Functional characterisation of Artemisia annua jasmonic acid carboxyl methyltransferase: a key enzyme enhancing artemisinin biosynthesis.

MAIN CONCLUSION: Overexpression of Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT) leads to enhanced artemisinin content in Artemisia annua. Artemisinin-based combination therapies remain the sole deterrent against deadly disease malaria and Artemisia annua remains the only natural producer of artemisinin. In this study, the 1101 bp gene S-adenosyl-L-methionine (SAM): Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT), was characterised from A. annua, which converts jasmonic acid (JA) to methyl jasmonate (MeJA). From phylogenetic analysis, we confirmed that AaJMT shares a common ancestor with Arabidopsis thaliana, Eutrema japonica and has a close homology with JMT of Camellia sinensis. Further, the Clustal Omega depicted that the conserved motif I, motif III and motif SSSS (serine) required to bind SAM and JA, respectively, are present in AaJMT. The relative expression of AaJMT was induced by wounding, MeJA and salicylic acid (SA) treatments. Additionally, we found that the recombinant AaJMT protein catalyses the synthesis of MeJA from JA with a Km value of 37.16 µM. Moreover, site-directed mutagenesis of serine-151 in motif SSSS to tyrosine, asparagine-10 to threonine and glutamine-25 to histidine abolished the enzyme activity of AaJMT, thus indicating their determining role in JA substrate binding. The GC-MS analysis validated that mutant proteins of AaJMT were unable to convert JA into MeJA. Finally, the artemisinin biosynthetic and trichome developmental genes were upregulated in AaJMT overexpression transgenic lines, which in turn increased the artemisinin content.

Expression, purification and X-ray crystal diffraction analysis of alcohol dehydrogenase 1 from Artemisia annua L.

Alcohol dehydrogenase 1 identified from Artemisia annua (AaADH1) is a 40 kDa protein that predominately expressed in young leaves and buds, and catalyzes dehydrogenation of artemisinic alcohol to artemisinic aldehyde in artemisinin biosynthetic pathway. In this study, AaADH1 encoding gene was subcloned into vector pET-21a(+) and expressed in Escherichia coli. BL21(DE3), and purified by Co2+ affinity chromatography. Anion exchange chromatography was performed until the protein purity reached more than 90%. Crystallization of AaADH1 was conducted for further investigation of the molecular mechanism of catalysis, and hanging-drop vapour diffusion method was used in experiments. The results showed that the apo AaADH1 crystal diffracted to 2.95 Å resolution, and belongs to space group P1, with unit-cell parameters, a = 77.53 Å, b = 78.49 Å, c = 102.44 Å, α = 71.88°, ß = 74.02°, γ = 59.97°. The crystallization condition consists of 0.1 M Bis-Tris pH 6.0, 13% (w/v) PEG 8000 and 5% (v/v) glycerol.

High level production of amorphadiene using Bacillus subtilis as an optimized terpenoid cell factory.

The anti-malarial drug artemisinin, produced naturally in the plant Artemisia annua, experiences unstable and insufficient supply as its production relies heavily on the plant source. To meet the massive demand for this compound, metabolic engineering of microbes has been studied extensively. In this study, we focus on improving the production of amorphadiene, a crucial artemisinin precursor, in Bacillus subtilis. The expression level of the plant-derived amorphadiene synthase (ADS) was upregulated by fusion with green fluorescent protein (GFP). Furthermore, a co-expression system of ADS and a synthetic operon carrying the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes was established. Subsequently, farnesyl pyrophosphate synthase (FPPS), a key enzyme in formation of the sesquiterpene precursor farnesyl pyrophosphate (FPP), was expressed to supply sufficient substrate for ADS. The consecutive combination of these features yielded a B. subtilis strain expressing chromosomally integrated GFP-ADS followed by FPPS and a plasmid encoded synthetic operon showing a stepwise increased production of amorphadiene. An experimental design-aided systematic medium optimization was used to maximize the production level for the most promising engineered B. subtilis strain, resulting in an amorphadiene yield of 416 ± 15 mg/L, which is 20-fold higher than that previously reported in B. subtilis and more than double the production in Escherichia coli or Saccharomyces cerevisiae on a shake flask fermentation level.

Sparse Epistatic Patterns in the Evolution of Terpene Synthases.

We explore sequence determinants of enzyme activity and specificity in a major enzyme family of terpene synthases. Most enzymes in this family catalyze reactions that produce cyclic terpenes-complex hydrocarbons widely used by plants and insects in diverse biological processes such as defense, communication, and symbiosis. To analyze the molecular mechanisms of emergence of terpene cyclization, we have carried out in-depth examination of mutational space around (E)-ß-farnesene synthase, an Artemisia annua enzyme which catalyzes production of a linear hydrocarbon chain. Each mutant enzyme in our synthetic libraries was characterized biochemically, and the resulting reaction rate data were used as input to the Michaelis-Menten model of enzyme kinetics, in which free energies were represented as sums of one-amino-acid contributions and two-amino-acid couplings. Our model predicts measured reaction rates with high accuracy and yields free energy landscapes characterized by relatively few coupling terms. As a result, the Michaelis-Menten free energy landscapes have simple, interpretable structure and exhibit little epistasis. We have also developed biophysical fitness models based on the assumption that highly fit enzymes have evolved to maximize the output of correct products, such as cyclic products or a specific product of interest, while minimizing the output of byproducts. This approach results in nonlinear fitness landscapes that are considerably more epistatic. Overall, our experimental and computational framework provides focused characterization of evolutionary emergence of novel enzymatic functions in the context of microevolutionary exploration of sequence space around naturally occurring enzymes.

Characterization of a class III peroxidase from Artemisia annua: relevance to artemisinin metabolism and beyond.

KEY MESSAGE: A class III peroxidase from Artemisia annua has been shown to indicate the possibility of cellular localization-based role diversity, which may have implications in artemisinin catabolism as well as lignification. Artemisia annua derives its importance from the antimalarial artemisinin. The -O-O- linkage in artemisinin makes peroxidases relevant to its metabolism. Earlier, we identified three peroxidase-coding genes from A. annua, whereby Aa547 showed higher expression in the low-artemisinin plant stage whereas Aa528 and Aa540 showed higher expression in the artemisinin-rich plant stage. Here we carried out tertiary structure homology modelling of the peroxidases for docking studies. Maximum binding affinity for artemisinin was shown by Aa547. Further, Aa547 showed greater binding affinity for post-artemisinin metabolite, deoxyartemisinin, as compared to pre-artemisinin metabolites (dihydroartemisinic hydroperoxide, artemisinic acid, dihydroartemisinic acid). It also showed significant binding affinity for the monolignol, coniferyl alcohol. Moreover, Aa547 expression was related inversely to artemisinin content and directly to total lignin content as indicated by its transient silencing and overexpression in A. annua. Artemisinin reduction assay also indicated inverse relationship between Aa547 expression and artemisinin content. Subcellular localization using GFP fusion suggested that Aa547 is peroxisomal. Nevertheless, dual localization (intracellular/extracellular) of Aa547 could not be ruled out due to its effect on both, artemisinin and lignin. Taken together, this indicates possibility of localization-based role diversity for Aa547, which may have implications in artemisinin catabolism as well as lignification in A. annua.

Substrate promiscuity of enzymes from the sesquiterpene biosynthetic pathways from Artemisia annua and Tanacetum parthenium allows for novel combinatorial sesquiterpene production.

The therapeutic properties of complex terpenes often depend on the stereochemistry of their functional groups. However, stereospecific chemical synthesis of terpenes is challenging. To overcome this challenge, metabolic engineering can be employed using enzymes with suitable stereospecific catalytic activity. Here we used a combinatorial metabolic engineering approach to explore the stereospecific modification activity of the Artemisia annua artemisinic aldehyde ∆11(13) double bond reductase2 (AaDBR2) on products of the feverfew sesquiterpene biosynthesis pathway (GAS, GAO, COS and PTS). This allowed us to produce dihydrocostunolide and dihydroparthenolide. For dihydroparthenolide we demonstrate that the preferred order of biosynthesis of dihydroparthenolide is by reduction of the exocyclic methylene of parthenolide, rather than through C4-C5 epoxidation of dihydrocostunolide. Moreover, we demonstrate a promiscuous activity of feverfew CYP71CB1 on dihydrocostunolide and dihydroparthenolide for the production of 3ß-hydroxy-dihydrocostunolide and 3ß-hydroxy-dihydroparthenolide, respectively. Combined, these results offer new opportunities for engineering novel sesquiterpene lactones with potentially improved medicinal value.

[Prokaryotic expression， purification and crystallization of CYP71AV1, key enzyme in artemisinin biosynthesis].

Malaria is a worldwide epidemic that extensively endangers health of human beings. Before artemisinin was developed to treat with malaria, about 400 million person-time of malaria infections and at least 1 million deaths from malaria were reported in the world every year. Thus malaria has been listed as one of the world's three major death diseases by the WHO. The discovery of artemisinin by Chinese scientists created a novel therapy approach to treat with malaria effectively. Amorpha-4,11-diene oxidase is a plant cytochrome P450 enzymes, i.e. CYP71AV1, which catalyzes each of the three oxidation steps from amorpha-4,11-diene to form artemisinic acid, the intermediate of artemisinin. CYP71AV1 is the key enzyme in artemisinin biosynthesis. By constructing the prokaryotic expression vector pCWOri(+)-CYP71AV1, functional expression and purification of complementary CYP71AV1 were performed. The enzyme activity was monitored by CO differential spectrum assay and the heme-based activity analysis. The preliminary crystallization condition was obtained by crystallization screening. These studies provide basis for resolving the crystal structure of CYP71AV1 and for producing artemisinin in large scale through biosynthetic biology approach, and will provide references for over expression, purification and crystallization of other plant P450 enzymes.

Expression of key genes affecting artemisinin content in five Artemisia species.

Artemisinin, an effective anti-malarial drug is synthesized in the specialized 10-celled biseriate glandular trichomes of some Artemisia species. In order to have an insight into artemisinin biosynthesis in species other than A. annua, five species with different artemisinin contents were investigated for the expression of key genes that influence artemisinin content. The least relative expression of the examined terpene synthase genes accompanied with very low glandular trichome density (4 No. mm-2) and absence of artemisinin content in A. khorassanica (S2) underscored the vast metabolic capacity of glandular trichomes. A. deserti (S4) with artemisinin content of 5.13 mg g-1 DW had a very high expression of Aa-ALDH1 and Aa-CYP71AV1 and low expression of Aa-DBR2. It is possible to develop plants with high artemisinin synthesis ability by downregulating Aa-ORA in S4, which may result in the reduction of Aa-ALDH1 and Aa-CYP71AV1 genes expression and effectively change the metabolic flux to favor more of artemisinin production than artemisinic acid. Based on the results, the Aa-ABCG6 transporter may be involved in trichome development. S4 had high transcript levels and larger glandular trichomes (3.46 fold) than A. annua found in Iran (S1), which may be due to the presence of more 2C-DNA (3.48 fold) in S4 than S1.

[Molecular cloning and characterization of CMK from Artemisia annua].

Artemisinin is a preferred medicine in the treatment of malaria. In this study, AaCMK, a key gene involved in the upstream pathway of artemisinin biosynthesis, was cloned and characterized from Artemisia annua for the first time. The full-length cDNA of AaCMK was 1 462 bp and contained an ORF of 1 197 bp that encoded a 399-anomo-acid polypeptide. Tissue expression pattern analysis showed that AaCMK was expressed in leaves, flowers, roots and stems, but with higher expression level in glandular secretory trichomes. In addition, the expression of AaCMK was markedly increased after MeJA treatment. Subcellular localization showed that the protein encoded by AaCMK was localized in chloroplast. Overexpression of AaCMK in Arabidopsis increased the contents of chlorophyll a, chlorophyll b and carotenoids. These results suggest that AaCMK plays an important role in the biosynthesis of terpenoids in A. annua and this research provids a candidate gene that could be used for engineering the artemisinin biosynthesis.

An in-silico insight into the substrate binding characteristics of the active site of amorpha-4, 11-diene synthase, a key enzyme in artemisinin biosynthesis.

The enzyme amorphadiene synthase (ADS) conducts the first committed step in the biosynthetic conversion of the substrate farnesyl pyrophosphate (FPP) to artemisinin, which is a highly effective natural product against multidrug-resistant strains of malaria. Due to the either low abundance or low turn-over rate of the enzyme, obtaining artemisinin from both natural and synthetic sources is costly and laborious. In this in silico study, we strived to elucidate the substrate binding site specificities of the ADS, with the rational that unraveling enzyme features paves the way for enzyme engineering to increase synthesis rate. A homology model of the ADS from Artemisia annua L. was constructed based on the available crystal structure of the 5-epiaristolochene synthase (TEAS) and further analyzed with molecular dynamic simulations to determine residues forming the substrate recognition pocket. We also investigated the structural aspects of Mg2+ binding. Results revealed DDYTD and NDLMT as metal-binding motifs in the putative active site gorge, which is composed of the D and H helixes and one loop region (aa519-532). Moreover, several representative residues including Tyr519, Asp444, Trp271, Asn443, Thr399, Arg262, Val292, Gly400 and Leu405, determine the FPP binding mode and its fate in terms of stereochemistry as well as the enzyme fidelity for the specific end product. These findings lead to inferences concerning key components of the ADS catalytic cavity, and provide evidence for the spatial localization of the FPP and Mg2+. Such detailed understanding will probably help to design an improved enzyme.

Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase.

Terpene synthases (TPSs) are responsible for the extremely diversified and complex structure of terpenoids. Amorpha-4,11-diene synthase (ADS) has a high (90%) fidelity in generating the sesquiterpene precursor for the biosynthesis of artemisinin, an antimalarial drug, however, little is known about how active site residues of ADS are involved in carbocation rearrangement and cyclization reactions. Here, we identify seven residues that are key to most of the catalytic steps in ADS. By structural modeling and amino acid sequence alignments of ADS with two functionally relevant sesquiterpene synthases from Artemisia annua, we performed site-directed mutagenesis and found that a single substitution, T296V, impaired the ring closure activity almost completely, and tetra-substitutions (L374Y/L404V/L405I/G439S) led to an enzyme generating 80% monocyclic bisabolyl-type sesquiterpenes, whereas a double mutant (T399L/T447G) showed compromised activity in regioselective deprotonation to yield 34.7 and 37.7% normal and aberrant deprotonation products, respectively. Notably, Thr296, Leu374, Gly439, Thr399, and Thr447, which play a major role in directing catalytic cascades, are located around conserved metal-binding motifs and function through impacting the folding of the substrate/intermediate, implying that residues surrounding the two motifs could be valuable targets for engineering TPS activity. Using this knowledge, we substantially increased amorpha-4,11-diene production in a near-additive manner by engineering Thr399 and Thr447 for product release. Our results provide new insight for the rational design of enzyme activity using synthetic biology.

Overexpression of a type-I isopentenyl pyrophosphate isomerase of Artemisia annua in the cytosol leads to high arteannuin B production and artemisinin increase.

We recently characterized a gene-terpene network that is associated with artemisinin biosynthesis in self-pollinated (SP) Artemisia annua, an effective antimalarial plant. We hypothesize that an alteration of gene expression in the network may improve the production of artemisinin and its precursors. In this study, we cloned an isopentenyl pyrophosphate isomerase (IPPI) cDNA, AaIPPI1, from Artemisia annua (Aa). The full-length cDNA encodes a type-I IPPI containing a plastid transit peptide (PTP) at its amino terminus. After the removal of the PTP, the recombinant truncated AaIPPI1 isomerized isopentenyl pyrophosphate (IPP) to dimethyl allyl pyrophosphate (DMAPP) and vice versa. The steady-state equilibrium ratio of IPP/DMAPP in the enzymatic reactions was approximately 1:7. The truncated AaIPPI1 was overexpressed in the cytosol of the SP A. annua variety. The leaves of transgenic plants produced approximately 4% arteannuin B (g g-1 , dry weight, dw) and 0.17-0.25% artemisinin (g g-1 , dw), the levels of which were significantly higher than those in the leaves of wild-type plants. In addition, transgenic plants showed an increase in artemisinic acid production of more than 1% (g g-1 , dw). In contrast, isoprene formation was significantly reduced in transgenic plants. These results provide evidence that overexpression of AaIPPI1 in the cytosol can lead to metabolic alterations of terpenoid biosynthesis, and show that these transgenic plants have the potential to yield high production levels of arteannuin B as a new precursor source for artemisinin.

Molecular cloning and characterization of the promoter of aldehyde dehydrogenase gene from Artemisia annua.

In recent years, although several related genes had been cloned and characterized, the role of aldehyde dehydrogenase 1 (ALDH1), the newly cloned gene involved in artemisinin biosynthesis pathway, is still not clear. In this study, a 2,100-bp ALDH1 promoter region fused with GUS reporter gene was stably transferred into Arabidopsis thaliana. Histochemical staining showed the methyl jasmonate (MeJA) and wounding treatment induced the GUS gene expression specifically in the trichomes of transgenic A. thaliana, consistent with the results that the expression level of ALDH1 gene was increased in the A. annua under MeJA and wounding treatments. Two RAA motifs (AP2/ERF binding site) but no W box (WRKY binding site) motif were identified in the ALDH1 promoter by the analysis through PLACE and plantCARE. Through the dual luciferase reporter assay, we revealed that both AaORA and AaERF2, rather than AaWRKY1, could activate the expression of ALDH1 promoter. Our study shed light on the in-depth understanding of the role of ALDH1 in artemisinin biosynthesis.

The T296V Mutant of Amorpha-4,11-diene Synthase Is Defective in Allylic Diphosphate Isomerization but Retains the Ability To Cyclize the Intermediate (3R)-Nerolidyl Diphosphate to Amorpha-4,11-diene.

The T296V mutant of amorpha-4,11-diene synthase catalyzes the abortive conversion of the natural substrate (E,E)-farnesyl diphosphate mainly into the acyclic product (E)-ß-farnesene (88%) instead of the natural bicyclic sesquiterpene amorphadiene (7%). Incubation of the T296V mutant with (3R,6E)-nerolidyl diphosphate resulted in cyclization to amorphadiene. Analysis of additional mutants of amino acid residue 296 and in vitro assays with the intermediate analogue (2Z,6E)-farnesyl diphosphate as well as (3S,6E)-nerolidyl diphosphate demonstrated that the T296V mutant can no longer catalyze the allylic rearrangement of farnesyl diphosphate to the normal intermediate (3R,6E)-nerolidyl diphosphate, while retaining the ability to cyclize (3R,6E)-nerolidyl diphosphate to amorphadiene. The T296A mutant predominantly retained amorphadiene synthase activity, indicating that neither the hydroxyl nor the methyl group of the Thr296 side chain is required for cyclase activity.

Functional Analysis of Amorpha-4,11-Diene Synthase (ADS) Homologs from Non-Artemisinin-Producing Artemisia Species: The Discovery of Novel Koidzumiol and (+)-α-Bisabolol Synthases.

The production of artemisinin, the most effective antimalarial compound, is limited to Artemisia annua. Enzymes involved in artemisinin biosynthesis include amorpha-4,11-diene synthase (ADS), amorpha-4,11-diene 12-monooxygenase (CYP71AV1) and artemisinic aldehyde Δ(11)13 reductase (DBR2). Although artemisinin and its specific intermediates are not detected in other Artemisia species, we reported previously that CYP71AV1 and DBR2 homologs were expressed in some non-artemisinin-producing Artemisia plants. These homologous enzymes showed similar functions to their counterparts in A. annua and can convert fed intermediates into the following products along the artemisinin biosynthesis in planta These findings suggested a partial artemisinin-producing ability in those species. In this study, we examined genes highly homologous to ADS, the first committed gene in the pathway, in 13 Artemisia species. We detected ADS homologs in A. absinthium, A. kurramensis and A. maritima. We analyzed the enzymatic functions of all of the ADS homologs after obtaining their cDNA. We found that the ADS homolog from A. absinthium exhibited novel activity in the cyclization of farnesyl pyrophosphate (FPP) to koidzumiol, a rare natural sesquiterpenoid. Those from A. kurramensis and A. maritima showed similar, but novel, activities in the cyclization of FPP to (+)-α-bisabolol. The unique functions of the novel sesquiterpene synthases highly homologous to ADS found in this study could provide insight into the molecular basis of the exceptional artemisinin-producing ability in A. annua.

RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua.

Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H.

Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L.

Flavonoids were found to synergize anti-malaria and anti-cancer compounds in Artemisia annua, a very important economic crop in China. In order to discover the regulation mechanism of flavonoids in Artemisia annua, the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from Artemisia annua for the first time by using RACE (rapid amplification of cDNA ends). The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. The recombinant protein of AaF3H was expressed in E. coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed naringenin to dihydrokaempferol in the present of Fe(2+). The Km for naringenin was 218.03 µM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35 °C. The AaF3H transcripts were found to be accumulated in the cultivar with higher level of flavonoids than that with lower level of flavonoids, which implied that AaF3H was a potential target for regulation of flavonoids biosynthesis in Artemisia annua through metabolic engineering.

Branch Pathway Blocking in Artemisia annua is a Useful Method for Obtaining High Yield Artemisinin.

There are many biosynthetic pathways competing for the metabolic flux with the artemisinin biosynthetic pathway in Artemisia annua L. To study the relationship between genes encoding enzymes at branching points and the artemisinin biosynthetic pathway, ß-caryophyllene, ß-farnesene and squalene were sprayed on young seedlings of A. annua. Transient expression assays indicated that the transcription levels of ß-caryophyllene synthase (CPS), ß-farnesene synthase (BFS) and squalene synthase (SQS) were inhibited by ß-caryophyllene, ß-farnesene and squalene, respectively, while expression of some artemisinin biosynthetic pathway genes increased. Thus, inhibition of these genes encoding enzymes at branching points may be helpful to improve the artemisinin content. For further study, the expression levels of four branch pathway genes CPS, BFS, germacrene A synthase (GAS) and SQS were down-regulated by the antisense method in A. annua. In anti-CPS transgenic plants, mRNA levels of BFS and ADS were increased, and the contents of ß-farnesene, artemisinin and dihydroartemisinic acid (DHAA) were increased by 212, 77 and 132%, respectively. The expression levels of CPS, SQS, GAS, amorpha-4,11-diene synthase (ADS), amorphadiene 12-hydroxylase (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1) were increased in anti-BFS transgenic plants and, at the same time, the contents of artemisinin and DHAA were increased by 77% and 54%, respectively, and the content of squalene was increased by 235%. In anti-GAS transgenic plants, mRNA levels of CPS, BFS, ADS and ALDH1 were increased. The contents of artemisinin and DHAA were enhanced by 103% and 130%, respectively. In anti-SQS transgenic plants, the transcription levels of BFS, GAS, CPS, ADS, CYP71AV1 and ALDH1 were all increased. Contents of artemisinin and DHAA were enhanced by 71% and 223%, respectively, while ß-farnesene was raised to 123%. The mRNA level of artemisinic aldehyde Δ11(13) reductase (DBR2) had changed little in almost all transgenic plants.

Molecular cloning and promoter analysis of the specific salicylic acid biosynthetic pathway gene phenylalanine ammonia-lyase (AaPAL1) from Artemisia annua.

Phenylalanine ammonia-lyase (PAL) is the key enzyme in the biosynthetic pathway of salicylic acid (SA). In this study, a full-length cDNA of PAL gene (named as AaPAL1) was cloned from Artemisia annua. The gene contains an open reading frame of 2,151 bps encoding 716 amino acids. Comparative and bioinformatics analysis revealed that the polypeptide protein of AaPAL1 was highly homologous to PALs from other plant species. Southern blot analysis revealed that it belonged to a gene family with three members. Quantitative RT-PCR analysis of various tissues of A. annua showed that AaPAL1 transcript levels were highest in the young leaves. A 1160-bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including W-box, TGACG-motif, and TC-rich repeats. Quantitative RT-PCR indicated that AaPAL1 was upregulated by salinity, drought, wounding, and SA stresses, which were corroborated positively with the identified cis-elements within the promoter region. AaPAL1 was successfully expressed in Escherichia. coli and the enzyme activity of the purified AaPAL1 was approximately 287.2 U/mg. These results substantiated the involvement of AaPAL1 in the phenylalanine pathway.

Expression of ß-glucosidase increases trichome density and artemisinin content in transgenic Artemisia annua plants.

Artemisinin is highly effective against multidrug-resistant strains of Plasmodium falciparum, the aetiological agent of the most severe form of malaria. However, a low level of accumulation of artemisinin in Artemisia annua is a major limitation for its production and delivery to malaria endemic areas of the world. While several strategies to enhance artemisinin have been extensively explored, enhancing storage capacity in trichome has not yet been considered. Therefore, trichome density was increased with the expression of ß-glucosidase (bgl1) gene in A. annua through Agrobacterium-mediated transformation. Transgene (bgl1) integration and transcript were confirmed by molecular analysis. Trichome density increased up to 20% in leaves and 66% in flowers of BGL1 transgenic plants than Artemisia control plants. High-performance liquid chromatography, time of flight mass spectrometer data showed that artemisinin content increased up to 1.4% in leaf and 2.56% in flowers (per g DW), similar to the highest yields achieved so far through metabolic engineering. Artemisinin was enhanced up to five-fold in BGL1 transgenic flowers. This study opens the possibility of increasing artemisinin content by manipulating trichomes' density, which is a major reservoir of artemisinin. Combining biosynthetic pathway engineering with enhancing trichome density may further increase artemisinin yield in A. annua. Because oral feeding of Artemisia plant cells reduced parasitemia more efficiently than the purified drug, reduced drug resistance and cost of prohibitively expensive purification process, enhanced expression should play a key role in making this valuable drug affordable to treat malaria in a large global population that disproportionally impacts low-socioeconomic areas and underprivileged children.