[show abstract][hide abstract] ABSTRACT: Artemisinin,a new and a very potent antimalarial drug, is produced by the plant Artemisia annua L. with a very low yield ranging from 0.01% to 0.8% on a dry-weight basis. This makes artemisinin an expensive drug. Several studies reported chemical synthesis of the artemisinin, but none of them seems a viable economical alternative compared with the isolation of artemisinin from the plant. Hence, a higher artemisinin concentration in the plant is necessary for cheap antimalarial drug production. Many types of cyclic sesquiterpenes in Artemisia annua have been characterized to date, each derived from the common cyclic precursor FDP in a reaction catalyzed by a sesquiterpene synthase. Sesquiterpene synthases are widely regarded as the rate-determining regulatory enzymes in the pathways they participate, and a number of sesquiterpene synthases have been cloned from Artemisia annua up to now. This report is a brief review on the following sesquiterpene synthases: epi-cedrol synthase, amorpha-4,11-diene synthase, beta-caryophyllene synthase, (E)-beta-farnesene synthase, germacrene A synthase, as well as a new sesquiterpene synthase whose function remains largely unknown. The report is of help for a better understanding of metabolic engineering of Artemisia annua.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2007; 23(6):976-81.
[show abstract][hide abstract] ABSTRACT: Artemisinin, a novel and highly potent antimalarial drug, is produced from the plant Artemisia annua L. with very low yields ranging from 0.01% to 0.8% on a dry-weight basis. This makes artemisinin an expensive drug. Several studies reported the chemical synthesis of artemisinin, but none of them seems to be a viable economical alternative compared with the isolation of artemisinin from the plant. Hence, a higher concentration of artemisinin in the plant is necessary for the cheap production of antimalarial drug. Several types of cyclic sesquiterpenes in Artemisia annua have been characterized so far, and each was derived from the common cyclic precursor FDP in a reaction catalyzed by a sesquiterpene synthase. Sesquiterpene synthases are widely regarded as the rate-determining regulatory enzymes in the pathways they participate in, and several sesquiterpene synthases have been cloned from Artemisia annua till date. This report gives a brief review of the following sesquiterpene synthases: epi-cedrol synthase, amorpha-4,11-diene synthase, β-caryophyllene synthase, (E)-β-farnesene synthase, germacrene A synthase, as well as a novel sesquiterpene synthase whose function remains largely unknown. This study provides a better understanding of the metabolic engineering of Artemisia annua.
[show abstract][hide abstract] ABSTRACT: Terpenoids are present in all organisms but are especially abundant in plants, with more than 30,000 compounds. Not only do they play an important role in the life of plant, but also have high commercial values. However, the content of many important terpenoids in plant is very low. Therefore, how to improve the inefficient production of terpenoids is an urgent task. Metabolic engineering has been one of the most potential technologies to improve terpenoids production in recent years, following the study of metabolic pathway and regulation mechanism of terpenoids. Although there are some breakthroughs, metabolic engineering of terpenoids is still full of challenges because of the lack of knowledge on metabolic control of most terpenoids. Functional genomics approaches, including transcriptomics, proteomics and metabolomics, are potential tools for exploring of metabolic engineering. Integrating transcriptomics and metabolomics is an effective way to discover new genes involved in metabolic pathway. In this paper, the representative research outcomes about the metabolic engineering of terpenoids in plant were reviewed concisely and then the application of functional genomics approaches to study metabolic pathway and regulation mechanism of terpenoids and the strategies for metabolic engineering of terpenoids were discussed.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 08/2007; 23(4):561-9.
[show abstract][hide abstract] ABSTRACT: Salidroside is a novel effective adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Because this plant is a rare resource and has low yield, there is great interest in enhancing the production of salidroside. In this study, a putative UDP-glucosyltransferase (UGT) cDNA, UGT73B6, was isolated from Rhodiola sachalinensis using a rapid amplification of cDNA ends (RACE) method. The cDNA was 1,598 bp in length encoding 480 deduced amino acid residues with a conserved UDP-glucose-binding domain (PSPG box). Southern blot analysis of genomic DNA indicated that UGT73B6 existed as a single copy gene in the R. sachalinensis genome. Northern blot analysis revealed that transcripts of UGT73B6 were present in roots, calli and stems, but not in leaves. The UGT73B6 under 35S promoter with double-enhancer sequences from CaMV-Omega and TMV-Omega fragments was transferred into R. sachalinensis via Agrobacterium tumefaciens. PCR, PCR-Southern and Southern blot analyses confirmed that the UGT73B6 gene had been integrated into the genome of transgenic calli and plants. Northern blot analysis revealed that the UGT73B6 gene had been expressed at the transcriptional level. High performance liquid chromatography (HPLC) analysis indicated that the overexpression of the UGT73B6 gene resulted in an evident increase of salidroside content. These data suggest that the cloned UGT73B6 can regulate the conversion of tyrosol aglycon to salidroside in R. sachalinensis. This is the first cloned glucosyltransferase gene involved in salidroside biosynthesis.
[show abstract][hide abstract] ABSTRACT: A rhabdovirus associated with a lethal hemorrhagic disease in cultured turbot Scophthalmus maximus Linnaeus was isolated. The virus induced typical cytopathogenic effects (CPE) in 9 of 15 fish cell lines examined and was then propagated and isolated from infected carp leucocyte cells (CLC). Electron microscopy observations revealed that the negatively stained virions had a typical bullet-shaped morphology with one rounded end and one flat base end. The bullet-shaped morphology was more obvious and clear in ultrathin sections of infected cells. Experimental infections also indicated that the S. maximus rhabdovirus (SMRV) was not only a viral pathogen for cultured turbot, but also had the ability to infect other fish species, such as freshwater grass carp. A partial nucleotide sequence of the SMRV polymerase gene was determined by RT-PCR using 2 pairs of degenerate primers designed according to the conserved sequences of rhabdovirus polymerase genes. Homology analysis, amino acid sequence alignment, and phylogenetic relationship analysis of the partial SMRV polymerase sequence indicated that SMRV was genetically distinct from other rhabdoviruses. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified SMRV revealed 5 major structural proteins, and their molecular masses were estimated to be about 250, 58, 47, 42, and 28 kDa. Significant serological reactivity differences were also observed between SMRV and its nearest neighbor, spring viremia of carp virus (SVCV). The data suggest that SMRV is likely a novel fish rhabdovirus, although it is closely related to rhabdoviruses in the genus Vesiculovirus.
Diseases of Aquatic Organisms 03/2007; 74(2):95-105. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Increasing demand of artemisinin in the treatment of malaria has placed substantial stress on the total artemisinin supplies world-wide, so more attention has been paid to increasing the content of artemisinin in the Artemisia annua L. plant. In this study, amorpha-4,11-diene synthase (ADS) cDNA (ads1) and genomics gene (gads1) were cloned from a high-yield A. annua strain 001. The activity of ADS1 was confirmed by heterogeneous overexpression of ads1 and in vitro enzymatic incubation. Reverse transcript-polymerase chain reaction results demonstrated that ads1 expressed in leaves, flowers and young stems, but not in roots. This organ-specific expression pattern of ads1 is consistent with that of artemisinin accumulation in the plant. The gads1 has a complex organization including seven exons and six introns, and belongs to class III terpene synthase. DNA gel blotting revealed that the ADS gene has at least four copies in the genome of strain 001. The higher copy numbers might be one of the reasons for its high artemisinin content.(Managing editor: Wei Wang)
[show abstract][hide abstract] ABSTRACT: Artemisinin is a novel effective antimalarial drug extracted from the medicinal plant Artemisia annua L. Owing to the tight market and low yield of artemisinin, there is great interest in enhancing the production of artemisinin. In the present study, farnesyl diphosphate synthase (FPS) was overexpressed in high-yield A. annua to increase the artemisinin content. The FPS activity in transgenic A. annua was two- to threefold greater than that in non-transgenic A. annua. The highest artemisinin content in transgenic A. annua was approximately 0.9% (dry weight), which was 34.4% higher than that in non-transgenic A. annua. The results demonstrate the regulatory role of FPS in artemisinin biosynthesis.(Managing editor: Wei Wang)
[show abstract][hide abstract] ABSTRACT: Artemisinin is a new effective antimalarial drug extracted from the medicinal plant Artemisia annua L. There is a great interest in enhancing the production of artemisinin by means of transgenic plants. In this work, experiments with different combinations of hormone concentration were performed to obtain higher frequency of shoot and root induction. In particular, the factors influencing A. tumefaciens-mediated transformation of A. annua were explored to optimize the transformation system, which included A. tumefaciens strain, plant genotype, preculture period, composition of infection bacterium suspension, methods of co-cultivation, and co-cultivation period. Finally, a system of high efficiency of genetic transformation and regeneration of A. annua was established.
[show abstract][hide abstract] ABSTRACT: The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.
Diseases of Aquatic Organisms 01/2004; 57(1-2):27-34. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Artemisinin, a new and a very potent antimalarial drug, is produced by the Chinese medicinal herb Artemisia annua L. It is a sesquiterpene lactone with an endoperoxide bridge and is active against chloroquine resistant forms of Plasmodium falciparum. The relatively low yield (0.01% - 0.6%) of artemisinin in A. annua is a serious limitation to the commercialization of the drug. Therefore, a through understanding of the biosynthetic pathway and the characterization of the involved enzymes are important for the biology production of artemisinin. This review is focused on the recent progress in the molecular regulation of artemisinin biosynthesis from the following aspects: the biosynthetic pathway of artemisinin, the key enzymes involved in artemisinin biosynthesis, and the molecular regulation of artemisinin biosynthesis. The biosynthetic pathway of artemisinin belongs to the isoprenoid metabolite pathway, the key enzymes involved in the biosynthesis of artemisinin include: 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FDPS), and amorpha-4, 11-diene synthase, of which amorpha-4, 11-diene synthase catalyzes the cyclisation of the ubiquitous precursor farnesyl diphosphate to the highly specific olefinic sesquiter-pene skeletons and has been postulated as the regulatory step in the biosynthesis of artemisinin. Recently the gene encoding of the amorpha-4, 11-diene synthase has been cloned and the functional expressions have been studied by several research teams, therefore, the breakthroughs in production of artemisinin could hopefully be achieved by metabolic engineering of the plant, in particular, by over-expressing enzyme(s) catalyzing the rate limiting step(s) of artemisinin biosynthesis or by inhibiting the enzyme(s) of other pathway competing for its precursors. Besides, the effects of the heterogenesis isoprenoid pathway related genes on artemisinin biosynthesis of the transformed plants were also discussed.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2003; 19(6):646-50.
[show abstract][hide abstract] ABSTRACT: A pathogenic virus (RGV), isolated from diseased pig frog Rana grylio with lethal syndrome, was investigated with regard to morphogenesis and cellular interactions in EPC cells, a cell line from fish. Different stages of virus amplification, maturation and assembly were observed at nucleus, cytoplasm and cellular membranes. The matured virus particles, were not only distributed diffusely in nucleus, cytoplasm and cellular surface, but also aggregated as pseudocrystalline arrays in the cytoplasm. Virions were released by budding from the plasma membranes, or following cell lysis. Various types of cell damage, such as small vacuoles, spherical inclusions, and swollen and empty mitochondria, were also found. Some typical characteristics of RGV, such as the symmetrical shape of the virions, replication process involving both nuclear and cytoplasmic phases, budding release from cellular membrane and intracellular membrane, viromatrix and paracrystalline aggregation in cytoplasm, and its acute pathogenic effects, were observed to be similar to that of other iridoviruses. Therefore, the RGV appears to be a member of the Iridoviridae based on these studies.