Abscisic acid (ABA) treatment increases artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> by enhancing the expression of genes in artemisinin biosynthetic pathway

Artemisinin, a sesquiterpene lactone endoperoxide derived from Artemisia annua L., is the most effective antimalarial drug. In an effort to increase the artemisinin production, abscisic acid (ABA) with different concentrations (1, 10 and 100 μM) was tested by treating A. annua plants. As a result, the artemisinin content in ABA-treated plants was significantly increased. Especially, artemisinin content in plants treated by 10 μM ABA was 65% higher than that in the control plants, up to an average of 1.84% dry weight. Gene expression analysis showed that in both the ABA-treated plants and cell suspension cultures, HMGR, FPS, CYP71AV1 and CPR, the important genes in the artemisinin biosynthetic pathway, were significantly induced. While only a slight increase of ADS expression was observed in ABA-treated plants, no expression of ADS was detected in cell suspension cultures. This study suggests that there is probably a crosstalk between the ABA signaling pathway and artemisinin biosynthetic pathway and that CYP71AV1, which was induced most significantly, may play a key regulatory role in the artemisinin biosynthetic pathway.     

青蒿素生物合成分子调控研究进展

青蒿素是目前世界上最有效的疟疾治疗药物。通过对青蒿素的生物合成途径,青蒿素生物合成途径的关键酶,青蒿素生物合成的分子调控的介绍,综述了青蒿素生物合成分子调控的最新研究进展。     

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.     

Isoprenoid biosynthesis in Artemisia annua: cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library

Artemisia annua (Asteraceae) is the source of the anti-malarial compound artemisinin. To elucidate the biosynthetic pathway and to isolate and characterize genes involved in the biosynthesis of terpenoids including artemisinin in A. annua, glandular trichomes were used as an enriched source for biochemical and molecular biological studies. The sequencing of 900 randomly selected clones from a glandular trichome plasmid cDNA library revealed the presence of many ESTs involved in isoprenoid biosynthesis such as enzymes from the methylerythritol phosphate pathway and the mevalonate pathway, amorpha-4,11-diene synthase and other sesquiterpene synthases, monoterpene synthases and two cDNAs showing high similarity to germacrene A synthases. Full-length sequencing of the latter two ESTs resulted in a 1686-bp ORF encoding a protein of 562 aa. Upon expression in Escherichia coli, the recombinant protein was inactive with geranyl diphosphate, but catalyzed the cyclization of farnesyl diphosphate to germacrene A. These results demonstrate the potential of the use of A. annua glandular trichomes as a starting material for studying isoprenoid biosynthesis in this plant species.     

Effect of wounding on gene expression involved in artemisinin biosynthesis and artemisinin production in <Emphasis Type="Italic">Artemisia annua</Emphasis>

Abstract-Effects of mechanical wounding on gene expression involved in artemisinin biosynthesis and artemisinin production in Artemisia annua leaves were investigated. HPLC-ELSD analysis indicated that there was a remarkable enhancement of the artemisinin content in 2 h after wounding treatment, and the content reached the maximum value at 4 h (nearly 50% higher than that in the control plants). The expression profile analysis showed that many important genes (HMGR, ADS, CPR, and CYP71AV1) involved in the artemisinin biosynthetic pathway were induced in a short time after wounding treatment. This study indicates that the artemisinin biosynthesis is affected by mechanical wounding. The possible mechanism of the control of gene expression during wounding is discussed.     

Functional genomics and the biosynthesis of artemisinin

Artemisinin, a sesquiterpene lactone endoperoxide derived from the glandular secretory trichomes (GSTs) of Artemisia annua, provides the basis for the most effective treatments of malaria. The biology and biochemistry of GSTs of the Asteraceae and their biosynthesis of isoprenoids is reviewed. Recent efforts to understand the biosynthesis of artemisinin in A. annua GSTs are discussed in detail. This includes the development in the authors' laboratory of an expressed sequence tag (EST) approach to identifying the relevant biosynthetic genes using isolated GST as a source of mRNA. This has lead to the isolation of a cDNA encoding CYP71AV1, a multifunctional cytochrome P450 which catalyzes multiple oxidations of the sesquiterpene intermediate amorpha-4,11-diene to artemisinic acid. Further biochemical and molecular genetic work is required to elucidate the precise route from artemisinic alcohol to artemisinin and to engineer more efficient low cost production of artemisinin-based antimalarial drugs.     

Promotion of artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> by overexpression of multiple artemisinin biosynthetic pathway genes

Artemisinin, isolated from an annual herbaceous plant Artemisia annua L., is an effective antimalarial compound. However, artemisinin is accumulated in small amounts (0.01–0.1% leaf dry weight) in A. annua, resulting in constant high artemisinin price. Although metabolic engineering of partial artemisinin metabolic pathway in yeast achieved great success, artemisinin from A. annua is still the important business resource. Here, we report on the generation of transgenic plants with simultaneously overexpressing four artemisinin biosynthetic pathway genes, amorpha-4,11-diene synthase gene (ADS), amorpha-4,11-diene 12-monooxygenase gene (CYP71AV1), cytochrome P450 reductase gene (CPR), and aldehyde dehydrogenase 1 gene (ALDH1) via Agrobacterium-mediated transformation. The qRT-PCR analysis demonstrated that the introduced four genes of the transgenic lines were all highly expressed. Through high-performance liquid chromatography analysis, the artemisinin contents were increased markedly in transformants, with the highest being 3.4-fold higher compared with non-converter. These results indicate that overexpression of multiple artemisinin biosynthetic pathway genes is a promising approach to improve artemisinin yield in A. annua.     

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.     

Computational identification of sweet wormwood (Artemisia annua) microRNA and their mRNA targets

Despite its efficacy against malaria, the relatively low yield (0.01%-0.8%) of artemisinin in Artemisia annua is a serious limitation to the commercialization of the drug. A better understanding of the biosynthetic pathway of artemisinin and its regulation by both exogenous and endogenous factors is essential to improve artemisinin yield. Increasing evidence has shown that microRNAs (miRNAs) play multiple roles in various biological processes. In this study, we used previously known miRNAs from Arabidopsis and rice against expressed sequence tag (EST) database of A. annua to search for potential miRNAs and their targets in A. annua. A total of six potential miRNAs were predicted, which belong to the miR414 and miR1310 families. Furthermore, eight potential target genes were identified in this species. Among them, seven genes encode proteins that play important roles in ar- temisinin biosynthesis, including HMG-CoA reductase (HMGR), amorpha-4,11-diene synthase (ADS), farnesyl pyrophosphate synthase (FPS) and cytochrome P450. In addition, a gene coding for putative AINTEGUMENTA, which is involved in signal transduction and development, was also predicted as one of the targets. This is the first in silico study to indicate that miRNAs target genes encoding enzymes involved in artemisinin biosynthesis, which may help to understand the miRNA-mediated regulation of artemisinin biosynthesis in A. annua.     

利用异源生物生产青蒿素及其前体的研究进展

青蒿素是从中药青蒿中分离出来的一种倍半萜内酯类化合物,也是目前最有效的抗疟疾药物,对人类健康意义重大。该文通过对青蒿素生物合成途径及其相关酶的介绍,综述了利用异源生物通过组合生物合成途径生产青蒿素及其前体的最新研究进展。     

青蒿素对蔬菜种子发芽和幼苗生长的化感效应

试验以菜豆、豇豆、大白菜和小白菜为对象,用不同浓度的青蒿素浸种,研究了黄花蒿产生的化感物质??青蒿素对蔬菜种子发芽及幼苗生长的影响。结果表明,青蒿素对蔬菜种子发芽和幼苗生长的化感作用表现出浓度效应和品种差异,即浓度越高,抑制作用愈强,尤以豇豆种子发芽率和小白菜生长的表现最为明显,前者的发芽率可降低75.00%,后者的苗高降幅高达88.37%,且胚根停止生长。青蒿素抑制同季和后季作物的种子发芽和幼苗生长,有利于扩大黄花蒿的生存空间,增强生存竞争优势。在黄花蒿?蔬菜种植体系中,选择抗化感作用较强的大白菜和菜豆可提高土地利用率和整体生产水平。用青蒿素浸种后,蔬菜幼苗的根系活力降低,菜豆和豇豆叶绿素含量提高,而大、小白菜降低,均可视为妨碍生长的生理原因。此外,青蒿素浸种还提高蔬菜种子可溶性糖和游离氨基酸含量,推测青蒿素对种子水解酶活性的影响较小,但抑制合成酶催化的生化反应,导致代谢紊乱,抑制幼苗生长。     

TLC-densitometric analysis of artemisinin for the rapid screening of high-producing plantlets of Artemisia annua L

A simple TLC-densitometric technique has been developed for the rapid and accurate analysis of artemisinin in a large number of Artemisia annua plantlets cultured in vitro. This new analytical method is based on the structural conversion of artemisinin on a silica gel layer by ammonia vapour to form 10-azadesoxyartemisinin, a chromophore-containing compound (lambdamax 320 nm) that can be detected by UV-based TLC densitometry. The TLC system was evaluated quantitatively in terms of product stability, precision, accuracy and calibration. Good linearity was obtained in the range of 0.01-0.12 microg artemisinin. The technique appeared to be accurate and sensitive as compared with the complicated pre-column reaction-HPLC technique. Among 90 samples of A. annua plantlets, the artemisinin content in the leaves appeared to be highly variable, ranging from 0.02 to 0.67% w/w dry weight. These results demonstrate that densitometric TLC can be a cheap and simple technique for the accurate screening of high-artemisinin-producing plants.     

Nitric oxide potentiates oligosaccharide-induced artemisinin production in Artemisia annua hairy roots

The purpose of the present study was to characterize the generation of nitric oxide （NO） in Artemisia annua roots induced by an oligosaccharide elicitor （OE） from Fusarium oxysporum mycelium and the potentiation role of NO in the elicitation of artemisinin accumulation. The OE （0.3 mg total sugar/mL） induced a rapid production of NO in cultures, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 8 h of OE treatment. Artemisinin content in 20-day-old hairy roots was increased from 0.7mg/g dry wt to 1.3 mg/g dry wt by using the OE treatment for 4d. In the absence of OE, the NO donor sodium nitroprusside （SNP） at 10, 50 ~1 and 100 ~1 enhanced the growth of hairy roots, but had no effect on artemisinin synthesis, The combination of SNP with OE increased artemisinin content from 1.2 mg/g drywt to 2.2 mg/g dry wt, whereas the maximum production of artemisinin in cultures was 28.5 mg/L, a twofold increase over the OE treatment alone. The effects of SNP on the OE-induced artemisinin were suppressed strongly by the NO scavenger 2-（4- carboxyphenyl）-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide （cPTIO）. The results suggest that NO can strongly potentiate elicitor-induced artemisinin synthesis in A. annua hairy roots.     

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.     

Concise synthesis of artemisinin from a farnesyl diphosphate analogue

Artemisinin is one of the most potent anti-malaria drugs and many often-lengthy routes have been developed for its synthesis. Amorphadiene synthase, a key enzyme in the biosynthetic pathway of artemisinin, is able to convert an oxygenated farnesyl diphosphate analogue directly to dihydroartemisinic aldehyde, which can be converted to artemisinin in only four chemical steps, resulting in an efficient synthetic route to the anti-malaria drug.