Abstract: The first-line drug artemisinin is widely used against malaria. Commercially available artemisinin is extracted from plants. However, the lack of sufficient raw material, artemisinin and the cost associated with the drug's manufacture have limited the supply of ACT to most malaria sufferers in the Developing World. As such, it is important to develop a low cost, fine to environment and high-quality method to supply sufficient and reliable quantities of artemisinin in the future. The field of synthetic biology, which utilizes cell factories to manipulate microbial metabolism to enhance the production of artemisinin and its intermediates, has a particularly strong impact by providing new platforms for chemical production. After a brief introduction of the artemisinin biosynthetic pathway, the present review focuses on the introduction of artemisinin biosynthetic genes, such as the genes encoding amorpha-4, 11-diene monooxygenase, NADPH: cytochrome P450 oxidoreductase, artemisinic aldehyde delta 11(13) reductase and aldehyde dehydrogenase. The review also addresses general considerations for potential contributions of synthetic biology to artemisinin production, with an emphasis on factors influencing interest compounds production in chassis cells.

Influenza A/H1N1 virus-encoded nonstructural, or NS1, protein inhibits the 3'-end processing of cellular pre-mRNAs by binding the cellular protein: the 30-kDa subunit of CPSF (cleavage and polyadenylation specificity factor, CPSF30). CPSF30 binding site of the NS1 protein is a potential target for the development of drugs against influenza A/H1N1 virus. A yeast two-hybrid screening system was constructed and used for screening Chinese medicines that inhibit the interaction of the A/H1N1 flu NS1 protein and human CPSF30 protein. The NS1 gene of A/H1N1 virus was amplified by consecutive polymerase chain reaction (PCR), and the human CPSF30 gene of HeLa cell cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). Then the two gene fragments confirmed by sequencing were subcloned into the yeast expression vectors pGBKT7 and pGADT7, respectively. The two constructs, bait vector pGBKNS1 and prey vector pGADCPSF, were co-transformed into yeast AH109. The eight individual yeast colonies were picked and subjected to verification by PCR/gel electrophoresis. The inhibition of the NS1-CPSF30 interaction was allowed the identification of selective inhibitors. The four of more than thirty identified Chinese medicines, including 'Shuanghuanglian oral liquid', showed the strong inhibition of the NS1-CPSF30 interaction.

The synthetic biology matures to promote the heterologous biosynthesis of the well-known drug paclitaxel that is one of the most important and active chemotherapeutic agents for the first-line clinical treatment of cancer. This review focuses on the construction and regulation of the biosynthetic pathway of paclitaxel intermediates in both Escherichia coli and Saccharomyces cerevisiae. In particular, the review also features the early efforts to design and overproduce taxadiene and the bottleneck of scale fermentation for producing the intermediates.

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.

Human enterovirus 71 (EV71) is one of the major etiological agents for the hand, foot, and month disease (HFMD) and is causing frequent, widespread occurrence in the mainland of China. The single positive-stranded RNA genome of EV71 is translated into a single polyprotein which is autocleavaged into structural and nonstructural proteins. The functions of many nonstructural proteins characterized in the life cycle of virus are potential targets for blocking viral replication. This article reviews the studies of the structures and functions of nonstructural proteins of EV71 and the anti-enterovirus 71 drugs targeting on these nonstructural proteins.

The cyclotides are a family of cyclic "mini" proteins that occur in Violaceae, Rubiaceae and Cucurbitaceae plant families and contain a head-to-tail cyclic backbone and a cystine knot arranged by three disulfide bonds. To study the natural cyclotides of V tianshanica, dried herb was extracted with 50% ethanol, and the concentrated aqueous extract was subjected to a solvent-solvent partitioning between water and hexane, ethyl acetate and n-butanol, separately. The n-butanol extract containing cyclotides was subjected to column chromatography over Sephadex LH-20, eluted with 30% methanol. The subfractions were directly reduced by DTT and analyzed by reverse-phase HPLC. The peaks with different retention times were shown on the profile of RP-HPLC and collected. The cyclotides were speculated based on masses range from 3 000 to 3 500 Da. The purified cyclotides were reduced with DTT, alkylated with iodoacetamide, and then were cleaved with endoproteinase Glu-C, endoproteinase Lys-C and Trypsin, separately. The digested peptides were purified on RP-HPLC and analyzed on MALDI TOF/TOF analyzer. A new cyclotide, cycloviolacin T1 and a reported cyclotide varv E were systemically determined using MALDI TOF/TOF system. So the method for the isolation and characterization of cyclotides was quickly built up in succession.

Millettia nitida var.hirsutissima is a Chinese herbal medicine used for the treatment of gynecological diseases.An HPLC/DAD/ESI-MSn method was established for the rapid separation and characterization of bioactive flavonoids in M.nitida var.hirsutissima.A total of 32 flavonoids were detected,of which 14 compounds were unambiguously characterized by comparing their retention time,UV,and MS spectra with those of the reference standards,and the others were tentatively identified based on their tandem mass spectrometry fragmentation data obtained in the negative ionization mode on line.Nineteen of these compounds characterized were reported from this plant for the first time.

Three cyclotides were isolated from the whole plant of Viola yedoensis in this study. The two, vary peptide E and cycloviolacin Y5, were previously reported, and a novel cycloviolacin VY1 was characterized according to the interpretation of MS/MS fragmentation of peptides which were produced from the reduced and alkylated parent peptide with the digestion of Endo Lys-C, trypsin and chymotrypsin, separately. The stability of remarkable resistance to proteolytic degradation by trypsin and chymotrypsin, and that of thermal denaturation was confirmed again. Besides, the IC50 value of cycloviolacin VYI against influenza A H1N1 virus was (2.27 +/- 0.20) microg x mL(-1). It is the first cyclotide reported with anti-influenza A H1N1 virus activity in vitro assay.

To construct an engineered Saccharomyces cerevisiae producing high titres of amorpha-4,11-diene, we investigated the possible synergistic effect of different vectors containing amorpha-4,11-diene synthase(ADS) gene within one yeast cell. We constructed the ADS recombinant plasmid pGADADS. This plasmid and another ADS recombinant plasmid pYeDP60/G/ADS were alone, or co-transformed into yeast Saccharomyces cerevisiae W303-1B and WK1, respectively, resulting in the following engineered yeasts, W303B[pGADADS], W303B[pYGADS], W303B[pYGADS+pGADADS], WK1[pGADADS], WK1[pYGADS] and WK1[pYGADS+pGADADS]. All of the six strains were cultured for GC-MS analysis of amorpha-4,11-diene. The results showed that all of the engineered yeasts could produce amorpha-4,11-diene. The yield of the product was improved with increasing ADS gene copies while no deleterious effect on the strain growth was found. Moreover, the product yield of the engineered yeast co-transformed with multiple plasmids was much higher than the total yield of the different engineered yeasts with only one plasmid, respectively. In conclusion, there was a distinct synergistic effect between different recombinant ADS plasmids within one cell. Our results facilitate the construction of the engineered yeast with high yield of amorpha-4,11-diene, the precursor of artemisinin.
Alkyl and Aryl Transferases ; biosynthesis ; genetics ; Artemisinins ; chemistry ; metabolism ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Recombination, Genetic ; Saccharomyces cerevisiae ; genetics ; metabolism