High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Mono and diterpene production in Escherichia coli.

Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.

Structure of human pro-chymase: a model for the activating transition of granule-associated proteases.

Human chymase is a protease involved in physiological processes ranging from inflammation to hypertension. As are all proteases of the trypsin fold, chymase is synthesized as an inactive "zymogen" with an N-terminal pro region that prevents the transition of the zymogen to an activated conformation. The 1.8 A structure of pro-chymase, reported here, is the first zymogen with a dipeptide pro region (glycine-glutamate) to be characterized at atomic resolution. Three segments of the pro-chymase structure differ from that of the activated enzyme: the N-terminus (Gly14-Gly19), the autolysis loop (Gly142-Thr154), and the 180s loop (Pro185A-Asp194). The four N-terminal residues (Gly14-Glu15-Ile16-Ile17) are disordered. The autolysis loop occupies a position up to 10 A closer to the active site than is seen in the activated enzyme, thereby forming a hydrogen bond with the catalytic residue Ser195 and occluding the S1' binding pocket. Nevertheless, the catalytic triad (Asp102-His57-Ser195) is arrayed in a geometry close to that seen in activated chymase (all atom rmsd of 0.52 A). The 180s loop of pro-chymase is, on average, 4 A removed from its conformation in the activated enzyme. This conformation disconnects the oxyanion hole (the amides of Gly193 and Ser195) from the active site and positions only approximately 35% of the S1-S3 binding pockets in the active conformation. The backbone of residue Asp194 is rotated 180 degrees when compared to its conformation in the activated enzyme, allowing a hydrogen bond between the main-chain amide of residue Trp141 and the carboxylate of Asp194. The side chains of residues Phe191 and Lys192 of pro-chymase fill the Ile16 binding pocket and the base of the S1 binding pocket, respectively. The zymogen positioning of both the 180s and autolysis loops are synergistic structural elements that appear to prevent premature proteolysis by chymase and, quite possibly, by other dipeptide zymogens.

Anisotropic dynamics of the JE-2147-HIV protease complex: drug resistance and thermodynamic binding mode examined in a 1.09 A structure.

The structure of HIV protease (HIV Pr) bound to JE-2147 (also named AG1776 or KNI-764) is determined here to 1.09 A resolution. This highest-resolution structure for HIV Pr allows refinement of anisotropic displacement parameters (ADPs) for all atoms. Clustering based on the directional information in ADPs defines two sets of subdomains such that within each set, subdomains undergo similar anisotropic motion. These sets are (a) the core of monomer A grouped with both substrate-binding flaps and (b) the core of monomer B coupled to both catalytic aspartates (25A/B). The four-stranded beta-sheet (1-4 A/B and 95-99 A/B) that forms a significant part of the dimer interface exhibits large anisotropic amplitudes that differ from those of the other sets of subdomains. JE-2147 is shown here to be a picomolar inhibitor (K(i) = 41 +/- 18 pM). The structure is used to interpret the mechanism of association of JE-2147, a second-generation inhibitor for which binding is enthalpically driven, with respect to first-generation inhibitors for which binding is predominantly entropically driven [Velazquez-Campoy, A., et al. (2001) Arch. Biochem. Biophys. 390, 169-175]. Relative to the entropically driven inhibitor complexes, the JE-2147-HIV Pr complex exhibits an approximately 0.5 A movement of the substrate flaps in toward the substrate, suggesting a more compatible enthalpically driven association. Domains of the protease identified by clustering of ADPs also suggest a model of enthalpy-entropy compensation for all HIV Pr inhibitors in which dynamic coupling of the flaps is offset by an increased level of motion of the beta-sheet domain of the dimer interface (1-4 A/B and 95-99 A/B).

Conformational change coupling the dimerization and activation of KSHV protease.

The mechanism of herpesviral protease activation upon dimerization was studied using two independent spectroscopic assays augmented by directed mutagenesis. Spectroscopic changes, attributable to dimer interface conformational plasticity, were observed upon dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr). KSHV Pr's dissociation constant of 585 +/- 135 nM at 37 degrees C was measured by a concentration-dependent, 100-fold increase in specific activity to a value of 0.275 +/- 0.023 microM product min(-1) (microM enzyme)(-1). A 4 nm blue-shifted fluorescence emission spectrum and a 25% increase in ellipticity at 222 nm were detected by circular dichroism upon dimer association. This suggested enhanced hydrophobic packing within the dimer interface and/or core, as well as altered secondary structures. To better understand the structure-activity relationship between the monomer and the dimer, KSHV Pr molecules were engineered to remain monomeric via substitution of two separate residues within the dimer interface, L196 and M197. These mutants were proteolytically inactive while exhibiting the spectroscopic signature and thermal stability of wild type, dissociated monomers (T(M) = 75 degrees C). KSHV Pr conformational changes were found to be relevant in vivo, as the autoproteolytic inactivation of KSHV Pr at its dimer disruption site [Pray et al. (1999) J. Mol. Biol. 289, 197-203] was detected in viral particles from KSHV-infected cells. This characterization of structural plasticity suggests that the structure of the KSHV Pr monomer is stable and significantly different from its structure in the dimer. This structural uniqueness should be considered in the development of compounds targeting the dimer interface of KSHV Pr monomers.

Functional consequences of the Kaposi's sarcoma-associated herpesvirus protease structure: regulation of activity and dimerization by conserved structural elements.

The structure of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr), at 2.2 A resolution, reveals the active-site geometry and defines multiple possible target sites for drug design against a human cancer-producing virus. The catalytic triad of KSHV Pr, (Ser114, His46, and His157) and transition-state stabilization site are arranged as in other structurally characterized herpesviral proteases. The distal histidine-histidine hydrogen bond is solvent accessible, unlike the situation in other classes of serine proteases. As in all herpesviral proteases, the enzyme is active only as a weakly associated dimer (K(d) approximately 2 microM), and inactive as a monomer. Therefore, both the active site and dimer interface are potential targets for antiviral drug design. The dimer interface in KSHV Pr is compared with the interface of other herpesviral proteases. Two conserved arginines (Arg209), one from each monomer, are buried within the same region of the dimer interface. We propose that this conserved arginine may provide a destabilizing element contributing to the tuned micromolar dissociation of herpesviral protease dimers.

Ion-channel-forming colicins.

Several features of ion-channel-forming colicins have been illuminated by recent revelations: its four-domain structure, the mechanism and thermodynamics of binding to the gating loop of outer membrane porins, the mechanism of translocation, competition for the transperiplasmic excursion facilitated by the Tol or Ton transperiplasmic proteins, and the formation of a waisted, funnel-shaped transmembrane channel of well-characterized shape.

The effects of different ozone exposures on three contrasting populations of Plantago major.

Plantago major grows throughout Britain in a range of ozone climates. Because populations have been shown to differ in ozone resistance, the aim of the experiment was to compare the reaction of populations from contrasting ozone climates to different types of ozone exposure. Three populations were grown under controlled conditions in five different ozone treatments (including controls for 10 wk. Development, growth, stomatal conductance and seed production were recorded. Populations were from the south coast of England (Lullington), near a mountain summit (Great Dun Fell) and lowland Scotland (Bush). Ozone treatments were: charcoal and Purafil filtered air (CF); 35 nl l(-1) for 24 h every day: 70 nl l(-1) h for 7 h everyday: CF then three episodes each week of 70 nl l(-1) for 7 h; and 35 nl l(-1) continuously plus three 7 h episodes each week of 70 nl l(-1) . The different ozone treatments resulted in different responses in each population. Ozone promoted senescence in the Great Dun Fell population but not in the others; it reduced root growth more in the Lullington population than in the others but those from Lullington and Great Dun Fell maintained seed production to a much greater extent than the Bush population. The reproductive effort (number of seeds g(-1) of vegetative weight) actually increased in ozone in the Lullington and Great Dun Fell populations. It is suggested that this might he a general stress response rather than being specifically related to ozone. Effects on stomatal conductance were similar to those previously reported and the converse of effects on seed production. The relative responses of the populations varied according to the ozone treatment. Continuous exposure to 35 n1 l(-l) reduced leaf size only in the Great Dun Fell population, but seed output was reduced in the Bush population. In some cases, giving 3-d episodes of 70 n1 l(-1) had a greater effect than giving the dose every day but the effects varied with the population. This greater effect was considered to be a result of the time it takes for a plant to develop maximum anti-oxidant defence, which is lost when the ozone decreases after the episode. A plant exposed to episodes might have to re-induce defence with each exposure. Although it is reported frequently that ozone favours allocation of resources to the shoot over the root, it is concluded that this is an over-simplification of the response. Even within a species there is a complex suite of responses that varies with the population and with ozone exposure. Describing a population as resistant or sensitive is also an over-simplification.

Effects of exposure to ozone at different stages in the development of Plantago major L. on chlorophyll fluorescence and gas exchange.

Similar ozone treatments were applied at different stages of growth to a population of Plantago major L., which is as sensitive to ozone as Bel-W3 tobacco. Plants were grown from seed for 8 wk in controlled-environment chambers and exposed to 70 µl O3 l-1 7 h d-1 for the whole period or for 2-wk episodes during weeks 1 + 2, 3 + 4, 5 + 6 or 7 + 8. Controls had charcoal-filtered air. Effects on stomatal conductance, chlorophyll fluorescence and net photosynthesis are described. The fluorescence character, t1/2 , proved to be very sensitive to ozone, and it responded at all stages of plant development, but the ratio of variable to maximum fluorescence decreased only when plants received ozone during the first 2 wk of growth. The reduction was caused by a drop in maximum fluorescence. Ozone had no effect on F0 . Whenever exposure occurred, O3 significantly reduced net CO2 assimilation and increased stomatal conductance. Unlike t1/2 , the effects were persistent, affecting leaves that were in bud or enshrouded by leaves when the exposure occurred. It is suggested that these persistent effects may have been caused by changes in resource allocation, flowering and senescence, which resulted in altered hormonal balance.

Interaction between ozone and winter stress.

In some countries, ozone (O3) is primarily a summer pollutant, but in much of Europe, elevated concentrations occur outside the growing season so perennials and over-wintering annuals may be subjected to the combined stresses of pollution, plus chilling, freezing, and winter desiccation. It is recognised that some air pollutants modify the response of plants to environmental stress, but little is known of interactions involving O3. This paper is part of a programme concerned with the effects of O3 on resistance to chilling, freezing, and winter desiccation. Pea (Pisum sativum L.) was used as a convenient model to confirm that O3 affects freezing resistance. The experiment also served as a further evaluation of the use of induced chlorophyll fluorescence kinetics to detect latent O3 injury. Two cultivars, 'Feltham First' and 'Conquest', were fumigated for 7 days, 7 h day(-1). Diffusive resistance and induced fluorescence were recorded daily during the period, then the plants were hardened at 4 degrees C day/2 degrees C night before exposure to 0, -2, -4, -6 and -8 degrees C. Ozone (0.075 ppm; 150 microg O3 m(-3)) caused stomatal closure in both cultivars, but the response was more rapid in 'Conquest'. There were also rapid effects on fluorescence kinetics, and it was concluded that FR, the rate of rise of induced fluorescence, is a useful parameter for indicating latent injury and for distinguishing between cultivars of different sensitivity. Exposure to O3 increased freezing injury and led to greater electrolyte leakage. The freezing resistance of 'Feltham First' was more affected than that of 'Conquest', probably because of the slower stomatal response to the pollutant leading to greater flux of O3 to the internal tissues. It is concluded that interactions involving pollutants and winter stress have implications for crop loss assessment. Perennials and over-wintering annuals should be exposed to the full range of environmental stresses.