Supply Of Artemisinin Research Articles

Manual correction of genome annotation improved alternative splicing identification of Artemisia annua.

Gene annotation is essential for genome-based studies. However, algorithm-based genome annotation is difficult to fully and correctly reveal genomic information, especially for species with complex genomes. Artemisia annua L. is the only commercial resource of artemisinin production though the content of artemisinin is still to be improved. Genome-based genetic modification and breeding are useful strategies to boost artemisinin content and therefore, ensure the supply of artemisinin and reduce costs, but better gene annotation is urgently needed. In this study, we manually corrected the newly released genome annotation of A. annua using second- and third-generation transcriptome data. We found that incorrect gene information may lead to differences in structural, functional, and expression levels compared to the original expectations. We also identified alternative splicing events and found that genome annotation information impacted identifying alternative splicing genes. We further demonstrated that genome annotation information and alternative splicing could affect gene expression estimation and gene function prediction. Finally, we provided a valuable version of A. annua genome annotation and demonstrated the importance of gene annotation in future research.

Elevation of artemisinin content by co-transformation of artemisinin biosynthetic pathway genes and trichome-specific transcription factors in Artemisia annua.

Artemisinin, derived from Artemisia annua, is currently used as the first-line treatment for malaria. However, wild-type plants have a low artemisinin biosynthesis rate. Although yeast engineering and plant synthetic biology have shown promising results, plant genetic engineering is considered the most feasible strategy, but it is also constrained by the stability of progeny development. Here we constructed three independent unique overexpressing vectors harboring three mainstream artemisinin biosynthesis enzymes HMGR, FPS, and DBR2, as well as two trichomes-specific transcription factors AaHD1 and AaORA. The simultaneous co-transformation of these vectors by Agrobacterium resulted in the successful increase of the artemisinin content in T0 transgenic lines by up to 3.2-fold (2.72%) leaf dry weight compared to the control plants. We also investigated the stability of transformation in progeny T1 lines. The results indicated that the transgenic genes were successfully integrated, maintained, and overexpressed in some of the T1 progeny plants' genomes, potentially increasing the artemisinin content by up to 2.2-fold (2.51%) leaf dry weight. These results indicated that the co-overexpression of multiple enzymatic genes and transcription factors via the constructed vectors provided promising results, which could be used to achieve the ultimate goal of a steady supply of artemisinin at affordable prices around the world.

Mapping Worldwide Environmental Suitability for Artemisia annua L.

Artemisinin, which is isolated from the naturally occurring plant Artemisia annua L. (A. annua; Qinghao in traditional Chinese medicine), is considered to be the active ingredient in the most effective treatment for malaria. Current malaria eradication plans rely on an affordable and robust supply of artemisinin, resulting in the demand to expand the area of A. annua under cultivation. However, there is no reliable assessment of the potential land resources suitable for planting A. annua at the global scale. By explicitly incorporating the assembled contemporary occurrence records of A. annua with various spatial predictor variables, a species distribution modelling procedure was adopted to produce the first global environmental suitability map for A. annua with high geographic detail (5 × 5 km2). The estimated map reveals that the total amount of potential land resources suitable for planting A. annua is approximately 1496.56 million hectares, mainly distributed in Asia (516.50 million hectares), Europe (378.82 million hectares), North America (354.56 million hectares) and South America (172.01 million hectares). The relationships between the relevant variables and A. annua were explored, and these illustrated that the most noteworthy predictor variables were meteorological factors, followed by solar radiation factors, soil factors and topographical factors. The map provides a rigorous environmental niche baseline to support the reasonable expansion of the A. annua cultivation area.

Debottlenecking mevalonate pathway for antimalarial drug precursor amorphadiene biosynthesis in Yarrowia lipolytica

World Health Organization reports that half of the population in developing countries are at risk of malaria infection. Artemisinin, the most potent anti-malaria drug, is a sesquiterpene endoperoxide extracted from the plant Artemisia annua. Due to scalability and economics issues, plant extraction or chemical synthesis could not provide a sustainable route for large-scale manufacturing of artemisinin. The price of artemisinin has been fluctuating from 200$/Kg to 1100$/Kg, due to geopolitical and climate factors. Microbial fermentation was considered as a promising method to stabilize the artemisinin supply chain. Yarrowia lipolytica, is an oleaginous yeast with proven capacity to produce large quantity of lipids and oleochemicals. In this report, the lipogenic acetyl-CoA pathways and the endogenous mevalonate pathway of Y. lipolytica were harnessed for amorphadiene production. Gene overexpression indicate that HMG-CoA and acetyl-CoA supply are two limiting bottlenecks for amorphadiene production. We have identified the optimal HMG-CoA reductase and determined the optimal gene copy number for the precursor pathways. Amorphadiene production was improved further by either inhibiting fatty acids synthase or activating the fatty acid degradation pathway. With co-expression of mevalonate kinase (encoded by Erg12), a push-and-pull strategy enabled the engineered strain to produce 171.5 ​mg/L of amorphadiene in shake flasks. These results demonstrate that balancing carbon flux and manipulation of precursor competing pathways are key factors to improve amorphadiene biosynthesis in oleaginous yeast; and Y. lipolytica is a promising microbial host to expand nature’s biosynthetic capacity, allowing us to quickly access antimalarial drug precursors.

Trichoderma asperellum ACCC30536 inoculation improves soil nutrition and leaf artemisinin production in Artemisia annua

Artemisia annua L. is the main source of artemisinin, currently the most effective treatment for malaria. However, an affordable and abundant supply of artemisinin remains elusive. Trichoderma is a biocontrol agent that stimulates plant growth and defense responses, and improves soil quality. To date, relatively few studies have focused on improving leaf biomass and artemisinin production in A. annua using Trichoderma. To investigate the role of T. asperellum ACCC30536 in improving the artemisinin yield of A. annua, field-grown A. annua was inoculated with T. asperellum conidia and grown for 60 days. The results showed that leaf artemisinin concentration and dry weight were increased significantly after inoculation. The optimal inoculation dose was 200 mL of conidia suspension at 1 × 107 colony-forming units (cfu)/mL, the highest artemisinin concentration was 8.83 mg/g, and the highest artemisinin production was 70.6 g on day 50. The results of qRT-PCR revealed that expression of genes encoding key enzymes for artemisinin biosynthesis, namely HMGR1, FPS, ADS, CYP71AV1, CPR, DBR, DXS1, and DXR1, was generally upregulated during days 20–50 following induction by Trichoderma. In addition, the moisture, pH stability, organic matter content, and availability of nitrogen, phosphorus, and kalium in inoculated soil were significantly improved. Thus, application of T. asperellum ACCC30536 may offer a novel approach for improving artemisinin production by upregulating the expression of key enzymes for artemisinin biosynthesis, increasing leaf yield, and improving soil fertility.

Biomass and Artemisinin Production of Artemisia annua L. on Several Altitudes

Increasing supply of artemisinin as a multipurpose medicinal compound, especially to cope with malaria, is a global problem that should be solved immediately. This research aimed to obtain high artemisinin production from Artemisia annua L. cultivated at a suitable altitude at Bengkulu Province of Indonesia. Experimental design used was a Split Plot in Randomized Complete Block Design with three replications, altitude (1000, 1100 and 1200 m above sea level altitudes each located at Sukasari, Bandung Baru, and Tangsi Duren, Kepahyang District Province Bengkulu of Indonesia) as main plot and bio-fertilizer application (control, arbuscular mycorrhizal fungi, and phosphate solubilizer fungi) as subplot. Each experimental unit received 10 ton ha-1 of cow-dung. Research result shows that altitude significantly affects artemisinin, essential oil, mycorrhizal and phosphate solubilizer fungus population, but not affect biomass production. Overall, Artemisia annua L. was successfully planted at the 1000 - 1200 m above sea level. Biomass and artemisinin production was higher compared to another experiment, i.e. 350 g per plant with 0.21 - 0.43% of artemisinin content.

Searching for cheaper malaria medicines

In 2016, malaria killed an estimated 445,000 people, 90% of them in Africa. That’s one death every 71 seconds, caused by a disease that is both preventable and treatable. Although that death toll has fallen by 25% since 2010, the World Health Organization recently warned that progress has stalled and called for a redoubled assault on this scourge. Some of the most crucial weapons in this battle are artemisinin-based combination therapies (ACTs), judged to be the most effective antimalarial medicines. They contain derivatives of artemisinin, a molecule found in the plant sweet wormwood (Artemisia annua) that is grown mostly in China and Vietnam. But over the past 15 years, the world’s supply of artemisinin has been wildly erratic. As more farmers planted A. annua to meet rising demand, artemisinin supplies rose and pushed prices down. So farmers switched to more lucrative crops. Artemisinin supplies then plummeted and prices soared again.

Stable Production of the Antimalarial Drug Artemisinin in the Moss Physcomitrella patens.

Malaria is a real and constant danger to nearly half of the world’s population of 7.4 billion people. In 2015, 212 million cases were reported along with 429,000 estimated deaths. The World Health Organization recommends artemisinin-based combinatorial therapies, and the artemisinin for this purpose is mainly isolated from the plant Artemisia annua. However, the plant supply of artemisinin is irregular, leading to fluctuation in prices. Here, we report the development of a simple, sustainable, and scalable production platform of artemisinin. The five genes involved in artemisinin biosynthesis were engineered into the moss Physcomitrella patens via direct in vivo assembly of multiple DNA fragments. In vivo biosynthesis of artemisinin was obtained without further modifications. A high initial production of 0.21 mg/g dry weight artemisinin was observed after only 3 days of cultivation. Our study shows that P. patens can be a sustainable and efficient production platform of artemisinin that without further modifications allow for industrial-scale production. A stable supply of artemisinin will lower the price of artemisinin-based treatments, hence become more affordable to the lower income communities most affected by malaria; an important step toward containment of this deadly disease threatening millions every year.

Transferring a bioactive

Plant Science![Figure][1] Once only found in small hairs on sweet wormwood plants, artemisinin can now be produced by tobacco plants. PHOTO: MANFRED RUCKSZIO / ALAMY STOCK PHOTO Artemisinin, which is critical in defense against malaria, was originally found in tiny hairs on the surface of leaves of the plant Artemisia annua. But this source plant does not support a sufficiently stable supply of artemisinin for worldwide use. Fuentes et al. have developed a synthetic biology approach termed COSTREL (combinatorial supertransformation of transplastomic recipient lines) that produces the immediate precursor of artemisinin, artemisinic acid, in widely cultivated tobacco ( Nicotiana tabacum cv. Petit Havana). The first stage of COSTREL involves transformation of tobacco chloroplasts with genes encoding the core enzymes of the artemisinin biosynthetic pathway. With that pathway established, the second stage involves combinatorial nuclear transformation to superimpose genes that regulate flux through the biosynthetic pathway. A transformation mix supports selection of the most optimal gene combination. The resulting tobacco plants produce artemisinic acid at up to ~4.8 kg per acre. eLife 10.7554/eLife.13664 (2016). [1]: pending:yes

ChemInform Abstract: Synthetic Chemistry Fuels Interdisciplinary Approaches to the Production of Artemisinin

AbstractReview: 59 refs.

Can plant still be a major and cost-effective source for the supply of artemisinin, the most potent anti-malaria drug

Can plant still be a major and cost-effective source for the supply of artemisinin, the most potent anti-malaria drug

Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua.

BackgroundThe medicinal plant Artemisia annua is covered with filamentous trichomes and glandular, artemisinin producing trichomes. A high artemisinin supply is needed at a reduced cost for treating malaria. Artemisinin production in bioreactors can be facilitated if a better insight is obtained in the biosynthesis of artemisinin and other metabolites. Therefore, metabolic activities of glandular and filamentous trichomes were investigated at the transcriptome level.ResultsBy laser pressure catapulting, glandular and filamentous trichomes as well as apical and sub-apical cells from glandular trichomes were collected and their transcriptome was sequenced using Illumina RNA-Seq. A de novo transcriptome was assembled (Trinity) and studied with a differential expression analysis (edgeR).A comparison of the transcriptome from glandular and filamentous trichomes shows that MEP, MVA, most terpene and lipid biosynthesis pathways are significantly upregulated in glandular trichomes. Conversely, some transcripts coding for specific sesquiterpenoid and triterpenoid enzymes such as 8-epi-cedrol synthase and an uncharacterized oxidosqualene cyclase were significantly upregulated in filamentous trichomes. All known artemisinin biosynthesis genes are upregulated in glandular trichomes and were detected in both the apical and sub-apical cells of the glandular trichomes. No significant differential expression could be observed between the apical and sub-apical cells.ConclusionsOur results underscore the vast metabolic capacities of A. annua glandular trichomes but nonetheless point to the existence of specific terpene metabolic pathways in the filamentous trichomes. Candidate genes that might be involved in artemisinin biosynthesis are proposed based on their putative function and their differential expression level.

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.

A Continuous‐Flow Process for the Synthesis of Artemisinin

Isolation of the most effective antimalarial drug, artemisinin, from the plant sweet wormwood, does not yield sufficient quantities to provide the more than 300 million treatments needed each year. The high prices for the drug are a consequence of the unreliable and often insufficient supply of artemisinin. Large quantities of ineffective fake drugs find a market in Africa. Semisynthesis of artemisinin from inactive biological precursors, either dihydroartemisinic acid (DHAA) or artemisinic acid, offers a potentially attractive route to increase artemisinin production. Conversion of the plant waste product, DHAA, into artemisinin requires use of photochemically generated singlet oxygen at large scale. We met this challenge by developing a one-pot photochemical continuous-flow process for the semisynthesis of artemisinin from DHAA that yields 65 % product. Careful optimization resulted in a process characterized by short residence times. A method to extract DHAA from the mother liquor accumulated during commercial artemisinin extractions, a material that is currently discarded as waste, is also reported. The synthetic continuous-flow process described here is an effective means to supplement the limited availability of artemisinin and ensure increased supplies of the drug for those in need.

Stabilizing supply of artemisinin and artemisinin-based combination therapy in an era of wide-spread scale-up

The global demand for artemisinin-based combination therapy (ACT) has grown sharply since its recommendation by the World Health Organization in 2002. However, a combination of financing and programmatic uncertainties, limited suppliers of finished products, information opacity across the different tiers in the supply chain, and widespread fluctuations in raw material prices have together contributed to a market fraught with demand and supply uncertainties and price volatility. Various short-term solutions have been deployed to alleviate supply shortages caused by these challenges; however, new mechanisms are required to build resilience into the supply chain. This review concludes that a mix of strategies is required to stabilize the artemisinin and ACT market. First, better and more effective pooling of demand and supply risks and better contracting to allow risk sharing among the stakeholders are needed. Physical and financial buffer stocks will enable better matching of demand and supply in the short and medium term. Secondly, physical buffers will allow stable supplies when there are procurement and supply management challenges while financial buffer funds will address issues around funding disruptions. Finally, in the medium to long term, significant investments in country level system strengthening will be required to minimize national level demand uncertainties. In addition a voluntary standard for extractors to ensure appropriate purchasing and sales practices as well as minimum quality and ethical standards could help stabilize the artemisinin market in the long term.

Artemisia herba alba & Artemisia monosperma: The Discovery of the first potential Egyptian plant sources for the Pharmaceutical Commercial Production of Artemisinin and Some of Its Related Analogues

Artemisinin has received considerable attention in the last few decades as the current drug of choice for treatment of malaria and a number of other diseases. Because of the development of resistance against other malarial drugs, the demand for artemisinin has rapidly increased during the past decade. However, the supply of artemisinin is troublesome as neither total nor semi-synthesis is economically feasible and the only plant species known to produce artemisinin, Artemisia annua L., contains only low amounts of this compound ranging from (0.01-0.6%) of dry weight. The wish to improve the overall supply of artemisinin at a reduced market price has encouraged interest to hit upon novel plant sources for artemisinin production as alternative to A. annua. In our current study a fingerprint profile method was developed for the detection and quantification of artemisinin and its related analogues in the methanolic extract of Artemisia monosperma and Artemisia herba alba using a high-performance liquid chromatography (HPLC) with diode-array detector (DAD) and ion trap mass spectrometry. Artemisinin, dihydroartemisinin (α and β isomers), artemisitene, dihydroartemisinic aldehyde, dihydroartemisinic acid, dihydroartemisinic alcohol were detected and quantified in the methanolic extract of Artemisia monosperma using LC-MS peak at concentrations of 3.6, 1.9, 0.27, 0.06, 0.08 and 1.95 % of dry plant weight respectively in addition to the detection of arteannuin B and artemisinic acid at a trace levels. While artemisinin, artemisitene, dihydroartemisinic acid, and artemisinic acid were detected and quantified in the methanolic extract of Artemisia herba alba at concentrations of 4.9, 0.35, 0.08 and 0.04 % of dry plant weight respectively . The high unexpected concentration of artemisinin and some of its related analogues detected in this study reported Artemisia herba alba and Artemisia monosperma for the first time as a novel potential plant sources for artemisinin and some of its related analogues that may be helpful for its commercial pharmaceutical production and could lead to the improvement of the overall supply of artemisinin at a reduced market price offering an acceptable price for most patients.especially that these Artemisia species are abundant in distribution in Egyptian desert.

Continuous‐Flow Synthesis of the Anti‐Malaria Drug Artemisinin

Malaria is a serious global health issue. Artemisinin combination treatments are the first-line drugs, but supplies are limited because artemisinin is obtained solely by extraction from Artemisia annua. A continuous-flow process that converts dihydroartemisinic acid into artemisinin (see scheme) was shown to be an inexpensive and scalable process that can ensure a steady, affordable supply of artemisinin.

Research achievements in the synthetic biology and metabolic engineering of artemisinin

Although the biosynthesis of artemisinin is unique to Artemisia annua L., the upstream pathway is ubiquitous among eukaryotes. Therefore, it should be possible to re-establish the downstream pathway for the de novo biosynthesis of artemisinin in eukaryotic microorganisms such as yeast. In the past decade, artemisinin biosynthetic genes have been cloned and introduced into yeast, resulting in the production of several artemisinin precursors including artemisinic acid and dihydroartemisinic acid. However, because of the lack of a suitable cellular environment, these artemisinin precursors failed to be converted into artemisinin. Consequently, A. annua remains the sole source of artemisinin and, therefore, there is a continuing need for research on the genetic improvement of A. annua germlines. Chinese scientists have obtained high-yielding transgenic A. annua plants or varieties using either the opening carbon flux strategy or the closing carbon flux strategy. These studies have shed light on the accelerated and sustainable production of artemisinin. In addition, the rate-limiting steps of artemisinin biosynthesis, particularly the final reaction mechanism, have been identified and characterized. This information is important to simulate the cellular environment in transgenic systems, to enable the successful production and accumulation of artemisinin. Together, these studies have paved the way towards a solution to the problem of limited artemisinin supply. Finally, we discuss the implications of patents for microorganisms that produce artemisinin precursors, and the ways in which China can avoid patent infringements.

Apollon, a new Artemisia annua variety with high artemisinin content

Artemisinin, a sesquiterpene lactone endoperoxide isolated from the herb Artemisia annua L. (Asteraceae), is a highly potent antimalarial compound, which is efficient againt multidrug-resistant strains of Plasmodium falciparum. The promotion of artemisinin-based combination therapies (ACTs) by the WHO during the past years lead to a strong pressure on the world market of artemisinin. The artemisinin world market is volatile and therefore efforts to improve performance of this culture are often limited. The use of varieties with high artemisinin content is a key factor for the development of such cultures. This should secure the supply of artemisinin, lower its cost of production and improve the competitiveness of this new culture with other commercial crops. After the variety Artemis, Mediplant launches a new variety called Apollon with about 20% yield increase. Performances of this new hybrid, with artemisinin content nearing 1.6%, are being presented.

Quantification of the Total Amount of Artemisinin in Leaf Samples by Thin Layer Chromatography

Artemisinin is a natural molecule highly active against malaria. At present, the extraction of this molecule from the leaves of Artemisia annua L. remains the only viable method to produce cheaply large quantities of artemisinin. Agronomic research on this plant species aims to improve agricultural yields, to decrease production costs and to ensure a steady global supply of artemisinin. These research activities require an easy, rapid, low cost, and reliable analytical technique to quantify the artemisinin content in the leaves. Thin layer chromatography (TLC) methods to quantify this molecule have already been published. However, this method does not allow the quantification of the total artemisinin content in the leaves. In order to validate the TLC method, results obtained with this method were related to results for the same samples obtained by accelerated solvent extraction and high pressure liquid chromatography with an evaporative light scattering detector (ASE-HPLC-ELSD). Using the Nernst partition law, a corrective factor of 1.21 is suggested to enable information about the true total amount of artemisinin in leaf samples to be obtained within a range of 0.25 to 3%. In conclusion, this study proposes for the first time a corrective factor in order to quantify the total artemisinin content of A. annua leaves with TLC.