A catchment scale evaluation of multiple stressor effects in headwater streams.

Mitigation activities to improve water quality and quantity in streams as well as stream management and restoration efforts are conducted in the European Union aiming to improve the chemical, physical and ecological status of streams. Headwater streams are often characterised by impairment of hydromorphological, chemical, and ecological conditions due to multiple anthropogenic impacts. However, they are generally disregarded as water bodies for mitigation activities in the European Water Framework Directive despite their importance for supporting a higher ecological quality in higher order streams. We studied 11 headwater streams in the Hove catchment in the Copenhagen region. All sites had substantial physical habitat and water quality impairments due to anthropogenic influence (intensive agriculture, urban settlements, contaminated sites and low base-flow due to water abstraction activities in the catchment). We aimed to identify the dominating anthropogenic stressors at the catchment scale causing ecological impairment of benthic macroinvertebrate communities and provide a rank-order of importance that could help in prioritising mitigation activities. We identified numerous chemical and hydromorphological impacts of which several were probably causing major ecological impairments, but we were unable to provide a robust rank-ordering of importance suggesting that targeted mitigation efforts on single anthropogenic stressors in the catchment are unlikely to have substantial effects on the ecological quality in these streams. The SPEcies At Risk (SPEAR) index explained most of the variability in the macroinvertebrate community structure, and notably, SPEAR index scores were often very low (<10% SPEAR abundance). An extensive re-sampling of a subset of the streams provided evidence that especially insecticides were probably essential contributors to the overall ecological impairment of these streams. Our results suggest that headwater streams should be considered in future management and mitigation plans. Catchment-based management is necessary because several anthropogenic stressors exceeded problematic thresholds, suggesting that more holistic approaches should be preferred.

Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L.

A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Delta11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1alpha was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.

Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli.

Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zingiber officinale were expressed in Escherichia coli under different conditions to optimize the yield of active soluble protein. The cDNAs of these enzymes were inserted into the pET28 vector (Novagen) and expressed in four different bacterial strains; BL21 (DE3), BL21 (DE3) Tuner, BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner using different inducing agents (IPTG, The Inducer). The effects of induction under osmotic stress in the presence of glycine betaine and sorbitol were investigated. Although background expression for ADS was reduced when using pLysS strains, no significant difference was noted for ADS activity in soluble whole cell lysates after induction with either IPTG or The Inducer. For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner, induced with IPTG, leads to a twofold increase in enzyme activity in the soluble fraction while a reduction in activity is observed when using the pLysS strains. The same doubling of activity is observed for GDS when the commonly used BL.21 (DE3) is induced with The Inducer. Addition of 2.5 mM glycine betaine and 660 mM sorbitol to the bacterial growth media resulted in reduction of growth rate and biomass yield but under these conditions the best overall protein production, for both enzymes, was obtained. Compared to the standard conditions previously used in our laboratory the yield of soluble active protein was increased 7- and 2.5-fold for ADS and GDS, using BL21 (DE3) pLysS Tuner and BL21 (DE3), respectively.

Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale.

A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA (AY860846) contains a 1650-bp open reading frame coding for 550 amino acids (63.8kDa) with a theoretical pI=5.59. The deduced amino acid sequence is 30-46% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a major product, (+)-germacrene D (50.2% of total sesquiterpenoids produced) and a co-product, germacrene B (17.1%) and a number of minor by-products. The optimal pH for the recombinant enzyme is around 7.5. Substantial (+)-germacrene D synthase activity is observed in the presence of Mg2+, Mn2+, Ni2+ or Co2+, while the enzyme is inactive when Cu2+ or Zn2+ is used. The Km- and kcat-values are 0.88 microM and 3.34 x 10(-3) s(-1), respectively. A reaction mechanism involving a double 1,2-hydride shift has been established using deuterium labeled substrates in combination with GC-MS analysis.

Production of the artemisinin precursor amorpha-4,11-diene by engineered Saccharomyces cerevisiae.

The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN.PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 microg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days' batch cultivation.