# Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
## 4 Strictsidine  
Terpene indole alkaloids (TIAs) constitute one of the largest and most diverse families of complex nitrogen-containing metabolites found in plants [38; 39]. Many TIAs, especially monoterene indole alkaloids (MIAs) and their analogs, have medicinal properties: camptothecin and vinblastine are approved as anticancer therapeutics, whereas reserpine is used as an antihypertensive [40]. Physostigmine is an antidote of anticholinergic toxicity and is used primarily for treating glaucoma [41]. However, these structurally complex compounds can be difficult to synthesize chemically [42; 43]. To supply these compounds through biosynthetic routes, the biosynthetic pathway for a target compound must be elucidated. Once all of the genes involved in the pathway are identified, a host organism that is more convenient to cultivate than a plant can be used for heterologous production of the desired compound. Furthermore, elucidation of the biosynthetic pathway and establishment of a heterologous system would allow engineering of the pathway for efficient analog production. From these viewpoints, the most important compound in this class of natural products to engineer a heterologous production system would be strictosidine (**11**), because it is the universal intermediate for the biosynthesis of all MIAs. Below, the biosynthetic pathway leading to the formation of **11** and recent efforts in the engineered biosynthesis of **11** using a yeast host system are discussed. The general mechanism of the biosynthesis of **11** has been reviewed elsewhere [38]. Briefly, **11** is formed with two starting substrates, geraniol and l-tryptophan. The first starting substrate geraniol is  
Figure 8: Proposed overall scheme for the pentacyclic core skeleton assembly during the biosynthesis of saframycin A **9**[35]. SfmA, SfmB and SfmC are three saframycin A nonribosomal peptide synthetases. C, condensation domain; A, adenylation domain; T, thiolation domain; Re, reductase domain. initially converted into a dihydroxy intermediate, 8-hydroxygeraniol, by geraniol 8-hydroxylase (G8H). Then, both hydroxyl groups are oxidized to form 8-oxogerani. Subsequent cyclization and oxidation of 8-oxogeranial yields loganic acid, and methylation of the carboxylic acid moiety of loganic acid results in the formation of loganin (**12**) (Figure 9). The following transformation of **12** into secologanin (**13**) is an interesting reaction that is catalyzed by secologanin synthase (SLS, a cytochrome P450). This step is thought to be the rate-limiting step in the biosynthetic pathway leading to the formation of **11**[44]. SLS is proposed to catalyze the opening of the 5-membered ring of **12** by a radical-mediated hydroxylation, followed by a dehydration to yield **13** (Figure 9) [45]. The other starting substrate L-tryptophan is modified through a decarboxylation step to afford tryptamine, which is combined with **13** to give **11** by the action of strictosidine synthase (STR, an amine lyase), a key enzyme in MIA biosynthesis. This transformation would be the most chemically interesting step in the strictosidine biosynthetic pathway; it is an another example of the Pictet-Spengler reaction applied toward the biosynthesis of alkaloids, as it was also mentioned during the discussion of the saframycin biosynthesis, (Figure 9). As to the formation of **11**, the reaction is proposed to occur between the aldehyde of **13** and the amine of tryptamine to couple the two substrates through the formation of a \(\beta\)-carboline moiety [46]. To develop a yeast strain that can biosynthesize **11** de novo, 21 gene additions and three gene deletions were introduced into the yeast genome [47]. Fourteen of the added genes were known genes from a MIA producer plant _Catharantl thus roseus_[48]. Of the 14 genes, 11 constituted the actual strictosidine biosynthesis pathway and the other three were involved in enhancing the activity of P450s.