# Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
## 7 Conclusions  
Over the past several decades, elucidation of metabolic pathways leading to the biosynthesis of various alkaloids has seen a tremendous advancement. Those efforts have been facilitated greatly by the rapid accumulation of genomic sequence information and the use of heterologous host organisms that allow efficient reconstitution of the biosynthetic pathway being investigated. For the heterologous host, _E. coli_, _S. cerevisiae_ and _A. nidulans_ are used most commonly. Those hosts are particularly attractive because of their fast and robust growth and ease of genetic manipulation. They also have a metabolic background that imposes low interference to the alkaloid biosynthesis being studied. In addition to those in vivo analyses, in vitro characterizations of the biosynthetic enzymes also provided deep insight into the mechanisms involved in the generation of representative members of alkaloids. Biosynthesis of some of those compounds, such as diketopiperazine and tetrahydrooisquinoline natural products, employ the use of NRPSs, while a great number of alkaloids rely on oxidoreductase, primarily cytochrome P450s and flavin-containing monooxygenases, for extensive modifications of the core scaffolds that are typically constructed from aromatic amino acids and isoprenes. Combining the current efforts in the discovery of alkaloid biosynthetic pathways with the knowledge and experiences acquired from previous successful incorporation and engineering of NRPSs, oxidoreductases and other auxiliary enzymes in bacteria, yeast and filamentous fungi could further generalize our approach for comprehensive characterizations of alkaloid biosynthetic pathways and mass-production of clinically and industrially valuable alkaloids that are typically produced in limited quantity by the original producers. Detailed knowledge of biosynthesis of those compounds will also facilitate application  
Figure 16: Heterologous biosynthetic pathway established in _E. coli_ for de novo production of (_R,S_)-reticuline from simple sugar using a three-step fermentation method [81; 82; 83]. The three fermentation steps are designated by three different colors: black for the dopamine production step, red for the (_R,S_)-THP production step and blue for the (_R,S_)-reticuline production step. The initial attempt to use the norocolaurine synthase (NCS) for the production of (_S_)-THP for the preparation of (_S_)-reticuline (shown in green) was replaced by the use of a spontaneous intracellular Pictet-Spengler reaction (shown in red) that resulted in the eventual formation of (_R,S_)-reticuline (shown in blue). Subsequent biosynthesis of thebaine and hydrocodone followed the same biosynthetic scheme shown in Figures 14 and 15, respectively. MAO, monoamine oxidase.  
of synthetic biological approaches in developing efficient and sustainable platforms for preparing semi-synthetic analogs of alkaloids.  
We wish to thank the financial support from the Japan Society for the Promotion of Science (JSPS) Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (No. G2604) (K.W.). This work was also supported in part by the Japan Society for the Promotion of Science (JSPS) (K.W., 15 KT0068, and 26560450), the Takeda Science Foundation (K.W.), the Institution of Fermentation at Osaka (K.W.), the Japan Antibiotics Research Association (K.W.), the Uehara Memorial Foundation (K.W.), and the Tokyo Biochemical Research Foundation (K.W.).  
The authors declare no conflict of interest.