# Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
## 6 Opiates
flavour_ canadine synthase gene (_CYP719A1_, a P450 gene) was established in the reticuline-producing yeast to produce BIA products found along the sanguinarine and berberine biosynthetic pathways. This yeast strain was able to produce (_S_)-scouberine (sanguinarine precursor), (_S_)-tetrahydrocolumbamine and (_S_)-tetrahydroberberine (also known as canadine, a berberine precursor) (Figure 13, in black) [71]. Subsequently, incorporation of the canadine oxidase (COX) from _Berberis wilsonae_[76] and optimization of the berberine biosynthetic pathway in yeast led to successful production of berberine from fed (_R_,_S_)-THP [72]. Furthermore, a successful production of the first intermediate of the morphinan alkaloid biosynthesis pathway, salutaridine, was also accomplished by introducing a human P450 gene _CYP2D6_ into the reticuline-producing yeast strain (Figure 13, in red). Related to this work, another group also established a biosynthetic pathway in yeast that converted fed (_R_,_S_)-THP into dihydrosanguinarine, an immediate precursor of sanguinarine [77], through the formation of (_R_,_S_)-reticuline and (_S_)-scouberine using a biosynthetic scheme that was practically identical to that developed by [PERSON] et al. [71]. Figure 14: Heterologous biosynthetic pathway constructed in _Saccharomyces cerevisiae_ for the production of morphinan alkaloids and related semi-synthetic opioids from thebaine, the exogenously provided starting material [73]. The biosynthetic pathway leading to the formation of morphine, the prominent member of the morphinan alkaloids, is shown in black. The biosynthetic pathways leading to the formation of semi-synthetic opioids, hydromorphone, hydrocodone and oxycodone, are shown in red. T6 ODM, thebaine 6-\(O\)-demethylase; COR, codeinone reductase; CODM, codeine \(O\)-demethylase; MorA, morphine dehydrogenase; MorB, morphinone reductase. One unique strategy developed by [PERSON] et al. for optimizing the performance of a heterologous biosynthetic pathway in yeast was to use a GAL1-10 promoter system that is tunable with galactose concentration. Using this tunable expression system, [PERSON] et al. determined the optimal expression level of each of the heterologous genes for the production of desired products in yeast. Once the optimal expression level for each of the genes was determined, promoter of appropriate strength was selected for each of the genes to fine-tune the overall performance of the biosynthetic pathway. This type of optimization may be crucial in this type of study, where genes from diverse sources are assembled together to form a cooperative metabolic pathway. Streamlining the identification of the bottleneck in a newly installed heterologous metabolic pathway can also be accomplished by using a systems biological approach to illuminate the source of stress that is imposed on the host cell by expressing a foreign gene. In one example, effect of overproducing a foreign P450 in yeast was analyzed by DNA microarray analyses to reveal that transcription factors associated with iron starvation and heme-dependent gene regulation were overrepresented when the foreign P450 gene was expressed. This result indicated heme depletion as a possible stress in the system, and the activity of the target P450 was elevated by engineering the host's heme biosynthesis to increase the intracellular heme level [79]. A similar approach can be taken to identify the source of stress in an engineered strain so that necessary measures can be implemented to eliminate the bottleneck in the exogenous biosynthetic pathway of interest. The [PERSON] group also developed engineered yeast strains that can convert fed thebaine into codeine, morphine, hydromorphone, hydrocodone and oxycodone (Figure 14) [73]. To establish the biosynthetic pathway that converts thebaine into morphine, three _P.