# Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
## 6 Opiates
somniferum_ genes, 6-\(O\)-demethylase (_T6 DDM_), codeinone reductase isoform 1.3 (\(COR1.3\)) and codeine \(O\)-demethylase (\(CODM\)), were assembled into a single yeast artificial chromosome (YAC) and introduced into yeast. In one pathway, thebaine is converted into codeinone by T6 DDM via the formation of neopinone. Then, COR reduces  
Figure 15: Heterologous biosynthetic pathway installed in _Saccharomyces cerevisiae_ for de novo production of morphinan alkaloids and their semi-synthetic compounds from simple sugar [78]. Only the pathway leading to the formation of the key biosynthetic intermediate thebaine is shown. For the biosynthetic pathway leading to the formation of morphine and semi-synthetic morphinan opioids, see Figure 14. 4-HPP, 4-hydroxyphenylpyruvate; 4-HPAA, 4-hydroxyphenylacetaldehyde; L-DOPA, L-3,4-dihydropyoxyphenylalanine; Aro8, _S. cerevisiae_ aromatic aminotransferase I; Aro9, _S. cerevisiae_ aromatic aminotransferase II; TyrH, tyrosine hydroxylase; DODC, L-DOPA decarboxylase; Aro10, _S. cerevisiae_ phenylpyruvate decarboxylase; NCS, norocol taurine synthase; CYP80B, _N_-methylcolaurine 3′-hydroxyylase; DRS-DRR, 1,2-dehydroreticline synthase–1,2-dehydroreticline reductase; SalSyn, salutaridine synthase; SalR, salutaridine reductase; SalAT, salutaridinol 7-\(O\)-acetyltransferase. the carbonyl of codeinone to yield codeine. Finally, codeine is demethyllated to morphine by CODM (Figure 14, in black). With the feeding of thebaine, the yeast strain carrying the YAC was able to produce codeine and morphine successfully. However, the pathway performance was improved by supplementing the culture medium with 2-oxoglutarate, a key co-substrate of dioxygenases T6 ODM and CODM, as well as the precursors of 2-oxoglutarate, L-glutamine and L-glutamic acid. The copy number of the three genes was also optimized, where the ratio of 2:1:3 for _T6 ODM:COR1.3:CODM_ copy number was determined to give the best result with 5.2 mg/L of morphine being produced. The morphine-producing yeast was further engineered with two genes, morphine dehydrogenase (_morA_) and morphinone reductase (_morB_) from _Pseudomonas putida_ M10 to biosynthetically prepare semi-synthetic morphine derivatives, hydromorphone, hydrocodone and oxycodone (Figure 14, in red). Most recently, the yeast engineering effort was able to develop a strain capable of producing thebaine and hydrocodone from simple sugar [78]. To eliminate the need for supplementation of THP or thebaine, steps leading to the formation of (_S_)-reticuline were established by incorporating five plant-derived genes, including _6 OMT_, _CNMT_ and _4'OMT_ from _P. somniferum_ that were also used in the previous study [71]. In this system, dopamine, which is formed from L-tyrosine by the combined action of a mammalian and a bacterial enzyme, and 4-hydroxyphenylacetaldehyde (4-HPAA), an intermediate in the aromatic amine degradation pathway [80], were combined together by _Coptis japonica_ (Japanese goldthread) norocolaurine synthase (NCS) to form (_S_)-norcocalaurine (Figure 15). Compared to THP, norocolaurine lacks one hydroxyl group. Thus, _CYP80B_, another gene from the _Eschchcholzia californica_ (California poppy) _N_-methylcocalaurine 3'-hydroxylase, also had to be included in the pathway to produce (_S_)-reticuline. Once (_S_)-reticuline became available, it was converted into (_R_)-reticuline by introducing 1,2-dehydroreticline synthase-1,2-dehydroreticline reductase (DRS-DRR), a natural fusion protein from _Papaver bracetatum_ (Iranian poppy), into the system to initiate the morphinan alkaloid biosynthesis. Unlike the previous study that used the human P450 CYP2D6 to convert (_R_)-reticuline into salutaridine (Figure 13, in red) [71], this study employed an engineered _P. bracetatum_ salutaridine synthase (SalSyn) that was fused to another plant P450 relianthifoline synthase to drive proper folding and glycosylation of _P. bracetatum_ SalSyn in yeast. This fusion enzyme attained an approximately six-fold greater activity than the native _P. bracetatum_ SalSyn [78].