Production of the antimalarial drug precursor artemisinic acid in engineered yeast

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=Production of the antimalarial drug precursor artemisinic acid in engineered yeast=
 
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This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history.
This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history.
The authors describe how they managed to prepare a yeast strain Saccharomyces cerevisiae, also known as Baker’s yeast, which was able to produce artemisinic acid. Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
The authors describe how they managed to prepare a yeast strain Saccharomyces cerevisiae, also known as Baker’s yeast, which was able to produce artemisinic acid. Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.
-
==Part one:==
+
==Malaria and treatment==
 +
=== Malaria===
 +
Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%)  are misled in Sub-Saharan Africa (Layne SP. 2006).
 +
It is caused by four species of sporozoa, but mostly by Plasmodium vivax and Plasmidium falciparum. This parasite performs part if its life cycle in human and part of it in the mosquito called Anopheles. Female mosquitoes of genus Anopheles transmit protozoan Plasmodium falciparum from person to person. The life cycle of Plasmidium falciparum is complex. When mosquito injects saliva together with Plasmidium falciparum sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and ifect erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This  asexual reproductin cycle takes approximately 48 hours. During this 48-hour period, malaria specific sindroms occur, such as chills, followed by fever up to 40◦C when Plasmidium falciparum cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia.
 +
Not all protozoal cells liberated from red blood cells are able to infect other erithrocytes. The protozoal cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito.When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zigote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006).
 +
Picture of Plasmodium life cycle:  http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html
 +
===Treatment===
 +
Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley e tal. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed[3]. Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .
 +
 
 +
In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or Artemisia annua. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu e tal. 1979) .
 +
Today World Health Organisation recommends artemisinin-based combination therapies.
 +
== Production of the antimalarial drug precursor artemisinic acid in engineered yeast==
 +
 
 +
===Mevalonate pathway and artemisinin synthesis pathway===
 +
 
 +
Even though artemisinin can be exctacted from A. annua plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) .
 +
In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing productin of artemisinic acid in yeast Saccharomyces cerevisiae. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. Saccharomyces cerevisiae in an eucariotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper.
 +
Artemisinin  has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .
 +
 
 +
Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.
 +
 
 +
In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate)  are synthesized from Acetyl-CoA via mevalonate pathway.
 +
 
 +
This mevalonate pathway is present in all organisms including Saccharomices cerevisiae. Here should be mentioned that in Saccharomices cerevisiae mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, Mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of Ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).
 +
 
 +
In the second stage cyclic trepenes  are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is Amorpha – 4, 11 diene from A. annua. This intermediate is in next steps transformed into Artemisinic acid.
 +
 
 +
The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of  artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS)  (Bouwmeester et al. 1999). Gene encoding this enzyme was already known.
 +
However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates.
 +
Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/
 +
amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.
 +
 
 +
=== isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in A. annua ===
 +
 
 +
Previous research demonstrated, that enzyme catalyzing  the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005).
 +
 
 +
Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is a insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).
 +
 
 +
In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs)  belonging to sunflower and lettuce  from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu).
 +
EST is abbrevation for An expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their  length is limited by current technology to approximately 500 to 800 nucleotides.They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).
 +
 
 +
With known amino acid sequences of  cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from A. annua.
 +
 
 +
At the same time total RNA from trichome enriched cells of A.anua was extracted. Using total RNA, cDNA pool was prepeared.
 +
 
 +
Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in A. annua, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.
 +
 
 +
Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an A. annua trichome-enriched complementaryDNA pool.
 +
 
 +
Comparing DNA sequences of those fragmets single A. annua P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this A. annua P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a sutable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.
 +
 
 +
With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from A. annua was possible.
 +
 
 +
As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used A. annua cytochrome P450 oxidoreductase (CPR)  as a redox partner.
 +
 
 +
===rewriting the genome of ordinary Saccharomyces cerevisiae to encourage it to make artemisinic acid===
 +
 
 +
With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid.
 +
What they needed to do was:
 +
 
 +
1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols,
 +
2. To introduce the amorphadiene synthase gene (ADS) from A. annua into the high FPP producer to convert FPP to amorphadiene, and
 +
3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from A. annua and expressing it in the amorphadiene producer
 +
 
 +
 
 +
Yeast Integrating plasmids (YIp): These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination.
 +
==References==
 +
Layne SP. "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15)
 +
Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark
 +
Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance
 +
and prospects for new agents. Pharmacol Ther 79:55–87
 +
Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144
 +
 
 +
Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143
 +
 
 +
Zeng Q, Qiu F, Yuan L. Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 2008;30:581–592.
 +
 
 +
Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London
 +
 
 +
Covello PS, Teoh  KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871
 +
 
 +
Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930
 +
 
 +
Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854
 +
 
 +
Bertea, C. M. et al. Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47 (2005).
 +
 
 +
Roland Sigel; Sigel, Astrid; Sigel, Helmut (2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley. ISBN 0-470-01672-8.
 +
 
 +
Adams MD, Kelley JM, Gocayne JD, et al. (Jun 1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873. PMID 2047873.

Revision as of 00:33, 20 January 2015

This coursework is my attempt to paraphrase and explain in simple words the article of Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD with the title Production of the antimalarial drug precursor artemisinic acid in engineered yeast. The article was published in 2006 in Nature and it is one of the most cited articles in history. The authors describe how they managed to prepare a yeast strain Saccharomyces cerevisiae, also known as Baker’s yeast, which was able to produce artemisinic acid. Artemisinic acid can be afterwards chemically transformed into artemisinin, which is today the first line treatment for malaria worldwide. I divided this coursework in three parts. In the first part some aspects of malaria as disease and history of treatment are described. Second part is trying to explain the work behind the article. The third part is highlighting what happened after happy ending of the article in 2006, in other words, how things went on until today.

Contents

Malaria and treatment

Malaria

Malaria is an emerging epidemic disease, occurring in warmer parts of the world. Because of its occurrence in tropical and subtropical areas where a lot of moisture is present its name is derived from Italian words for “bad air” [2]. It is endemic in a broad band around the equator in America, Asia and Africa. Most of deaths (85–90%) are misled in Sub-Saharan Africa (Layne SP. 2006). It is caused by four species of sporozoa, but mostly by Plasmodium vivax and Plasmidium falciparum. This parasite performs part if its life cycle in human and part of it in the mosquito called Anopheles. Female mosquitoes of genus Anopheles transmit protozoan Plasmodium falciparum from person to person. The life cycle of Plasmidium falciparum is complex. When mosquito injects saliva together with Plasmidium falciparum sporozoites into a human host, those small, elongated cells travel through the bloodstream to the liver, where they convert into larger cells called schizont. Those cells divide into many small cells called merozoites, which enter bloodstream and ifect erythrocytes. In red blood cells merozites grow, divide and exit cell by cell lysis. This asexual reproductin cycle takes approximately 48 hours. During this 48-hour period, malaria specific sindroms occur, such as chills, followed by fever up to 40◦C when Plasmidium falciparum cells are released from cells. Because of the loss of red blood cells, malaria generally causes anemia. Not all protozoal cells liberated from red blood cells are able to infect other erithrocytes. The protozoal cells, that cannot infect red blood cells are named gametocytes and are infective only for mosquito.When another mosquito feeds with infected blood gametocytes enter its digestive tract. In mosquito sexual production occurs and zigote is formed, which forms number of sporozoites. Some of these reach the salivary gland of the mosquito, and are injected into next human reservoir by the bite of mosquito (Madigan et al. 2006). Picture of Plasmodium life cycle: http://www.uni-tuebingen.de/modeling/Mod_Malaria_Cycle_en.html

Treatment

Until now no antimalarial vaccine had been developed. Consequently all the treatment of malaria relies upon antilamarial drugs. First drug against malaria was quinine. It was extracted from the bark of the Cinchona tree in 1820 and was the only drug used for malaria treatment until 1930s (Foley e tal. 1998) . Due to the occurrence of resistance of plasmodium species against quinine, search of new active pharmaceutical substances against malaria started. In the context of those researches chloroquinine and other synthetic guinoline antimalarials such as mefloquine were developed[3]. Unfortunately, plasmodium species developed resistance against those drugs, too (Warhurst 2001) .

In China another antimalarial drug was discovered in glandular trichomes on leaves and floral buds of plant called sweet wormwood or Artemisia annua. It was called artemisinin. Molecules of artemisinin contain a peroxide (O-O) group. In the presence of iron from damaged blood cells, the peroxide group is assumed to generate reactive free radicals which could destroy the DNA of the plasmodium (Liu e tal. 1979) . Today World Health Organisation recommends artemisinin-based combination therapies.

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

Mevalonate pathway and artemisinin synthesis pathway

Even though artemisinin can be exctacted from A. annua plants, this way of obtaining the drug faces multiple obstacles. One of the major obstacle in direct extraction from plants lies in dependence of artemisinin assay in plant tissue upon natural environmental variability. Contamination by other plant trepenes, can cause problems in steps of purification (Zeng et al. 2008) . In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby make it unaffordable for third world patients, Ro and coworkers published an article describing productin of artemisinic acid in yeast Saccharomyces cerevisiae. Their idea was to rewrite the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid. Saccharomyces cerevisiae in an eucariotic microorganism and can be easily grown in large bioreactors in industrial environment, thus making artemisinin production and especially purification cheaper. Artemisinin has been categorized as a terpenoid or isoprenoid (Connolly et al. 1991) .

Ro and coworkers knew, that artemisinin biosynthesis pathway consists of two stages.

In the first stage linear isoprene precursors, such as GPP ( geranyl pyrophosphate ) later converted into FPP (farnesyl pyrophosphate) are synthesized from Acetyl-CoA via mevalonate pathway.

This mevalonate pathway is present in all organisms including Saccharomices cerevisiae. Here should be mentioned that in Saccharomices cerevisiae mevalonate pathway which starts from Acetyl-CoA and goes through intermediates such as HMG-CoA, Mevalonate, IPP, GPP and FPP continues into synthesis of Squalene, which is then used for the synthesis of Ergosterol (Covello et al. 2008) . Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible (Madigan et al. 2006).

In the second stage cyclic trepenes are synthesized from linear isoprene precursors ( GPP, FPP ). In case of artemisinic acid synthesis cyclic trepene is Amorpha – 4, 11 diene from A. annua. This intermediate is in next steps transformed into Artemisinic acid.

The involvement of mevalonate pathway in artemisinin biosynthesis has been already proven (Akhila et al. 1987). It was also known, that amorpha – 4, 11 diene, the first intermediate in the second stage of artemisinin is cyclized FPP by enzyme amorpha – 4, 11 diene synthase (ADS) (Bouwmeester et al. 1999). Gene encoding this enzyme was already known. However, the gene and amino acid sequence of the next enzyme in pathway was still unknown. This at the time unknown enzyme should be able to catalyze reaction of oxidation of amorpha – 4, 11 diene into artemisinic acid via artemisinic aldehyde and artemisinic alcohol intermediates. Ro and coworkers made great breakthrough and identified a cytochrome P450 monooxygenase (CYP71AV1)/ amorpha-4,11-diene oxidase (AMO) for the oxidation of amorpha - 4,11-diene.

isolating genes encoding enzymes responsible for oxidizing amorphadiene to artemisinic acid in A. annua

Previous research demonstrated, that enzyme catalyzing the first regiospecific hydroxylation of amorphadiene in A. annua belongs to group on enzymes called cytochrome P450 monooxygenase (P450) (Bertea et al. 2005).

Cytochrome P450 enzymes (CYPs) are, in general, oxidase enzymes. The most common reaction catalyzed by those enzymes is a insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water. Most CYPs require a protein partner to deliver one or more electrons (Siegel et al. 2007).

In order to find this specific enzyme they collected P450-expressed-sequence tags (ESTs) belonging to sunflower and lettuce from the Asteraceae EST-database (http://www.cgpdb.ucdavis.edu). EST is abbrevation for An expressed sequence tag. They are short sub-sequences of a cDNA sequence. ESTs have a relatively low quality of sequence. Their length is limited by current technology to approximately 500 to 800 nucleotides.They may be used to identify gene transcripts, to unravel new genes or to determine gene sequences. They also represent portions of expressed genes (Adams et al. 1991).

With known amino acid sequences of cytochrome P450 monooxygenases from sunflowers and lettuce (CYP71 subfamily), they designed degenerated primers, which would in PCR reaction amplify even cytochrome P450 monooxygenases from different species, in our case from A. annua.

At the same time total RNA from trichome enriched cells of A.anua was extracted. Using total RNA, cDNA pool was prepeared.

Researchers were hoping to find enzymes from P450 family that existed and were transcribed and translated in A. annua, because there was a strong possibility , that one of those enzymes was the enzyme they were looking for.

Retrieved cDNA pool and synthesized degenerative primers were used in PCR reaction. Result of this pcr reaction was the isolation of several unique P450 fragments from an A. annua trichome-enriched complementaryDNA pool.

Comparing DNA sequences of those fragmets single A. annua P450 gene fragment was spotted. It had 85–88% identity at the amino-acid level to ESTs of unknown function from both sunflower and lettuce. Sequence identity of this A. annua P450 fragment to other P450 fragments outside the Asteraceae family was much lower. This P450 gene was therefore a sutable candidate for a cytochrome P450 monooxygenase (CYP71AV1)/amorpha-4,11-diene oxidase (AMO) enzyme.

With DNA sequence, isolation of open reading frame of 495 amino acids of CYP71AV1 enzyme from A. annua was possible.

As previously stated CYP71AV1 as all cytochrome P450 enzyme needs its native redoxs partner. They used A. annua cytochrome P450 oxidoreductase (CPR) as a redox partner.

rewriting the genome of ordinary Saccharomyces cerevisiae to encourage it to make artemisinic acid

With missing genes discovered Ro and coworkers could start developing synthetic yeast strain which could produce atremisinic acid. What they needed to do was:

1. To engineer the farnesyl pyrophosphate (FPP) biosynthetic pathway to increase FPP production and decrease its use for sterols, 2. To introduce the amorphadiene synthase gene (ADS) from A. annua into the high FPP producer to convert FPP to amorphadiene, and 3. To clone a novel cytochrome P450 that performs a three-step oxidation of amorphadiene to artemisinic acid from A. annua and expressing it in the amorphadiene producer


Yeast Integrating plasmids (YIp): These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination.

References

Layne SP. "Principles of Infectious Disease Epidemiology" (PDF). EPI 220. UCLA Department of Epidemiology. Archived from the original on 2006-02-20. Retrieved 2007-06-15) Brock Biology of Microorganisms (11th Edition) (2006) by Michael T. Madigan, John M. Martinko, David Stahl, David P. Clark Foley M, Tilley L (1998) Quinoline antimalarials:Mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87 Warhurst D. (2001) New developments: Chloroquine-resistance in Plasmodium falciparum. Drug Resistance Updates 4:141–144

Liu JM, Ni MY, Fan JF, Tu YY, Wu ZH, Wu YL, Chou WS (1979) Structure and reaction of arteannuin. Acta Chim Sin 37:129–143

Zeng Q, Qiu F, Yuan L. Production of artemisinin by genetically-modified microbes. Biotechnol Lett. 2008;30:581–592.

Connolly JD, Hill RA (1991) Dictionary of terpenoids. Vol 1, Mono- and Sesquiterpenoids. Chapman and Hall, London

Covello PS, Teoh KH, Polichuk DR, Reed DW, Nowak G (2007) Functional genomics and the biosynthesis of artemisinin. Phytochemistry 68:1864–1871

Akhila A, Thakur RS, Popli SP (1987) Biosynthesis of artemisininin Artemisia annua. Phytochemistry 26:1927–1930

Boumeester HJ, Wallaart TE, Janssen MH, van Loo B,Jansen BJ, Posthumus MA, Schmidt CO, de Kraker JW, Knig WA, Franssen MC (1999) Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis. Phytochemistry 52:843–854

Bertea, C. M. et al. Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med. 71, 40–-47 (2005).

Roland Sigel; Sigel, Astrid; Sigel, Helmut (2007). The Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences. New York: Wiley. ISBN 0-470-01672-8.

Adams MD, Kelley JM, Gocayne JD, et al. (Jun 1991). "Complementary DNA sequencing: expressed sequence tags and human genome project". Science 252 (5013): 1651–6. doi:10.1126/science.2047873. PMID 2047873.

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