Abstract:The tropane and granatane alkaloids belong to the larger pyrroline and piperidine classes of plant alkaloids, respectively. Their core structures share common moieties and their scattered distribution among angiosperms suggest that their biosynthesis may share common ancestry in some orders, while they may be independently derived in others. Tropane and granatane alkaloid diversity arises from the myriad modifications occurring to their core ring structures. Throughout much of human history, humans have cultivated tropane-and granatane-producing plants for their medicinal properties. This manuscript will discuss the diversity of their biological and ecological roles as well as what is known about the structural genes and enzymes responsible for their biosynthesis. In addition, modern approaches to producing some pharmaceutically important tropanes via metabolic engineering endeavors are discussed.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca . We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/ N -methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

Tropane alkaloids are found in a scattered distribution among the angiosperm families including members within the Solanaceae, Erythroxylaceae, Convolvulaceae, and Brassicaceae. Recent studies regarding the origins of tropane production provide strong evidence for a polyphyletic origin, suggesting that novel enzymes from different gene families have been recruited during the course of flowering plant evolution. Tropane alkaloid biosynthesis is best documented on the molecular genetic and biochemical level from solanaceous species. Regardless of the system chosen, there are

The lack of data concerning the enzymes and genes responsible for the production of tropane and granatane alkaloids has hindered endeavors to metabolically engineer important pharmacologically active specialized metabolites. It is important to understand how biochemical pathways perform when they originate from evolutionarily diverse families. It is hypothesized that a type III polyketide synthase enzyme is involved in the extension and subsequent cyclization of the second ring in the bicyclic tropane and grantane core structure. This represents a new class of type III polyketide synthases in which the heterocyclic starter molecule is charged and also lacks a Coenzyme A thioester. Putative polyketide synthase enzymes were identified in the Erythroxylum coca and Punica granatum cDNA transcriptome database and heterologously expressed in Pichia pastoris. LC‐MS/MS analysis has revealed the formation of a 4‐(1‐methyl‐2‐pyrrolidinyl)‐3‐oxobutanoate intermediate that is hypothesized to lead to the second ring formation. Understanding how evolutionary changes have occurred in type III polyketide synthases from different metabolic pathways that determine the development and expansion of chemical diversity to produce high valued compounds will be a powerful tool for bioengineering pharmaceuticals.Support or Funding InformationNational Science FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.