Production of artemisinin by genetically-modified microbes

Artemisinin, an endoperoxidized sesquiterpene originally extracted from the medicinal plant Artemisia annua L., is a potent malaria-killing agent. Due to the urgent demand and short supply of this new antimalarial drug, engineering enhanced production of artemisinin by genetically-modified or transgenic microbes is currently being explored. Cloning and expression of the artemisinin biosynthetic genes in Saccharomyces cerevisiae and Escherichia coli have led to large-scale microbial production of the artemisinin precursors such as amorpha-4,11-diene and artemisinic acid. Although reconstruction of the complete biosynthetic pathway toward artemisinin in transgenic yeast and bacteria has not been achieved, artemisinic acid available from these transgenic microbes facilitates the subsequent partial synthesis of artemisinin by either chemical or biotransformational process, thereby providing an attractive strategy alternative to the direct extraction of artemisinin from A.annua L. In this review, we update the current trends and summarize the future prospects on genetic engineering of the microorganisms capable of accumulating artemisinin precursors through heterologous and functional expression of the artemisinin biosynthetic genes.     

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 Δ11(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 1α 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.     

Salicylic acid activates artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

This paper provides evidence that salicylic acid (SA) can activate artemisinin biosynthesis in Artemisia annua L. Exogenous application of SA to A. annua leaves was followed by a burst of reactive oxygen species (ROS) and the conversion of dihydroartemisinic acid into artemisinin. In the 24 h after application, SA application led to a gradual increase in the expression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene and a temporary peak in the expression of the amorpha-4,11-diene synthase (ADS) gene. However, the expression of the farnesyl diphosphate synthase (FDS) gene and the cytochrome P450 monooxygenase (CYP71AV1) gene showed little change. At 96 h after SA (1.0 mM) treatment, the concentration of artemisinin, artemisinic acid and dihydroartemisinic acid were 54, 127 and 72% higher than that of the control, respectively. Taken together, these results suggest that SA induces artemisinin biosynthesis in at least two ways: by increasing the conversion of dihydroartemisinic acid into artemisinin caused by the burst of ROS, and by up-regulating the expression of genes involved in artemisinin biosynthesis.     

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.

Abstract

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.

     

Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor,artemisinic acid

Background  

Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required.     

Exogenous GA<Subscript>3</Subscript> and flowering induce the conversion of artemisinic acid to artemisinin in <Emphasis Type="Italic">Artemisia annua</Emphasis> plants

The contents of artemisinin and artemisinic acid were monitored in the Artemisia annua plants treated with GA₃ at vegetative and flowering initiation stages. The highest artemisinin content was observed at full bloom. The decrease in artemisinic acid content occurred during the transition from the vegetative stage to the beginning of flowering. Endogenous GA₃ content in the leaves peaked at full bloom. At the vegetative stage, in plants treated with various concentrations of GA₃ , the content of artemisinin increased while that of artemisinic acid decreased. Apparently, the rate-limiting step in artemisinin biosynthesis was from artemisinic acid to artemisinin. The bottleneck of artemisinin biosynthesis was probably unlocked during the flowering or in the vegetative plants treated with GA₃ , which triggered off the conversion of artemisinic acid to artemisinin.From Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 68–73.Original English Text Copyright © 2005 by Zhang, Ye, Liu, Wang, Li.This article was presented by the authors in English.     

Artemisinin production by transformed roots of Artemisia annua

Summary Transformed root cultures of several strains of Artemisia annua were obtained by infection with Agrobacterium rhizogenes ATCC 15834. Production of artemisinin, measured by HPLC, ranged from 0–0.42 % of dry weight (DW) in 10 different clones. Artemistene, artemisinic acid, and arteannuin B were also measured. Comparisons to literature reports suggest that the commercial production of artemisinic compounds using transformed roots is feasible.     

Transgenic approach to increase artemisinin content in Artemisia annua L.

Artemisinin, the endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from the Chinese medicinal plant Artemisia annua L. Due to its effectiveness against multi-drug-resistant cerebral malaria, it becomes the essential components of the artemisinin-based combination therapies which are recommended by the World Health Organization as the preferred choice for malaria tropica treatments. To date, plant A. annua is still the main commercial source of artemisinin. Although semi-synthesis of artemisinin via artemisinic acid in yeast is feasible at present, another promising approach to reduce the price of artemisinin is using plant metabolic engineering to obtain a higher content of artemisinin in transgenic plants. In the past years, an Agrobacterium-mediated transformation system of A. annua has been established by which a number of genes related to artemisinin biosynthesis have been successfully transferred into A. annua plants. In this review, the progress on increasing artemisinin content in A. annua by transgenic approach and its future prospect are summarized and discussed.     

Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin

Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua, forms the basis of the most important treatments of malaria in use today. In an effort to elucidate the biosynthesis of artemisinin, an expressed sequence tag approach to identifying the relevant biosynthetic genes was undertaken using isolated glandular trichomes as a source of mRNA. A cDNA clone encoding a cytochrome P450 designated CYP71AV1 was characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of the proposed biosynthetic intermediates amorpha-4,11-diene, artemisinic alcohol and artemisinic aldehyde. The identification of the CYP71AV1 gene should allow for the engineering of semi-synthetic production of artemisinin in appropriate plant or microbial hosts.     

Enhanced artemisinin production from engineered yeast precursors upon biotransformation

Abstract

Production of artemisinin in genetically modified microorganisms is an attractive option to enable sufficient supply of the effective antimalarial agent. Although a sundry of artemisinin precursors are available from engineered bacteria or yeast, no artemisinin has been manufactured by engineering any microbial platforms due to inaccessibility to unidentified steps. To this end, it is essential to consider how to convert artemisinin precursors to artemisinin, either biochemically or chemically. To establish a novel procedure of artemisinin production, we incubate the mixture of artemisinin precursors from engineered Sacchromyces cerevisiae with the cell-free enzyme extract of Artemisia annua. For the single gene-expressing strain INVScI (pYES-ADS), amorpha-4,11-diene accumulation within 48 h or 14 days led to higher artemisinin content than the control. In the multiple gene-expressing strain YPH501 (pYES-ADS:: pESC-CYP71AV1-DBR2), artemisinin accumulation from the 14-day-induced yeast precursor mixture was nearly equivalent between the single gene-transferred strain and the multiple gene-transferred strain. Alternatively, biotransformation of 48-hour-induced yeast amorpha-4,11-diene mixture by the cold-acclimated A. annua cell-free extract that possesses the abundant enzymes relevant to artemisinin biosynthesis gave rise to considerable elevation of artemisinin content up to 0.647% in maximum, accounting to 15-folds increase as the A. annua cell-free extract without cold-acclimation (0.045%), thereby providing a practical protocol for artemisinin overproduction through the interplay of engineered microbial artemisinin precursors with upregulated plant enzymes.     

Comparative quantitative analysis of artemisinin by chromatography and qNMR

Introduction – Since the discovery of artemisinin in the 1970s, many techniques based on diverse chromatography techniques have been developed to detect and quantify this important antiplasmodial compound. The accurate quantification of this compound in the Artemisia annua plant material is mainly needed for breeding purposes in order to cultivate higher yielding varieties. It is also important for the quality control of herbal preparations containing A. annua plant material. Objective – To evaluate the most common validated quantification techniques (LC‐MS, HPLC‐ELSD and TLC) and compare the results to quantitative nuclear magnetic resonance spectroscopy (qNMR) in eight different A. annua samples collected from around the world. Methodology – The leaf material were extracted according to standard procedures and analysed with the validated quantification techniques. For the qNMR analysis we did not employ a standard curve but instead used an internal standard (maleid acid) which is not chemically related to artemisinin. Results – We found a significant difference between the results in this study. Compared with the qNMR results the HPLC‐ELSD corresponded closely, followed by LC‐MS. Quantitation with TLC led to an estimation range of ?0.5 to +3.2 mg artemisinin/g of A. annua. Conclusion – These results imply that qNMR, with the addition of an internal standard, can be used to quantify artemisinin in A. annua samples in a rapid and reproducible manner. Copyright © 2010 John Wiley & Sons, Ltd.     

Glucose-6-phosphate dehydrogenase plays critical role in artemisinin production of <Emphasis Type="Italic">Artemisia annua</Emphasis> under salt stress

Artemisinin, a natural sesquiterpenoid isolated from Artemisia annua L., is regarded as the most efficient drug against malaria in the world. Artemsinin production in NaCl-treated A. annua seedlings and its relationships with the glucose-6-phosphate dehydrogenase (G6PDH) activity and generation of H₂O₂ and nitric oxide (NO) were investigated. Results revealed that artemisinin content in the seedlings was increased by 79.3 % over the control after 1-month treatment with 68 mM NaCl. The G6PDH activity was enhanced in the presence of NaCl together with stimulated generation of H₂O₂ and NO. Application of 1.0 mM glucosamine (GlcN), an inhibitor of G6PDH, blocked the increase of NADPH oxidase and nitrate reductase (NR) activities, as well as H₂O₂ and NO production in A. annua seedlings under the salt stress. The induced H₂O₂ was found to be involved in the upgrading gene expression of two key enzymes in the later stage of artemisinin biosynthetic pathway: amorphadiene synthase (ADS) and amorpha-4,11-diene monooxygenase (CYP71AV1). The released NO being attributed mainly to the increase of NR activity, negatively interacted with H₂O₂ production and enhanced gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Inhibition of NO generation partly blocked NaCl-induced artemisinin accumulation, and NO donor strongly rescued the decreased content of artemisinin caused by GlcN. These results suggest that G6PDH could play a critical role in NaCl-induced responses and artemisinin biosynthesis in A. annua.     

Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin

The sesquiterpenoid artemisinin, isolated from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight. Received: 8 June 2000 / Accepted: 21 August 2000     

The molecular cloning of artemisinic aldehyde Delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Delta11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Delta11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small alpha,beta-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.