# Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
## 3 Saframycin  
Saframycin A (**9**) isolated from _Streptomyces laverndulae_ is a representative member of the tetrahydroroisoquinoline alkaloids (Figure 7) [21]. This class of natural products has been isolated from various organisms, including soil bacteria and marine invertebrates, such as sponges and ascidians [22]. These compounds exhibit various antibiotic activities but also possess a potent antitumor activity, which is thought to be effected through covalent modification of DNA by iminium ions via carbinolamine moiety and its equivalents [23]. Among the saframycin analogs, ecteinascidin 743 (**10** in Figure 7) [24] showed a remarkably potent antitumor activity against not only common tumor cell lines but also resistant cell lines [25]. Recently, **10** was approved as an anticancer drug in the European Union for patients with soft tissue sarcoma [26]. Because of the limited supply from natural sources, **10** is currently produced by a semisynthetic process [27]. However, this synthesis requires more than 25 chemical transformation steps, making it a suboptimal method for a commercial production of **10**. Engineered biosynthesis for generating this important alkaloid as an alternative strategy is strongly desired, especially from the perspective of sustainability and environmental burden  
Figure 6: Proposed mechanism of DtpC-catalyzed formation of dimerization products (\(-\))-dibreviianamide F **5**[9], a new compound **7**[14] and naseaseazine B **8**[15]. Radical-mediated formation of **7** and **8** is thought to be initiated by DtpC forming a radical at N1 instead of N10 as proposed for the formation of **5**. The radical species are labeled by the position of the radical. To differentiate between radical species derived from hydrogen extraction at N10 (**left column**) vs. N1 (**right column**), those formed via the N1 hydrogen extraction are given a label with a superscript N1. Chemical structures of related known natural products are shown in the box. The monomer-bridging bonds are shown in red. of the synthetic manufacturing process. To put this bioproduction approach into practice, detailed understanding of the biosynthetic mechanism of this class of natural products is essential. Therefore, there has been a considerable study on the subject involving gene isolation [28; 29; 30], substrate feeding experiments [31; 32], biochemical characterizations [30; 33] and bioinformatic analyses [30]. In this review, the current understanding of the mechanism of biosynthesis of **9** is described. Investigation of the biosynthesis of **9** and related compounds has been ongoing for quite some time after the initial discovery of **9** in 1977 [21]. By 1985, it was determined that the main framework of **9** was comprised of one molecule each of L-alanine and glycine and two molecules of L-tyrosine [31]. The alanyl-glycyl terminal moiety was found to be modifiable by feeding of particular amino acids, such as 2-amino-_n_-butylic acid [32]. The initial discovery of a saframycin biosynthetic gene cluster was made in _Myxococcus xanthus_, where two NRPS-encoding genes and an \(O\)-methyltransferase gene responsible for the formation of the core structure were identified [28]. The findings from this study confirmed that the main framework of **9** was biosynthesized by NRPS. The subsequent study on the gene cluster for the biosynthesis of safracin, a compound closely related to **9**, revealed additional genes proposed to be involved in the modification of building block amino acids and post-NRPS intermediates [29]. The most recent study provided the most complete information on the saframycin biosynthetic gene cluster with a total of 30 genes that are proposed to be involved in the biosynthetic process [30]. Based on the bioinformatically predicted functions of the genes that were identified as described above, various biosynthetic schemes were hypothesized [29; 31; 34]. However, no actual experimental data were available to verify the proposed mechanisms.