CH391L/S14/Artemisinin

=Artemisinin= Artemisinin is a natural plant product whose derivatives form the basis for antimalarial medication.

History
Also known as wormwood or quinghao, artemisinin was used in ancient China to treat fevers and what is now known as malaria. The compound was rediscovered in 1970s as part of an effort to fight strains of malaria that had become resistant to contemporary medications. Artemisinin derivatives with improved pharmacokinetic properties were developed, and by 2002 the World Health Organization had named these compounds the first line in defense against uncomplicated malaria caused by Plasmodium falciparum. Paddon2014

Artemisinin is traditionally isolated in very low yields from the plant Artemesia annua. Additionally, approximately 18 months pass between planting and isolation of artemisinin. Fluctuations in supply and high costs threaten the availability of the compound in the developing countries where it is most needed. Paddon2014 Artemisinin therefore represents an important synthetic target. A successful synthesis would produce artemsinin, or a precursor, on the order of 25 g/L to be able to supplement global supply in a meaningful and feasible way. Paddon2014 The most successful strategy thus far, outlined below, has been to engineer microbes to produce an intermediate on the path to artemisinin which is converted by chemical reactions into artemisnin.

Semisynthetic Route
The pathway to artemesinin utilized in A. annua is shown in Figure 2. By manipulating certain key features of this route, the Keasling lab was able to achieve artemisinic acid concentrations of 25 g/L. Artemisinic acid was chosen as the target because the route to the more direct precursor, dihydroartemisinic acid, was not well understood and conversion between the two is simple.

Optimization of amorphadiene production
The first committed intermediate in the route to artemesinin is amorphadiene, which is derived from the isoprenoid farnesyl diphosphate (FPP). To increase overall flux into the artemisinin synthesis pathway, enzymes of the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway, which produces FPP in E. coli, were overexpressed in that organism. This strategy increased production of pathway intermediates, but only to low mg/L amounts. Reiling2004

In yeast, FPP is produced from an alternate route- the mevalonate pathway. Martin and coworkers Martin2003 split the genes encoding the mevalonate pathway onto two plasmids (MevT and MevB) and expressed them in E. coli, as shown on the right side of Figure 3, below. They also expressed an ADS enzyme optimized for E. coli, which as shown in Figure 2 converts FPP to the amorphadiene intermediate. Strains expressing the mevalonate plasmids and the ADS, with optimized fermentation conditions, produced 0.5 g/L amorphadiene.

Studies by Pitera and coworkers showed that increased expression of the mevT operon caused inhibition of growth. Pitera2007   More specifically, accumulation of an intermediate in the part of the mevalonate path that was encoded by MevT plasmid inhibited fatty acid biosynthesis. To address this issue, the expression of enzymes just upstream and downstream from the intermediate was reduced and increased, respectively. Along with that change, replacement of the downstream enzymes with genes from other species resulted in production of amorphadiene at concentrations of about 25g/L. Pfleger2006 Tsuruta2009

Optimization of artemisinic acid production
Once acceptable amorphadiene production levels were achieved, efforts were made to increase production of the more direct precursor artemisinic acid. In A. annua, amorphadiene is converted to more oxidized compounds, including artemisinic acid, by a cytochrome P450 (CYP71AV1). When expressed in amorphadiene-producing S. cerevisae, CYP71AV1 was shown to accomplish the conversion. Ro2006   However, E. coli is generally unable to express eukaryotic P450 enzymes. Parallel tests of E. coli and S. cerevisae found that S. cerevisae produced higher concentrations of artemisinic acid (2.5 g/L vs 1 g/L for E. coli) under conditions suitable for scaling Lenihan2008  and was therefore chosen as the host organism.

Once yeast was determined to be the best organism, a strain with desirable properties (CEN.PK2) was chosen and its mevalonate pathway was overexpressed to increase amorphadiene production. Further process improvements led to a strain producing 40 g/L of amorphadiene. Westfall2012 However, expression of CYP71AV1 did not lead to increased artemisinic acid levels.

Cells that expressed CYP71AV1 were found to exhibit decreased viability. The possibility that poor interaction between CYP71AV1 and its cognate reductase was creating destructive reactive oxygen species merited a decrease in the expressed reductase. Paddon2014 Paddon2013 Additionally, cytochrome b5 from A. annua was expressed to increase the rate of P450. Zhang2007 These changes were beneficial in amount of artemisinic acid but produced high levels of the presumably toxic intermediate artemisinic aldehyde. Paddon2013

Drastic improvement in the production of artemisinic acid was achieved through coexpression of artemisinic aldehyde dehydrogenase and artemisinic alcohol dehydrogenase, both from A. annua. When the mevalonate pathway was constitutively expressed in this strain, Paddon et al report that the target concentration of 25 g/L arteminisic acid was achieved. Paddon2013

Conversion of artemisinic acid to artemisinin
The route from artemisinic acid to artemisinin is shown in Figure 4. The four major steps include reduction of artemisinic acid to the more direct precursor dihydroartemisinic acid. Esterification at the acid protects that moiety during the subsequent formation of the hydroperoxide. Hock fragmentation and rearrangement gives the artemisinin in 40-45% yield. Paddon2013

Future directions
This method of utilizing reaction pathways in nature to achieve complex chemical transformations only becomes more exciting as new synthetic biology tools are developed. More efficient ways to study and manipulate genetic systems will allow access to much more difficult and complicated structures. Paddon Paddon2014 illustrates other molecules whose synthesis might be aided by a similar semisynthetic route, including vincristine, morphine, and prostratin. The artemisinin semisynthesis was successful in part due to thorough understanding of the pathway used. Targets with well known biosynthesis pathways may be more likely to succeed.

Other strategies
While the semisynthetic route has been most successful, there are other strategies. For example, Zhu et al completed a total synthesis of artemisinin. Zhu2012 While their starting material was inexpensive, it is not ready to be scaled up to global supply. Others have attempted to use transgenic approaches to improve artemisinin yields in A. annua or other plants, such as tobacco. These efforts seem to mimic the same strategies embraced the semisynthetic Keasling method in a plant host (ie overexpress key enzymes). While improvements have been made, they are not on the order of the semisynthetic route. Tang2014

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VCU (2011) attempted to introduce isoprenoid pathways into Synechococcus elongatus for possible use as host organisms. Success in this endeavor was not clear.