## 3 The Biosynthesis of Artemisinin (Qinghaosu)
### Plant Breeding Programmes  
Plant selection and breeding programmes have often sought to combine the properties of artemisinin-rich clones of _A. annua_ (such as the Chinese and Vietnamese varieties), with more vigorous but lower-yielding clones (such as some of the European varieties) in order to achieve a robust hybrid with a high yield of artemisinin (typically 1% or more - corresponding to an agricultural yield of approximately 200 Kg/ha for dry leaves) [322-324]. There is increasing evidence that artemisinin production in _A. annua_ is localized in the biseriate glandular trichomes: specialized structures, found predominantly on the surface of leaves and flowers [244,325]. Several authors have reported that the artemisinin content is highest just before flowering [326-328] (interestingly, this trend is apparently not followed by other metabolites: the content of artemisinin ketone (**276**) decreases before flowering and then increases afterwards; caryophyllene (**405**) shows the opposite trend; while levels of monoterenes, such as cineole (**326**), bornyl acetate (**335**) and camphor (**341**) all remain relatively constant throughout growth [112]) and it has also been noted that the content of artemisinin can be up to ten-fold higher in flowers, as compared with leaves [19,329,330]. These observations may simply reflect an increased density of glandular trichomes in the flowers [301], and hence a correspondingly higher yield of artemisinin. By contrast, artemisinin acid (**473**) seems to be obtained in maximum yield at the late vegetative stage (however, the concentration of artemisinin acid in _A. annua_ (**473**) is strongly dependant on chemotype - it can be up to ten-fold higher than that of artemisinin in European and Chinese varieties, although this is reversed in Vietnamese strains).  
It has also been observed that increased illumination or sunlight results in a higher yield of artemisinin [331-333]. This would be consistent with the hypothesis that the final steps in the biosynthesis of artemisinin proceed in the trichomes by spontaneous autoxidation reactions of dihydroartemisinin acid (**480**), requiring \({}^{1}\)O\({}_{2}\), which is produced in the presence of light and a photosensitizer (e.g., chlorophyll - see step i) in Scheme 12). Several researchers have endeavoured to quantify not just the amount of artemisinin (**495**) [334], but also its presumed biosynthetic precursors, dihydroartemisinic acid (**480**) [335] and artemisinin acid (**473**) [335-337], when studying the developmental biology of _A. annua_. One particularly interesting study has concluded that correlated variations in all three natural products prove the existence of chemotypes of _A. annua_. Thus, plants with a high artemisinin (**495**) level were found also to have a high dihydroartemisinic acid (**480**) level, but a relatively low concentration of artemisinin acid (**473**); while chemotypes with low levels of artemisinin (**495**) and dihydroartemisinic acid (**480**) contained a correspondingly high concentration of artemisinin acid (**473**) [338]. These findings were explained [20] on the assumption that the enzymatic reduction of artemisinin acid (**473**) to dihydroartemisinic acid (**480**) might represent a \"bottle neck\" in the biosynthetic pathway to artemisinin (**495**). However, given that it seems now to be proven that artemisininic acid (**473**) is not a precursor to dihydroartemisinic acid (**480**) (see crossed arrow in Scheme 11) [155,185], the existence of these two chemotypes might instead be explained on the assumption that artemisininic acid (**473**) is a \"dead end\" metabolite, leading \"away\" from artemisinin (**495**) in phase 2 of the biosynthetic route, as is implied in Scheme 11.  
Finally, there have been several recent attempts to create transgenic _A. annua_ plants with elevated levels of artemisinin [339-341] by transferring genes for various enzymes in the biosynthetic pathway to artemisinin (e.g. HMGR, FPPS, ADS and CYP71 AV1 - see Scheme 13).