Production of artemisinin by hairy root cultures of Artemisia annua L

Using a combination of sucrose (70 g/L), nitrate (30 mM), inorganic phosphate (1.5 mM), gibberellic acid (5 mg/L) and the ratio of N (NH ) to N - (NO ) (1:5), artemisinin production was increased to 550 mg/L when the cultures of Artemisia annua L hairy root were elicited with a homogenate of Aspergillus oryzae.     

Production of artemisinin by hairy rot cultures of Artemisia annua L in bioreactor

Hairy root cultures of Artemisia annua L were cultivated in four different culture systems: a flask, a bubble column, a modified bubble column and a modified inner-loop airlift bioreactor. The artemisinin contents of hairy root cultures in the bubble column and the modified inner-loop airlift bioreactor were higher than that in the modified bubble column. The growth rate and hairy root distribution in the modified inner-loop airlift bioreactor were better than those in other bioreactors, and dry weight and artemisinin production reached to 26.8 g/L and 536 mg/L after 20 days.     

Enhanced production of artemisinin by Artemisia annua L hairy root cultures in a modified inner-loop airlift bioreactor

Hairy root cultures of Artemisia annua L were cultured in a modified inner-loop airlift bioreactor for achieving maximum artemisinin production. The effects of initial pH, air flow rate, cycle of light irradiation and temperature on growth and artmisinin production in Artemisia annua L hairy root cultures were investigated. Under the optimum conditions, the maximum production of artemisinin reached to 577.5?mg/l after 20 days.     

Improved artemisinin accumulation in hairy root cultures of Artemisia annua by (22S, 23S)-homobrassinolide

When (22S, 23S)-homobrassinolide (SSHB) was added at 1 g l^–1 to hairy root cultures of Artemisia annua, the production of artemisinin reached to 14 mg l^–1, an increment of 57% over the control. SSHB treatment led concomitantly to an increased biomass production of 15 g l^–1. A stimulatory activity of SSHB on nucleic acids and soluble protein content in hairy roots was also observed at the growth stage.     

Isolation and production of artemisinin and stigmasterol in hairy root cultures of Artemisia annua

Scaled-up hairy root culture of Artemisia annua L. was established in three-liter Erlenmeyer flask. Both artemisinin and stigmasterol that derive from the common precursors of isopentenyl diphosphate and farnesyl pyrophosphate were isolated from hairy roots. The production rate of artemisinin isolated by column chromatography from hairy root cultures was 0.54% (mg.gDW⁻¹). Stigmasterol was identified by mass spectrometry and nuclear magnetic resonance analysis. The production of stigmasterol isolated by column chromatography from hairy root cultures was 108.3% (mg.gDW⁻¹). In hairy root cultures, the production rate of stigmasterol was estimated to be 201 times greater than that of artemisinin. Our results suggest that investigation of secondary metabolites may provide a new insight to study artemisinin production in hairy root cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

青蒿毛状根生长、青蒿素合成以及 营养物消耗的动力学

诱导产生的青蒿毛状根培养物置于MS培养基(含30 g/L蔗糖)进行悬浮培养,并对悬浮培养过程中毛状根生长、青蒿素合成、蔗糖、磷酸盐和不同氮源的消耗、pH和电导率的动力学过程进行分析。经30 d培养,生物量干重和青蒿素产量分别达到13.7 g/L和0.23 g/L,碳源和氮源在培养过程中被逐渐利用,而磷酸盐的利用速率最快,培养至15 d所有的磷酸盐均被吸收,pH在培养初期降低,后又逐渐上升,电导率由于毛状根生长对无机离子的吸收而逐渐减低。     

温度对青蒿毛状根生长和青蒿素生物合成的影响

本实验研究了不同温度(15℃～35℃)对青蒿毛状根生长和青蒿素生物合成的影响,发现25℃有利于毛状根生长,30℃促进了青蒿素生物合成。通过温度改变的二步培养技术(培养前20d温度控制在25℃,后10d温度提高到30℃),青蒿素的产量得到明显提高,高于在恒温培养时(25℃或30℃)的结果。     

Modulation of artemisinin biosynthesis by elicitors,inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.     

Effects of gibberellic acid on hairy root cultures of Artemisia annua: Growth and artemisinin production

Summary Artemisinin (AN), a potent antimalarial drug that has been used for centuries as a folk remedy in China, is an effective treatment against quinine-resistant strains of Plasmodium. It can be produced through the in vitro culture of genetically transformed (hairy) roots. The effect of gibberellic acid (GA₃) on the growth and secondary metabolite production of hairy roots of Artemisia annua was investigated. Six different concentrations of GA₃ were tested in shaker flasks to determine the optimum concentration. GA₃ levels of 0.01–0.001 mg/l (28.9–2.89 μM) provided the most significant increase in biomass, and 0.01 mg/l (28.9 μM) produced the highest amount of AN. Investigation of growth kinetics showed that the use of GA₃ at 0.01 mg/l (28.9 μM) increased the growth rate of hairy roots of A. annua by 24.9%. Thus, the cultures treated with GA₃ reached stationary phase faster than control cultures.     

Stimulation of artemisinin production in Artemisia annua hairy roots by the elicitor from the endophytic Colletotrichum sp.

Artemisinin content in hairy roots of Artemisia annua was increased from 0.8 mg g^–1 dry wt to 1 mg g^–1 dry wt by using elicitor treatment of mycelial extracts from the endophytic fungus Colletotrichum sp. The increase of artemisinin was dependent on the growth stage of hairy roots as well as on the dose of the elicitor applied. When hairy roots of 23-day-old cultures (later growth phase) were exposed to the elicitor at 0.4 mg total sugar ml^–1 for 4 days, the maximum production of artemisinin reached to 13 mg l^–1, a 44% increase over the control. This is the first report on the stimulation of artemisinin production in hairy roots by the elicitor from an endophytic fungus of A. annua.     

Factors influencing artemisinin production from shoot cultures of Artemisia annua L.

Artemisinin, an anti-malarial drug isolated from the annual wormwood Artemisia annua L., has a marked activity against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum, and is useful in treatment of cerebral malaria. Shoot cultures of Artemisia annua L. were established on Murashige and Skoog basal medium which contained (per litre) 30 g sucrose, 0.5 mg 6-benzyladenine and 0.05 mg naphthaleneacetic acid. Using an optimized combination of sucrose (30 g/l), nitrate (45 mM), inorganic phosphate (200 mg/l), gibberellic acid (7 mg/l) and the ratio of NH₄ ⁺-N to NO₃ ^–-N of 1:3, artemisinin production reached 26.7 mg/l after 30 days. This procedure provides a potential alternative for production of artemisinin from in vitro tissue cultures.     

Tetraploid Artemisia annua hairy roots produce more artemisinin than diploids

Hairy root cultures of diploid Artemisia annua L. (clone YUT16) grow rapidly and produce the antimalarial sesquiterpene artemisinin. Little is known about how polyploidy affects the growth of transformed hairy roots and the production of secondary metabolites. Using colchicine, we produced four stable tetraploid clones of A. annua L. from the YUT16 hairy root clone. Analysis showed major differences in growth and artemisinin production compared to the diploid clone. Tetraploid clones produced up to six times more artemisinin than the diploid parent. This study provides an initial step in increasing our understanding of the role of polyploidy in secondary metabolite production, especially in hairy roots.     

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.     

Improved growth of Artemisia annua L hairy roots and artemisinin production under red light conditions

Hairy root cultures of Artemisia annua L were cultivated for 30 days under either white, red, blue, yellow or green light. Red light at 660 nm gave the highest biomass of hairy roots (5.73 g dry wt cells l^–1 medium) and artemisinin content (31 mg arteminsinin g^–1 dry cells) which were, respectively, 17% and 67% higher than those obtained under white light.     

Effect of fungal elicitor on thiophene production in hairy root cultures of Tagetes patula

Summary The effect of fungal elicitor, derived from mycelial extracts of Fusarium conglutinans, on thiophene production in hairy-root cultures of Tagetes patula was studied. Various concentrations of elicitor were added to the culture media. Time-course experiments were carried out using a defined concentration of elicitor. Thiophene production increased with the addition of elicitor. The major thiophenes produced were 5-(4-aceoxy-1-butenyl)-2,2-bithiophene and 5-(buten-1-enyl)-2,2'bithiophene.On leave from Department of Biological Sciences, R. J. College, Bombay 86, India Offprint requests to: M. A. Hjortso     

Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua

Artemisinin, isolated from the shrub-Artemisia annua, is a sesquiterpene lactone used to treat multi-drug resistant strains of falciparum malaria. It is also effective against a wide variety of cancers such as leukemia and colon cancer. To counter the present low content in leaves and uneconomical chemical synthesis, alternate ways to produce artemisinin have been sought. But this compound remains elusive in cell cultures of A. annua despite the extensive studies undertaken. This work reports the first successful approach for production of artemisinin by cell cultures of Indian variety of A. annua. In the present study, an integrated yield enhancement strategy, developed by addition of selected precursor (mevalonic acid lactone) and elicitor (methyl jasmonate) at optimized concentrations, resulted in 15.2g/l biomass and 110.2mg/l artemisinin, which was 5.93 times higher in productivity in comparison to control cultures.     

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l^-1 BAP, 0.05 mg l^-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l^-1 gibberellic acid, 0.5 g l^-1 casein hydrolysate, 10 mg l^-1 or 20 mg l^-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.Abbreviations BAP benzylaminopurine - DW dry weight - FW fresh weight - GA₃ gibberellic acid - MS Murashige & Skoog basal medium - NAA naphthaleneacetic acid     

Metabolic engineering of artemisinin biosynthesis in Artemisia annua L.

Artemisinin, a sesquiterpene lactone isolated from the Chinese medicinal plant Artemisia annua L., is an effective antimalarial agent, especially for multi-drug resistant and cerebral malaria. To date, A. annua is still the only commercial source of artemisinin. The low concentration of artemisinin in A. annua, ranging from 0.01 to 0.8% of the plant dry weight, makes artemisinin relatively expensive and difficult to meet the demand of over 100 million courses of artemisinin-based combinational therapies per year. Since the chemical synthesis of artemisinin is not commercially feasible at present, another promising approach to reduce the price of artemisinin-based antimalarial drugs is metabolic engineering of the plant to obtain a higher content of artemisinin in transgenic plants. In the past decade, we have established an Agrobacterium-mediated transformation system of A. annua, and have successfully transferred a number of genes related to artemisinin biosynthesis into the plant. The various aspects of these efforts are discussed in this review.     

Biosynthesis of artemisinin in Artemisia annua

The isotope ratios (³H:¹⁴C) in arteannuin B and artemisinin biosynthesized in Artemisia annua from [4R-³H₁,2-¹⁴C]-, [5-³H₂,2-¹⁴C]- and [2-³H₂,2-¹⁴C](3RS)- mevalonate have revealed that two specific 1,2-hydride shifts take place during the oxidation and lactonization of the germacrane skeleton to yield dihydrocostunolide. The gem-methyls of DMAPP retain their identity until the final steps of artemisinin biosynthesis. Arteannuin B is considered to be a late precursor of artemisinin and the following biosynthetic sequence is suggested: farnesylpyrophosphate → germacrane skeleton → dihydrocostunolide → cadinanolide → arteannuin B → artemisinin.