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Archangium gephyra KYC5002 produces tubulysins during the death phase. In this study, we aimed to determine whether dead cells produce tubulysins. Cells were cultured for three days until the verge of the death phase, disrupted via ultrasonication, incubated for 2 h, and examined for tubulysin production. Non-disrupted cells produced 0.14 mg/L of tubulysin A and 0.11 mg/L of tubulysin B. Notably, tubulysin A production was increased by 4.4-fold to 0.62 mg/L and that of tubulysin B was increased by 6.7-fold to 0.74 mg/L in the disrupted cells. The same increase in tubulysin production was observed when the cells were killed by adding hydrogen peroxide. However, when the enzymes were inactivated via heat treatment of the cultures at 65 °C for 30 min, no significant increase in tubulysin production due to cell death was observed. Reverse transcription-quantitative polymerase chain reaction analysis of tubB mRNA revealed that the expression levels of tubulysin biosynthetic enzyme genes increased during the death phase compared to those during the vegetative growth phase. Our findings suggest that A. gephyra produces biosynthetic enzymes and subsequently uses them for tubulysin production in the cell death phase or during cell lysis by predators.

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Carbon tetrachloride (CCl4) is a widely used hepatotoxin for the studies of liver fibrosis and cirrhosis, and taurine has function to abate liver fibrosis induced by CCl4. But the interacting mechanisms between taurine and CCl4 in liver are still largely unknown. These made us to hypothesize that CCl4 may induce liver fibrosis by affecting the expressions of taurine biosynthetic enzymes and taurine synthesis. We thus assayed the expressions of hepatic cysteine dioxygenase (CDO), cysteine sulfonate acid decarboxylase (CSAD) and taurine transporter (TauT) in the progression of mouse liver fibrosis induced by CCl4. The results demonstrated that CCl4 treatment markedly decreased hepatic CSAD, CDO expressions, and taurine levels in hepatic tissue, although TauT expression did not exhibit significant decline. It was contrasting that hepatic α-SMA, serum AST, ALT, ALP kept increasing, which were accompanied by the pathological characters of liver, whereas taurine supplement attenuated the progression of liver fibrosis induced by CCl4. These results demonstrate that CCl4 may induce liver fibrosis by inhibiting hepatic CSAD and CDO expressions and taurine synthesis, which are crucial for our understanding the mechanisms of liver fibrosis induced by CCl4, and also potential for establishing therapeutic strategies of liver fibrosis and related diseases.

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Melatonin is widely involved in plant disease resistance through modulation of immune responses. Pathogenesis-related (PR) proteins play important roles in plant immune responses. However, the direct association between melatonin biosynthetic enzyme and PR protein remains elusive in plants. In this study, we found that N-acetylserotonin O-methyltransferase 2 (MeASMT2) physically interacted with MePR1 in vitro and in vivo, thereby promoting the anti-bacterial activity of MePR1 against Xanthomonas axonopodis pv. manihotis (Xam). Consistently, MeASMT2 improved the effect of MePR1 on positively regulating cassava disease resistance. In addition, we found that type 2C protein phosphatase 1 (MePP2C1) interacted with MeASMT2 to interfere with MePR1-MeASMT2 interaction, so as to inhibiting the effect of MeASMT2 and MePR1 on positively regulating cassava disease resistance. In contrast to the increased transcripts of MeASMT2 and MePR1 in response to Xam infection, the transcript of MePP2C1 was decreased upon Xam infection. Therefore, disease activated MeASMT2 was released from disease inhibited MePP2C1, so as to improving the anti-bacterial activity of MePR1, resulting in improved immune response. In summary, this study illustrates the dynamic modulation of the MePP2C1-MeASMT2-MePR1 module on cassava defense response against cassava bacterial blight (CBB), extending the understanding of the correlation between melatonin biosynthetic enzyme and PR in plants.

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Flavonoids are an important class of secondary metabolites widely found in plants, contributing to plant growth and development and having prominent applications in food and medicine. The biosynthesis of flavonoids has long been the focus of intense research in plant biology. Flavonoids are derived from the phenylpropanoid metabolic pathway, and have a basic structure that comprises a C15 benzene ring structure of C6-C3-C6. Over recent decades, a considerable number of studies have been directed at elucidating the mechanisms involved in flavonoid biosynthesis in plants. In this review, we systematically summarize the flavonoid biosynthetic pathway. We further assemble an exhaustive map of flavonoid biosynthesis in plants comprising eight branches (stilbene, aurone, flavone, isoflavone, flavonol, phlobaphene, proanthocyanidin, and anthocyanin biosynthesis) and four important intermediate metabolites (chalcone, flavanone, dihydroflavonol, and leucoanthocyanidin). This review affords a comprehensive overview of the current knowledge regarding flavonoid biosynthesis, and provides the theoretical basis for further elucidating the pathways involved in the biosynthesis of flavonoids, which will aid in better understanding their functions and potential uses.

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Cannabis sativa L. is an annual herb oldest cultivated plants as a source of fiber since about 5000 B.C. On the other hand, the cannabis flower and seed are listed in Shennong's classic Materia Medica approximately 2000 years ago. The formulas prescribed with cannabis in Kampo medicine have been summarized. Cannabidiol (CBD) and tetrahydrocannabinol (THC) are the major neurological and psychiatric cannabinoids, and develop to drugs. It becomes evident that the therapeutic CBD and/or THC are the important candidate of anti-dementia drugs having different mechanism for Alzheimer's patients. Two receptors and endocannabinoids are also discussed for underlying mechanism of action. In order to promote the breeding of cannabis plant containing higher concentration of target cannabinoid the biosynthetic enzymes were isolated, cloning and the tertiary structure of THCA synthase determined by x-ray analysis resulting in the possibility of molecular breeding for cannabinoids.

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The role of melatonin biosynthetic enzymes has been well studied. However, the transcriptional regulation of melatonin biosynthetic enzymes and their integrative crosstalk with other signaling pathways remain elusive. Here, we summarize recent progress in the functional analysis of melatonin biosynthetic enzymes and the major sites of melatonin synthesis in plants. We focus on the dual roles of melatonin biosynthetic enzymes in melatonin biosynthesis and in the crosstalk between melatonin and autophagy, antioxidant signaling, and stress responses in cassava. We highlight the transcriptional regulation and integrative protein complex of melatonin biosynthetic enzymes, and then raise the challenge of uncovering their precise regulation and crosstalk.

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Acute intermittent porphyria (AIP) is a rare hereditary metabolic disease with an autosomal dominant mode of inheritance. Germline mutations of HMBS gene causes AIP. Mutation of HMBS gene results into the partial deficiency of the heme biosynthetic enzyme hydroxymethylbilane synthase. AIP is clinically manifested with abdominal pain, vomiting, and neurological complaints. Additionally, an extreme phenotypic heterogeneity has been reported in AIP patients with mutations in HMBS gene. Here, we investigated a Chinese patient with AIP. The proband is a 28-year-old Chinese male manifested with severe stomach ache, constipation, nausea and depression. Proband's father and mother is normal. Proband's blood sample was collected and genomic DNA was extracted. Whole exome sequencing and Sanger sequencing identified a heterozygous novel single nucleotide deletion (c.809delC) in exon 12 of HMBS gene in the proband. This mutation leads to frameshift followed by formation of a truncated (p.Ala270Valfs∗2) HMBS protein with 272 amino acids comparing with the wild type HMBS protein of 361 amino acids. This mutation has not been found in proband's unaffected parents as well as in 100 healthy normal control. According to the variant interpretation guidelines of American College of Medical Genetics and Genomics (ACMG), this variant is classified as "likely pathogenic" variant. Our findings expand the mutational spectra of HMBS gene related AIP which are significant for screening and genetic diagnosis for AIP.

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The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.

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California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active benzylisoquinoline alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3'-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis.

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Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

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Anthocyanins are important pigments and are responsible for red coloration in plums. However, little is known about the molecular mechanisms underlying anthocyanin accumulation in plum fruits. In this study, the RNA-seq technique was used to analyze the transcriptomic changes during fruit ripening in the red-fleshed plum (Prunus salicina Lindl.) cultivar 'Furongli'. Over 161 million high-quality reads were assembled into 52,093 unigenes and 49.4% of these were annotated using public databases. Of these, 25,681 unigenes had significant hits to the sequences in the NCBI Nr database, 17,203 unigenes showed significant similarity to known proteins in the Swiss-Prot database and 5816 and 8585 unigenes had significant similarity to existing sequences in the Kyoto Encyclopedia of Genes and Genomes and the Cluster of Orthologous Groups databases, respectively. A total of 3548 unigenes were differentially expressed during fruit ripening and 119 of these were annotated as involved in "biosynthesis of other secondary metabolites." Biological pathway analysis and gene ontology term enrichment analysis revealed that 13 differentially expressed genes are involved in anthocyanin biosynthesis. Furthermore, transcription factors such as MYB and bHLH, which may control anthocyanin biosynthesis, were identified through coexpression analysis of transcription factors, and structural genes. Real-time qPCR analysis of candidate genes showed good correlation with the transcriptome data. These results contribute to our understanding of the molecular mechanisms underlying anthocyanin biosynthesis in plum flesh. The transcriptomic data generated in this study provide a basis for further studies of fruit ripening in plum.

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AIMS: One of six heparin biosynthetic enzymes, cloned and expressed in Escherichia coli as a soluble fusion protein, requires large-scale preparation for use in the chemoenzymatic synthesis of heparin, an important anticoagulant drug. METHODS AND RESULTS: The 6-O-sulfotransferase isoform-3 (6-OST-3) can be conveniently prepared at mg/L levels in the laboratory by culturing E. coli on Luria-Bertani medium in shake flasks and inducing with isopropyl ß-D-1-thiogalactopyranoside at an optical density of 0·6-0·8. The production of larger amounts of 6-OST-3 required fed-batch cultivation of E. coli in a stirred tank fermenter on medium containing an inexpensive carbon source, such as glucose or glycerol. The cultivation of E. coli on various carbon sources under different feeding schedules and induction strategies was examined. Conditions were established giving yields (5-20 mg g-cell-dry weight(-1)) of active 6-OST-3 with excellent productivity (2-5 mg l(-1) h(-1)). CONCLUSIONS: The production of 6-OST-3 in a fed-batch fermentation on an inexpensive carbon source has been demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to scale-up the production of heparin biosynthetic enzymes, such as 6-OST-3, is critical for scaling-up the chemoenzymatic synthesis of heparin. The success of this project may someday lead to a commercially viable bioengineered heparin to replace the animal-sourced anticoagulant product currently on the market.

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Gibberellins (GAs) are diterpenoid phytohormones that regulate various aspects of plant growth. Tetracyclic hydrocarbon ent-kaurene is a biosynthetic intermediate of GAs, and is converted from geranylgeranyl diphosphate, a common precursor of diterpenoids, via ent-copalyl diphosphate (ent-CDP) through successive cyclization reactions catalysed by two distinct diterpene synthases, ent-CDP synthase and ent-kaurene synthase. Rice (Oryza sativa L.) has two ent-CDP synthase genes, OsCPS1 and OsCPS2. It has been thought that OsCPS1 participates in GA biosynthesis, while OsCPS2 participates in the biosynthesis of phytoalexins, phytocassanes A-E, and oryzalexins A-F. It has been shown previously that loss-of-function OsCPS1 mutants display a severe dwarf phenotype caused by GA deficiency despite possessing another ent-CDP synthase gene, OsCPS2. Here, experiments were performed to account for the non-redundant biological function of OsCPS1 and OsCPS2. Quantitative reverse transcription-PCR (qRT-PCR) analysis showed that OsCPS2 transcript levels were drastically lower than those of OsCPS1 in the basal parts, including the meristem of the second-leaf sheaths of rice seedlings. qRT-PCR results using tissue samples prepared by laser microdissection suggested that OsCPS1 transcripts mainly localized in vascular bundle tissues, similar to Arabidopsis CPS, which is responsible for GA biosynthesis, whereas OsCPS2 transcripts mainly localized in epidermal cells that address environmental stressors such as pathogen attack. Furthermore, the OsCPS2 transgene under regulation of the OsCPS1 promoter complemented the dwarf phenotype of an OsCPS1 mutant, oscps1-1. The results indicate that transcripts of the two ent-CDP synthase genes differentially localize in rice plants according to their distinct biological roles, OsCPS1 for growth and OsCPS2 for defence.

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