## 3 The Biosynthesis of Artemisinin (Qinghaosu)
### Genetic Engineering  
Metabolic engineering has attracted increasing attention over recent years as an alternative means for the production of plant-derived drugs. Both artemisinin from _A. annua_ and taxol from _Taxus brevifolium_ are attractive targets for this emerging science of synthetic biology since both drugs are available in only limited quantities from the natural source and are also difficult to synthesize chemically [364]. Research into the production of artemisinin from such genetically modified microorganisms has been well reviewed [365-369]. The goal of synthetic biology is to reprogram a microorganism for the efficient production of a natural product by establishing new metabolic pathways, which lead to the formation of the desired metabolite, whilst simultaneously removing existing metabolic pathways which detract from the formation of such a product. To date, investigators have concentrated on expressing the enzymes from phases 1 and 2 of the biosynthetic route to artemisinin in transgenic organisms (see Scheme 9 in Section 3). These have included: HMG-CoA reductase (HMGR) [370]; famesyl diphosphate synthase (FPPS) [371-374]; amorpha-4,11-diene synthase (ADS) [375-377]; and the P\({}_{450}\) enzyme which oxidises amorpha-4,11-diene to artemisinin acid (CYP71 AV1) [378] (Scheme 13). Both _Saccharomyces cerevisiae_[379,380] and _Escherichia coli_[381-383] have been used as hosts when attempting to establish a viable recombinant microbial pathway for the biosynthesis of artemisinin. The approach outlined in Scheme 13 has succeeded in producing high levels of amorpha-4,11-diene (**451**) from _E. coli_[384] and artemisinin acid (**473**) from _S. cerevisiae_ in quantities which exceed those from _A. annua_ itself (100 mg\({}^{-1}\cdot\)g\(\cdot\)L\({}^{-1}\)) [385,386]. The semi-synthesis of artemisinin (**495**) from microbially-produced artemisinin acid (**473**) then requires two further chemical steps: reduction of the exocyclic double bond; and photo-sensitized oxidation of the endocytic double bond to produce the 1,2,4-trioxane ring in (**495**) [156,237]. Fortunately, the genetically-engineered yeast transports artemisininic acid (**473**) outside of the cell, where it is retained on the cell wall, and can easily be released simply by altering the pH. It is then necessary to purify microbially-produced artemisininic acid (**473**) prior to these chemical transformations. **Scheme 13. ** Genetic engineering of enzymes for the production of artemisinin acid (**473**) from _A. annua_ in to a microbial host. In order to achieve the heterologous production of artemisinin (**495**) by a large-scale fermentation process, it will be necessary to express every step in the biosynthetic pathway to artemisinin in a microbe. Keasling has observed that \"the production of drugs _via_ heterologous pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved, and the enzymes lack detailed characterization\" [387]. These comments are certainly pertinent to the biosynthesis of artemisinin in _A. annua_, as is evident from the foregoing discussions in Sections 3.2 and 3.3. The author would argue that the prerequisite for the reconstruction of the complete biosynthetic pathway to artemisinin (**495**) in a transgenic yeast or bacterium is the elucidation of the full details of phase 2 of the biosynthesis in _A. annua_, in which amorpha-4,11-diene (**451**) is converted to dihydroartemisinic acid (**480**); as well as a better understanding of phase 3, in which **480** is then converted to **495** (see Scheme 9).