# Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
## 6 Opiates  
The last section will look at opiates, which is a group of alkaloids that has been known to humans for about 6000 years and played a significant role as analgesic therapeutics and as abused recreational substances [66]. Opiates are naturally occurring members of opioids that can be isolated from the latex collected from opium poppy _Papaver somifierum_. The representative examples of opiates include reticuline, thebaine, codeine and morphine (Figures 12-15). Opioids also include semi-synthetic and synthetic compounds, for example oxycodone and fentanyl, that interact with opioid receptors to effect physiological responses, such as analgesia, euphoria and respiratory depression [67]. Currently, the pharmaceutical industry relies heavily on plants as the predominant source of opiates and opiate-derived compounds. However, plant cultivation is labor intensive, difficult to scale up and hard to maintain yield consistency. While metabolic engineering of plants has been attempted to improve growth parameters and opiate productivity, the efforts have generally been met with difficulties thus far [68]. More recently, metabolic engineering of microbes has made a significant progress and is quickly becoming a viable alternative strategy for improving the opiate production process. Not only microbe cultivation is lower in cost and easier to scale up than plant cultivation, the product can be purified more easily from microbial extracts than from plant extracts due to lack of similar metabolites [47; 69; 70]. Below, we will discuss recent successes in developing microbe-based biosynthetic systems that produce a type of opiates called benzylisoquinoline alkaloids (BIAs). Figure 12: Heterologous biosynthetic pathway constructed in yeast (_Saccharomyces cerevisiae_) for the production of (\(R\),\(S\))-reticulate from the exogenously provided substrate, (\(R\),\(S\))-tetrahydropropanazepine (THIP, also known as norlaudanosoline) [71]. 6 OMT, norocolaurine 6-O-methyltransferase; CNMT, coclaurine \(N\)-methyltransferasease; 4’OMT, 3’-hydroxy-N-methylicolaurine 4’-O-methyltransferase. Figure 13: Extension of the _Saccharomyces cerevisiae_ heterologous reticuline biosynthetic pathway for the production of precursors of the berberine- and sanguinarine-type benzylisoquinoline alkaloids (in black) and the morphinan alkaloids (in red). Compounds that are actually biosynthesized by the engineered yeast strains [71; 72] are shown in solid, whereas downstream products are shown in semi-transparent. BBE, berberine bridge enzyme; SMT, (S)-scouberine 9-O-methyltransferasease; CYP19A, canadine synthase cytochrome P450 719A; COX, canadine oxidase; CYP2D6, human cytochrome P450 2D6. In one approach toward developing microbes that can biosynthesize opiates, [PERSON] et al. engineered yeast _S. cerevisiae_ to produce a series of BIAs. First, they developed a yeast strain harboring a set of biosynthetic genes that successfully produced (_R_,_S_)-reticuline upon feeding of a racemic mixture of tetrahydropropagaveroline (THP), also known as norlaudanosoline (Figure 12) [71]. To produce reticuline, the key starting intermediate in the BIA biosynthetic pathway, a pair of plasmids carrying three biosynthetic genes encoding norcocal taurine 6-\(O\)-methyltransferase (6 OMT), cocalurine-N-methyltransferase (CNMT) and 3\({}^{\prime}\)-hydroxy-\(N\)-methylcocalurine 4\({}^{\prime}\)-\(O\)-methyltransferase (4\({}^{\prime}\)OMT) from _Thalictrum flavour_ (yellow meadow-rue) or _P. somiferum_ were introduced to _S. cerevisiae_. All three enzymes were known to accept both (_R_)-and (_S_)-enantiomers of their substrates [74; 75]. The yeast strain carrying those three genes was able to biotransform the fed (_R_,_S_)-THP into (_R_,_S_)-reticuline (Figure 12). Then, a plasmid-born biosynthetic pathway comprised of the _P. somiferum_ berberine bridge enzyme gene (_BBE_), the _T. flavour_ (_S_)-scouberine 9-\(O\)-methyltransferase gene (_SMT_) and the _T.