# Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids
## 5 Ergotamine  
Ergot alkaloids form another family of alkaloids under the structurally diverse group of TIAs. Unlike MIAs described in the previous section that use GPP and l-tryptophan as their building blocks, ergot alkaloids are derived from dimethylallyl pyrophosphate (DMAPP) and l-tryptophan [52]. The ergot type of natural products exerts biological activities by acting as either an agonist or an antagonist toward various receptors for a number of neurotransmitters, such as dopamine, serotonin, adrenaline and noradrenaline [53]. Certain members of ergot alkaloids have also been shown to exhibit anti-tumor activities [54]. The potent bioactivities of ergot alkaloids have led people to use them in various medical applications since early in the human history, with the oldest record dating back to around 1000 B.C.E. [55]. The ergot alkaloids are produced by fungi and plants, but the Claviicipitaceae family of fungi is most well known for ergot production. Among the Claviicipitaceae family, _Claviiceps purpurea_, an endophytic parasite that is also known as the ergot fungus of rye, and _Neotyphodium lolii_, an endophytic symbiont, are the two most-studied ergot-producing fungi [52]. Another fungus known for production of the ergot family of natural products, primarily clavine-type alkaloids, is _Aspergillus fumigatus_, an opportunistic pathogen of mammals [56]. Below, we will focus on the biosynthetic pathway of ergot alkaloids, and in particular discuss the mechanism involved in the formation of the interesting backbone core of 6,8-dimethylergoline tetracyclic ring structure (**14**) that is shared by all ergot alkaloids (Figure 10). Biosynthesis of ergot alkaloids was initially investigated through feeding experiments that used isotopically labeled substrates in the cultures of _C. purpurera_[53]. Based on those experiments, the ergot biosynthetic pathway was determined to be initiated by the prenylation of l-tryptophan with DMAPP that forms 4-(\(\gamma\),\(\gamma\)-dimethylallyl) tryptophan (**15**) (Figure 10) [57; 58]. The next step involves the \(N\)-methylation of **15** to yield 4-dimethyl-l-abrine (**16**) [59]. Subsequently, a series of successive oxidation steps is proposed to take place, resulting in the intramolecular cyclization of the prenyl and indole moieties to form the tricyclic chanoclavine-I (**17**) along with ring C of the tetracyclic  
Figure 10: Proposed biosynthetic pathway of d-lysergic acid **20**[53], having the principal 6,8-dimethylergoline tetracyclic ring structure **14** of ergot alkaloids. core of ergot alkaloids [60]. Compound 17 is then oxidized to form chanoclavine-I-aldehyde (**18**) from which ergot alkaloid biosynthesis can diverge. In one pathway, 18 can undergo intramolecular cyclization to form ring D to complete the tetracycline ergoline core structure, yielding agroclavine (**19**). Subsequently, **19** is converted to d-lysergic acid (**20**) via an oxidation of a methyl group as illustrated in Figure 10. In another pathway, **18** undergoes a similar cyclization to form a closely related tetracycline intermediate, festuclavine, which becomes the core molecule for the clavine-type fumigaclavines and related compounds produced by _A. fumigatus_. In this section, we will focus on the biosynthesis of ergotamine (**21**) from **20**, which takes place in _C. purpurea_ and _N. lolii_. Further steps in the biosynthesis of ergotamine (**21**) have been elucidated predominantly by [PERSON] et al. [61; 62; 63; 64]. Those steps mainly concern the formation and the modification of the peptide-derived segment of **21** (Figure 11). The peptide extension of **20** is initiated by LPS2 (lysergic peptide synthetase **2**, NRPS). The adenylation domain of LPS2 accepts **20** as the starter unit, and the condensation domain couples it with L-alanine, which is activated by the first adenylation domain of LPS1 (lysergic peptide synthetase **1**, NRPS), through an amide linkage formation.