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

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.

Part one:

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 [1]. 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 [2]. 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 [3]. 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 [4].

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[5]. Today World Health Organisation recommends artemisinin-based combination therapies. Part two: 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 [6]. In order to avoid those problems, which in effect cause major fluctuations in artemisinin price and thereby making it unaffordable for third world patients, Ro and coworkers published an article describing productin of artemisinic acid in yeast Saccharomyces cerevisiae. 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 [7].

Ro and coworkers knew, that artmisinin 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. We should mention 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 [8]. Ergosterol is found in cell membranes of fungi and protozoa where stabilizes the membrane an makes it less flexible [2].

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.  


Malaria patients can be treated with highly effective Artemisinin-based Combination Therapies (ACTs), but cultivating and extracting artemisinin, which comes from the Chinese Sweet Wormwood plant, is expensive and time consuming. Lack of access to this vital compound prevents millions of people in the developing world from receiving critical ACTs. http://amyris.com/products/artemisinin/ Semi-synthetic artemisinin has been in the pipeline since 2006, when Jay Keasling’s group at the Lawrence Berkeley National Laboratory in California, US, reported rewriting the genome of ordinary brewer’s yeast to encourage it to make artemisinic acid.1 But piecing together a practical route for making the drug precursor in yeast and then transforming it into the finished product has proved tricky. It has remained cheaper and more straightforward to extract the drug from its natural source. EST Shivashankar H. Nagaraj, Robin B. Gasser, Shoba Ranganathan, A hitchhiker's guide to expressed sequence tag (EST) analysis Brief Bioinform (2007) 8 (1): 6-21 first published online May 23, 2006 doi:10.1093/bib/bbl015

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

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