ZCCHC3 Binds RNA and Facilitates Viral RNA Sensing and Activation of the RIG-I-like Receptors.

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiates innate antiviral immune response. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified ZCCHC3 as a positive regulator of the RLRs including RIG-I and MDA5. ZCCHC3 deficiency markedly inhibited RNA virus-triggered induction of downstream antiviral genes, and ZCCHC3-deficient mice were more susceptible to RNA virus infection. ZCCHC3 was associated with RIG-I and MDA5 and functions in two distinct processes for regulation of RIG-I and MDA5 activities. ZCCHC3 bound to dsRNA and enhanced the binding of RIG-I and MDA5 to dsRNA. ZCCHC3 also recruited the E3 ubiquitin ligase TRIM25 to the RIG-I and MDA5 complexes to facilitate its K63-linked polyubiquitination and activation. Thus, ZCCHC3 is a co-receptor for RIG-I and MDA5, which is critical for RLR-mediated innate immune response to RNA virus.

Gene identification and semisynthesis of the anti-inflammatory oleanane-type triterpenoid wilforlide A.

Wilforlide A is one of the main active constituents produced in trace amounts in Tripterygium wilfordii Hook F, which has excellent anti-inflammatory and immune suppressive effects. Despite the seeming structural simplicity of the compound, the biosynthetic pathway of wilforlide A remains unknown. Gene-specific expression analysis and genome mining were used to identify the gene candidates, and their functions were studied in vitro and in vivo. A modularized two-step (M2S) technique and CRISPR-Cas9 methods were used to construct engineering yeast. Here, we identified a cytochrome P450, TwCYP82AS1, that catalyses C-22 hydroxylation during wilforlide A biosynthesis. We also found that TwCYP712K1 to K3 can further oxidize the C-29 carboxylation of oleanane-type triterpenes in addition to friedelane-type triterpenes. Reconstitution of the biosynthetic pathway in engineered yeast increased the precursor supply, and combining TwCYP82AS1 and TwCYP712Ks produced abrusgenic acid, which was briefly acidified to achieve the semisynthesis of wilforlide A. Our work presents an alternative metabolic engineering approach for obtaining wilforlide A without relying on extraction from plants.

A novel sterol glycosyltransferase catalyses steroidal sapogenin 3-O glucosylation from Paris polyphylla var. yunnanensis.

BACKGROUND: Paris polyphylla var. yunnanensis is an important medicinal plant, and the main active ingredient of the plant is polyphyllin, which is a steroid saponin with pharmacological activities. The central enzyme genes participating in the biosynthesis of polyphyllin are increasingly being uncovered; however, UGTs are rarely illustrated. METHODS AND RESULTS: In this study, we cloned a new sterol glycosyltransferase from Paris polyphylla var. yunnanensis and identified its catalytic function in vitro. PpUGT6 showed the ability to catalyse the C-3 glycosylation of pennogenin sapogenin of polyphyllin, and PpUGT6 showed catalytic promiscuity towards steroids at the C-17 position of testosterone and methyltestosterone and the triterpene at the C-3 position of glycyrrhetinic acid. Homology modelling of the PpUGT6 protein and virtual molecular docking of PpUGT6 with sugar acceptors and donors were performed, and we predicted the key residues interacting with ligands. CONCLUSIONS: Here, PpUGT6, a novel sterol glycosyltransferase related to the biosynthesis of polyphyllin from P. polyphylla, was characterized. PpUGT6 catalysed C-3 glycosylation to pennogenin sapogenin of polyphyllin, which is the first glycosylation step of the biosynthetic pathway of polyphyllins. Interestingly, PpUGT6 demonstrated glycodiversification to testosterone and methyltestosterone at C-17 and triterpene of glycyrrhetinic acid at the C-3 position. The virtual molecular docking of PpUGT6 protein with ligands predicted the key residues interacting with them. This work characterized a novel SGT glycosylating pennogenin sapogenin at C-3 of polyphyllin from P. polyphylla and provided a reference for further elucidation of the phytosterol glycosyltransferases in catalytic promiscuity and key residues interacting with substrates.

CYP72D19 from Tripterygium wilfordii catalyzes C-2 hydroxylation of abietane-type diterpenoids.

KEY MESSAGE: CYP72D19, the first functional gene of the CYP72D subfamily, catalyzes the C-2 hydroxylation of abietane-type diterpenoids. The abietane-type diterpenoids, e.g., triptolide, tripdiolide, and 2-epitripdiolide, are the main natural products for the anti-tumor, anti-inflammatory, and immunosuppressive activities of Tripterygium wilfordii, while their biosynthetic pathways are not resolved. Here, we cloned and characterized the CYP72D19-catalyzed C-2 hydroxylation of dehydroabietic acid, a compound that has been proven to be a biosynthetic intermediate in triptolide biosynthesis. Through molecular docking and site-directed mutagenesis, L386, L387, and I493 near the active pocket were found to have an important effect on the enzyme activity, which also indicates that steric hindrance of residues plays an important role in function. In addition, CYP72D19 also catalyzed a variety of abietane-type diterpenoids with benzene ring, presumably because the benzene ring of the substrate molecule stabilized the C-ring, allowing the protein and the substrate to form a relatively stable spatial structure. This is the first demonstration of CYP72D subfamily gene function. Our research provides important genetic elements for the structural modification of active ingredients and the heterologous production of other 2-hydroxyl abietane-type natural products.

[Gene clone and functional identification of sterol glycosyltransferases from Paris polyphylla var. yunnanensis].

In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl ß-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.

A cycloartenol synthase from the steroidal saponin biosynthesis pathway of Paris polyphylla.

Steroidal saponins named polyphyllin are the major active components of Paris polyphylla. Cycloartenol synthase (CAS) is a key enzyme that catalyzes the formation of the sterol scaffold. In this study, we cloned a putative CAS gene from Paris polyphylla. Heterologous expression in yeast indicated that PpCAS can convert 2,3-oxidosqualene into cycloartenol. qRT-PCR analysis showed that the expression of PpCAS was highest in leaves and lowest in roots. To our best knowledge, this is the first report of the functional characterization of cycloartenol synthase from Paris polyphylla, which lays the foundation for further analysis of the biosynthesis pathway of polyphyllins.[Formula: see text].

[Effect of ERK/JNK cell signaling pathway in carbon black induced cytokine IL-6 and IL-8 expression changes].

OBJECTIVE: To investigate the roles of extracellular signal-regulated kinase(ERK)/c-Jun amino-terminal kinase(JNK) signaling pathway on the expression of interleukin-6(IL-6) and interleukin-8(IL-8) in human embryonic lung fibroblasts(HELF) induced by carbon black. METHODS: HELFs were cultured in RPMI-1640 medium containing 0, 15, 30, 60, 120 or 240 µg/mL carbon black for 24 h, and the appropriate dose of carbon black was determined by MTT assay result HELFs were divided into three groups: HELFs, HELFs transfected with ERK dominant negative mutant plasmid(DN-ERK) and HELFs transfected with JNK dominant negative mutant plasmid(DN-JNK). 100 µg/mL carbon black was used to treat HELFs(CB), DN-ERK HELFs(CB-DN-ERK), DN-JNK HELFs(CB-DN-JNK), and HELFs without any black carbon treatment were considered as control group. At 16 h after carbon black treatment, scanning electron microscope(SEM) was used to observe HELFs morphology and whether there were carbon black particless. At 0, 1, 2, 4, 8, 12, 24 and 36 h, the enzyme linked immunosorbent assay(ELISA) was used to detect CB and control groups HELFs IL-6 and IL-8 expression levels, whereas CB-DN-ERK and CB-DN-JNK HELFs were detected only at 24 h. RESULTS: SEM result showed no carbon black particles were observed in CB group HELFs, whereas their surface projections were increased. The CB group HELFs IL-6 expression levels at 2 h(44. 86±3. 65 ng/L) and 4 h(76. 52±3. 15 ng/L) were significantly lower than those of the control group(96. 78±2. 82 and 147. 32±3. 26 ng/L)(P<0. 05), whereas the IL-6 expression levels were significantly higher than those of the control group(105. 54±6. 10, 101. 27±5. 84 and 97. 15±5. 12 ng/L) at 16 h(202. 64±7. 20 ng/L), 24 h(200. 38±6. 20 ng/L) and 36 h(183. 54±4. 54 ng/L)(P<0. 001). At 24 h(136. 75±3. 81 ng/L) and 36 h(149. 12±2. 74 ng/L), the CB group IL-8 expression levels were significantly higher than those of the control group(75. 16±2. 84 and 73. 44±2. 15 ng/L)(P<0. 001). Compared with CB group HELFs, CB-DN-ERK and CB-DN-JNK groups HELFs had significantly lower IL-6 and IL-8 expression levels(P<0. 05). CONCLUSION: While carbon black induced HELFs IL-6 and IL-8 expression levels changes, ERK and JNK may upregulate IL-6 and IL-8 expression levels.

[Roles of ERK/JNK in carbon black induced AP-1 cell signaling pathway changes].

OBJECTIVE: To investigate the role of ERK/JNK in the alteration of activator protein-1(AP-1) signaling pathway in human embryonic lung fibroblasts(HELFs) induced by carbon black. METHODS: HELFs were cultured in RPMI 1640 medium containing 0, 15, 30, 60, 120 or 240 µg/mL carbon black for 24 h, and the appropriate dose of carbon black was determined by MTT assay result. HELFs were divided into three groups: HELFs, HELFs transfected with ERK dominant negative mutant plasmid(DN-ERK) and HELFs transfected with JNK dominant negative mutant plasmid(DN-JNK). 100 µg/mL carbon black was used to treat HELFs(CB), DN-ERK HELFs(CB-DN-ERK), DN-JNK HELFs(CB-DN-JNK), and HELFs without any treatment were considered as control group. At 0, 1, 2, 4, 8, 12, 24 and 36 h of CB and control groups HELFs, the western blot was used to detect ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels, and AP-1 activity was detected by luciferase method. Whereas CB-DN-ERK and CB-DN-JNK HELFs were detected only at 24 h. RESULTS: Compared with the protein expression levels at 0 h, CB group HELFs ERK and p-ERK protein expression increased at each time point, whereas p38 protein expression decreased. AP-1 activity of CB group HELFs was declined to the lowest at 8 h(0.72±0.12), and upregulated to the peak at 36 h(1.38±0.11). CB group HELFs c-Fos, p-c-Fos and c-Jun protein expression levels at each time point from 1 h to 24 h were greater than those of 0 h, and p-c-Jun protein expression levels at 1 h, 2 h, 4 h, 8 h, 36 h were also greater than those of 0 h. CB group HELFs AP-1 activity, ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels changes followed biphasic patterns. There were no statistically significant differences in AP-1 activity between CB group HELFs(1.03±0.10) and CB-DN-ERK group(1.02±0.04) or CB-DN-JNK group(1.09±0.10) HELFs(t=0.16, P=0.88; t=0.73, P=0.50). However, compared with CB group HELFs, c-Fos(t=5.31, P=0.01), p-c-Fos(t=4.33, P=0.01), p-c-Jun(t=10.95, P& lt; 0.01)in CB-DN-JNK group, and c-Fos protein expression levels in CB-DN-ERK group(t=42.72, P& lt; 0.01)were significantly decreased. CONCLUSION: While carbon black induces HELFs increased protein expression levels of ERK, p-ERK, c-Jun, p-c-Jun, c-Fos and p-c-Fos, JNK may upregulate c-Fos, p-c-Fos, p-c-Jun protein expression levels, and ERK may upregulate c-Fos protein expression level.

SNX8 modulates innate immune response to DNA virus by mediating trafficking and activation of MITA.

MITA (also called STING) is a central adaptor protein in innate immune response to cytosolic DNA. Cellular trafficking of MITA from the ER to perinuclear microsomes after DNA virus infection is critical for MITA activation and onset of innate antiviral response. Here we found that SNX8 is a component of DNA-triggered induction of downstream effector genes and innate immune response. Snx8-/- mice infected with the DNA virus HSV-1 exhibited lower serum cytokine levels and higher viral titers in the brains, resulting in higher lethality. Mechanistically, SNX8 recruited the class III phosphatylinositol 3-kinase VPS34 to MITA, which is required for trafficking of MITA from the ER to perinuclear microsomes. Our findings suggest that SNX8 is a critical component in innate immune response to cytosolic DNA and DNA virus.

Utility of CT assessment in hematology patients with invasive aspergillosis: a post-hoc analysis of phase 3 data.

BACKGROUND: Pulmonary computed tomography (CT) scans are commonly used as part of the clinical criteria in diagnostic workup of invasive fungal diseases like invasive aspergillosis, and may identify radiographic abnormalities, such as halo signs or air-crescent signs. We assessed the diagnostic utility of CT assessment in patients with hematologic malignancies or those who had undergone allogeneic hematopoietic stem cell transplantation in whom invasive aspergillosis was suspected. METHODS: This post-hoc analysis assessed data from a prospective, multicenter, international trial of voriconazole (with and without anidulafungin) in patients with suspected invasive aspergillosis (IA; proven, probable, or possible, using 2008 European Organisation for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group criteria) [NCT00531479]. Eligible patients received at least one baseline lung CT scan. RESULTS: Of 395 patients included in this post-hoc analysis, 240 patients (60.8%) had 'confirmed' proven (9/240, 3.8%) or probable (231/240, 96.3%) invasive aspergillosis (cIA) and 155 patients (39.2%) had 'non-confirmed' invasive aspergillosis (all nIA; all possible IA (de Pauw et al., Clin Infect Dis 46:1813-21, 2008)). Mean age was 52.3 and 50.5 years, 56.3 and 60.0% of patients were male, and most patients were white (71.7 and 71.0%) in the cIA and nIA populations, respectively. Median baseline galactomannan was 1.4 (cIA) and 0.2 (nIA), mean Karnofsky score was 65.3 (cIA) and 66.8 (nIA), and mean baseline platelet count was 48.0 (cIA) and 314.1 (nIA). Pulmonary nodules (46.8% of all patients), bilateral lung lesions (37.5%), unilateral lung lesions (28.4%), and consolidation (24.8%) were the most common radiographic abnormalities. Ground-glass attenuation (cIA: 24.2%; nIA: 11.6%; P < 0.01) and pulmonary nodules (cIA: 52.5%; nIA: 38.1%; P < 0.01) were associated with cIA. Other chest CT scan abnormalities (including halo signs and air-crescent signs) at baseline in patients with hematologic malignancy or hematopoietic stem cell transplantation, and suspected IA, were not associated with cIA. CONCLUSIONS: These findings highlight the limitations in the sensitivity of chest CT scans for the diagnosis of IA, and reinforce the importance of incorporating other available clinical data to guide management decisions on individual patients, including whether empirical treatment is reasonable, pending full evaluation. TRIAL REGISTRATION: NCT00531479 (First posted on ClinicalTrials.gov on September 18, 2007).

Eudesmane-type sesquiterpene diols directly synthesized by a sesquiterpene cyclase in Tripterygium wilfordii.

Cryptomeridiol, a typical eudesmane diol, is the active principle component of the antispasmodic Proximol. Although it has been used for many years, the biosynthesis pathway of cryptomeridiol has remained blur. Among terpenoid natural products, terpenoid cyclases are responsible for cyclization and generation of hydrocarbon backbones. The cyclization is mediated by carbocationic cascades and ultimately terminated via deprotonation or nucleophilic capture. Isoprene precursors are, respectively, converted into hydrocarbons or hydroxylated backbones. A sesquiterpene cyclase in Tripterygium wilfordii (TwCS) was determined to directly catalyze (E,E)-farnesyl pyrophosphate (FPP) to unexpected eudesmane diols, primarily cryptomeridiol. The function of TwCS was characterized by a modular pathway engineering system in Saccharomyces cerevisiae The major product determined by NMR spectroscopy turned out to be cryptomeridiol. This unprecedented production was further investigated in vitro, which verified that TwCS can directly produce eudesmane diols from FPP. Some key residues for TwCS catalysis were screened depending on the molecular model of TwCS and mutagenesis studies. As cryptomeridiol showed a small amount of volatile and medicinal properties, the biosynthesis of cryptomeridiol was reconstructed in S. cerevisiae Optimized assays including modular pathway engineering and the CRISPR-cas9 system were successfully used to improve the yield of cryptomeridiol in the S. cerevisiae The best engineered strain TE9 (BY4741 erg9::Δ-200-176 rox1::mut/pYX212-IDI + TwCS/p424-tHMG1) ultimately produced 19.73 mg/l cryptomeridiol in a shake flask culture.

Comparison of Phytochemical Differences of the Pulp of Different Peach [Prunus persica (L.) Batsch] Cultivars with Alpha-Glucosidase Inhibitory Activity Variations in China Using UPLC-Q-TOF/MS.

In order to fully understand the variation of the fruit alpha-glucosidase inhibitory activity-related phytochemical basis in the Chinese peach [Prunus persica (L.) Batsch], mature fruit from 33 cultivars was used for the investigation of fruit phenolic phytochemical attributes, including total phenolics, flavonoids, anthocyanins, and procyanidins, as well as the alpha-glucosidase inhibitory activity in vitro. Alpha-glucosidase inhibitory activity varied significantly among tested peach cultivars and was strongly correlated with total phenolics, total procyanidins, and total flavonoids. Untargeted UPLC-Q-TOF/MS-based metabolomics were used to comprehensively discriminate between peaches with different inhibitory activity on alpha-glucosidase. Principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA) were used for this process. Twenty-three differential compounds were identified between peach cultivars with high and low alpha-glucosidase inhibitory activity, and nine, including procyanidin C1, procyanidin trimer isomer 1, procyanidin trimer isomer 2, procyanidin B1, procyanidin dimer, epicatechin-epicatechin-epicatechin, phloridzin, kaempferol 3-(2'',6''-di-(E)-p-coumarylglucoside), and luteolin 3'-methyl ether 7-malonylglucoside, were identified as marker compounds responsible for the discrimination. Overall, variations in metabolites in peach pulp reflect the diversity in peach germplasm, and these nine compounds are good candidate markers for future genetic breeding of peach fruit with high alpha-glucosidase inhibitory activity.

[Cloning and characterization of a monoterpene synthase gene from Tripterygium wilfordii].

Tripterygium wilfordii is a medicinal plant commonly used in the treatment of rheumatoid arthritis,and with pharmacological activities in anti-tumor and obesity treatment. The known active ingredients in T. wilfordii are mainly terpenoids,but with very low content. Therefore,the analysis of the biosynthesis pathway of terpenoids in T. wilfordii has become a research hotspot to solve the problem of its resources. Terpenoid synthase( TPS) is a key enzyme that catalyzes the formation of a wide variety of terpenoid skeletons. In this study,a gene fragment with an ORF of 1 785 bp was cloned from T. wilfordii. Bioinformatics analysis was performed using NCBI's BLASTP,ProtParam and Interpro online tools and MEGA 6.0 software. The response of this gene to methyl jasmonate was also detected by real-time fluorescent quantitative PCR,and its catalytic function was verified by prokaryotic expression and in vitro enzymatic assay. Bioinformatics analysis indicated that the amino acid sequence encoded by this gene had both N-terminal domain and C-terminal domain of TPS,as well as the DDxx D conserved domain of the class I of TPS family. And Tw MTS gathered together with TPS-b subfamily in the Neighbor-Joining Tree constructed with known homologous TPSs. The results of RT-PCR showed that 50 µmol·L-1 MeJA 12 h could increase the expression of Tw MTS to 735 times in the control group at 12 h,and 1 644 times at 24 h. In addition,in vitro enzymatic reaction results showed that Tw MTS can catalyze the production of ß-citronellol with GPP as substrate,indicating that Tw MTS was a monoterpene synthase. The above results provided a new element for the synthetic biology database of T. wilfordii terpenoids,and laid the foundation for future biosynthesis research.

Cloning and functional analysis of two sterol-C24-methyltransferase 1 (SMT1) genes from Paris polyphylla.

The biosynthetic pathways of phytosterols and steroidal saponins are located in two adjacent branches which share cycloartenol as substrate. The rate-limiting enzyme S-adenosyl-L-methionine-sterol-C24-methyltransferase 1 (SMT1) facilitates the metabolic flux toward phytosterols. It catalyzes the methylation of the cycloartenol in the side chain of the C24-alkyl group, to generate 24(28)-methylene cycloartenol. In this study, we obtained two full-length sequences of SMT1 genes from Pari polyphylla, designated PpSMT1-1 and PpSMT1-2. The full-length cDNA of PpSMT1-1 was 1369 bp long with an open reading frame (ORF) of 1038 bp, while the PpSMT1-2 had a length of 1222 bp, with a 1005 bp ORF. Bioinformatics analysis confirmed that the two cloned SMTs belong to the SMT1 family. The predicted function was further validated by performing in vitro enzymatic reactions, and the results showed that PpSMT1-1 encodes a cycloartenol-C24-methyltransferase, which catalyzes the conversion of cycloartenol to 24-methylene cycloartenol, whereas PpSMT1-2 lacked this catalytic activity. The tissue expression patterns of the two SMTs revealed differential expression in different organs of Paris polyphylla plants of different developmental stage and age. These results lay the foundation for detailed genetic studies of the biosynthetic pathways of steroid compounds, which constitute the main class of active substances found in P. polyphylla.

[DNA molecular identification of Manis pentadactyla].

The study was aimed to establish a stable, accurate site specific PCR identification system to identify Manis pentadactyla and its adulterants using DNA molecular identification. The genomic DNA was extracted from experimental samples using the DNA extraction kit. The Cytb and CO Ⅰ genes were amplified using PCR and sequenced bi-directionally. Obtained sequences were assembled using the BioEdit software. The neighbor-joining tree was constructed by MEGA 6.0. Specific identification primers were designed according to the specific allelets, and PCR reaction system was optimized. The results indicated that the Cytb and CO Ⅰ sequence both were able to be used to identify M. pentadactyla and its adulterants. With the specific primers CO Ⅰ-S10/A5, the M. pentadactyla could be amplified a 400 bp DNA band when the annealing temperature ranged from 55 to 60 ℃ and the amount of DNA template ranged from 3 to 100 ng within 35 PCR cycles. However, other adulterants displayed no relevant bands. So that primers CO Ⅰ- S10 / A5 can be used to identify the M. pentadactyla with the adulterants.

[Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii].

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

Cloning and functional characterization of an isopentenyl diphosphate isomerase gene from Tripterygium wilfordii.

Tripterygium wilfordii Hook.F. is one of the most valuable medicinal plants because it contains a large variety of active terpenoid compounds, including triptolide, celastrol, and wilforlide. All of the pharmacologically active secondary metabolites are synthesized from the 2-C-methyl-d-erythritol 4-phosphate and mevalonate pathway in the isoprenoid biosynthetic system. The key step in this pathway is the isomerization of dimethylallyl diphosphate and isopentenyl diphosphate, which is catalyzed by isopentenyl diphosphate isomerase (IPI). In the present study, a full-length cDNA encoding IPI (designate as TwIPI, GenBank accession no.KT279355) was cloned from a suspension of cultured cells from T. wilfordii. The full-length cDNA of TwIPI was 1,564 bp and encoded a polypeptide of 288 amino acids. The bioinformatics analysis showed that the deduced TwIPI sequence contained the TNTCCSHPL and WGEHELDY motif. The transcription level of the TwIPI in the suspension cells increased almost fivefold after treatment with methyl jasmonate as an elicitor. A functional color assay in Escherichia coli indicated that TwIPI could promote the accumulation of lycopene and encoded a functional protein.

CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.

Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stable-isotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.

Purification of Flavonoids from Chinese Bayberry (Morella rubra Sieb. et Zucc.) Fruit Extracts and α-Glucosidase Inhibitory Activities of Different Fractionations.

Chinese bayberry (Morella rubra Sieb. et Zucc.) fruit have a diverse flavonoid composition responsible for the various medicinal activities, including anti-diabetes. In the present study, efficient simultaneous purification of four flavonoid glycosides, i.e., cyanidin-3-O-glucoside (1), myricetin-3-O-rhamnoside (2), quercetin-3-O-galactoside (3), quercetin-3-O-rhamnoside (4), from Chinese bayberry pulp was established by the combination of solid phase extract (SPE) by C18 Sep-Pak(®) cartridge column chromatography and semi-preparative HPLC (Prep-HPLC), which was followed by HPLC and LC-MS identification. The purified flavonoid glycosides, as well as different fractions of fruit extracts of six bayberry cultivars, were investigated for α-glucosidase inhibitory activities. The flavonol extracts (50% methanol elution fraction) of six cultivars showed strong α-glucosidase inhibitory activities (IC50 = 15.4-69.5 µg/mL), which were higher than that of positive control acarbose (IC50 = 383.2 µg/mL). Four purified compounds 1-4 exerted α-glucosidase inhibitory activities, with IC50 values of 1444.3 µg/mL, 418.8 µg/mL, 556.4 µg/mL, and 491.8 µg/mL, respectively. Such results may provide important evidence for the potential anti-diabetic activity of different cultivars of Chinese bayberry fruit and the possible bioactive compounds involved.

[Cloning and protein expression of sterol-C-24-methyl transferase 2 in Tripterygium wilfordii].

24-Alkyl sterols are the major players in the control of membrane component and plant growth. In this paper, we cloned an important rate-limiting enzyme: sterol-C-24-methyl transferase (SMT) in the sterol biosynthetic pathway according to the transcriptome data of Tripterygium wilfordii. suspension cells, whose full-length cDNA was 1 631 bp with an open reading frame of 1 080 bp, encoding a protein of 359 amino acids. It was estimated that theoretical isoelectric point (p I) was 6.43 and the molecular mass was 40.0 kDa. Bioinformatics analysis attributed the SMT gene to SMT2 family. The expression vector was constructed as the pMAL-c2x-TwSMT2 plasmid and the recombinant protein was expressed in E. coil BL21(DE3) competent cells. After methyl jasmonate treatment, the relative expression level of Tw SMT2 has improved significantly in 24 h. SDS-PAGE electrophoresis and Western Blot showed that protein of TwSMT2 in BL21 (DE3) strain was expressed after induction by IPTG. In this study, TwSMT2 was cloned for the first time and the recombinant protein was expressed, which lay the foundation for elucidation of the sterol biosynthetic pathway of Tripterygium wilfordii in the future.