## 3 The Biosynthesis of Artemisinin (Qinghaosu)
### Genetic Engineering
If this can be achieved, then it should - at the very least - be possible to produce dihydroartemisinic acid (**480**), rather than artemisinin acid (**473**), by the fermentation of simple sugars (in fact, the production of dihydroartemisinic acid (**480**) in yeast using the Dbr2 gene has now been described [298] - see also discussion at the end of Section 3.2 - and the use of substrate-promiscuous enzymes as an alternative means for the microbial production of (**480**) is also under investigation [387]). If the final stages in the conversion of dihydroartemisinic acid (**480**) to the 1,2,4-trioxane ring in artemisinin (**495**) (phase 3 in Section 3.3) do turn out to be enzymatically catalysed, then it may indeed be possible to genetically engineer all the enzymes for the complete biosynthetic pathway to artemisinin (**495**) into a microbial system. The current evidence, however, points to the final steps in the biosynthesis of artemisinin (**495**) being non-enzymatic - most probably proceeding by spontaneous autoxidation reactions, which occur in the hydrophobic environment of a glandular trichome. If this is the case, then the simplest approach might be to engineer a microorganism to produce dihydroartemisinic acid (**480**) which is exported out of the cell, where it can more easily be isolated and purified [in much the same way that genetically-engineered yeast accumulates artemisinin acid (**473**) on the cell surface]. Inevitably, a chemical oxidation step would still then be required in order to convert microbially-produced dihydroartemisinic acid (**480**) to artemisinin (**495**), but this would certainly represent a saving in time and cost by comparison with the two-step chemical transformation (both reduction and oxidation), which is required for microbially-produced artemisinin acid (**473**). An alternative more radical approach would be to attempt to mimic within the microbial fermentation medium the spontaneous autoxidation reactions of dihydroartemisinic acid (**480**) which are believed to occur in the glandular trichomes of _A. annua_ plants. This would require the inclusion of some kind of hydrophobic phase in intimate association with the fermentation medium. Previously, dodecane has been incorporated into the culture medium, in order to provide a separate hydrophobic layer in which volatile amorpha-4,11-diene (**451**) produced by microbial fermentation can be trapped [382, 385]. It would be interesting to investigate whether alternative hydrophobic phases can be found which provide a lipophilic environment similar to that of the glandular trichome, in which spontaneous autoxidation is favoured. (Such a phase would also need to be able to efficiently \"trap\" dihydroartemisinic acid and to be non-toxic to the fermenting organism). It should be noted that our understanding of how the spontaneous autoxidation of natural products such as **480** occurs in the absence of a photosensitizer [388] (or, indeed, any other additional chemical reagents [218]), leading to highly oxidized products such as **495**, is still incomplete. This is a relatively unexplored area which requires fundamental research, but the ultimate prize would be the production of artemisinin, both cheaply and reliably, by a single fermentation process, which incorporates an \"in-built\" spontaneous autoxidation step.